Advertisement

The Major Replicative Histone Chaperone CAF-1 Suppresses the Activity of the DNA Mismatch Repair System in the Cytotoxic Response to a DNA-methylating Agent*

  • Lyudmila Y. Kadyrova
    Affiliations
    From the Department of Biochemistry and Molecular Biology, Southern Illinois University, School of Medicine, Carbondale, Illinois 62901
    Search for articles by this author
  • Basanta K. Dahal
    Affiliations
    From the Department of Biochemistry and Molecular Biology, Southern Illinois University, School of Medicine, Carbondale, Illinois 62901
    Search for articles by this author
  • Farid A. Kadyrov
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Neckers Bldg., 1245 Lincoln Dr., Carbondale, IL 62901. Tel.: 618-453-6405; Fax: 618-453-6440; E-mail: .
    Affiliations
    From the Department of Biochemistry and Molecular Biology, Southern Illinois University, School of Medicine, Carbondale, Illinois 62901
    Search for articles by this author
  • Author Footnotes
    * This work was supported by NIGMS, National Institutes of Health, Grant R01GM095758. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Open AccessPublished:November 21, 2016DOI:https://doi.org/10.1074/jbc.M116.760561
      The DNA mismatch repair (MMR) system corrects DNA mismatches in the genome. It is also required for the cytotoxic response of O6-methylguanine-DNA methyltransferase (MGMT)-deficient mammalian cells and yeast mgt1Δ rad52Δ cells to treatment with Sn1-type methylating agents, which produce cytotoxic O6-methylguanine (O6-mG) DNA lesions. Specifically, an activity of the MMR system causes degradation of irreparable O6-mG-T mispair-containing DNA, triggering cell death; this process forms the basis of treatments of MGMT-deficient cancers with Sn1-type methylating drugs. Recent research supports the view that degradation of irreparable O6-mG-T mispair-containing DNA by the MMR system and CAF-1-dependent packaging of the newly replicated DNA into nucleosomes are two concomitant processes that interact with each other. Here, we studied whether CAF-1 modulates the activity of the MMR system in the cytotoxic response to Sn1-type methylating agents. We found that CAF-1 suppresses the activity of the MMR system in the cytotoxic response of yeast mgt1Δ rad52Δ cells to the prototypic Sn1-type methylating agent N-methyl-N′-nitro-N-nitrosoguanidine. We also report evidence that in human MGMT-deficient cell-free extracts, CAF-1-dependent packaging of irreparable O6-mG-T mispair-containing DNA into nucleosomes suppresses its degradation by the MMR system. Taken together, these findings suggest that CAF-1-dependent incorporation of irreparable O6-mG-T mispair-containing DNA into nucleosomes suppresses its degradation by the MMR system, thereby defending the cell against killing by the Sn1-type methylating agent.

      Introduction

      The DNA mismatch repair (MMR)
      The abbreviations used are:MMR,DNAmismatch repair; PCNA, proliferating cell nuclear antigen; RFC, replication factor C; RPA, replication protein A; Pol, DNA polymerase; O6-mG, O6-methylguanine; nt, nucleotide(s).
      system has been conserved from bacteria to humans as a consequence of its importance for the maintenance of genome stability (
      • Modrich P.
      Methyl-directed DNA mismatch correction.
      ,
      • Kunkel T.A.
      • Erie D.A.
      DNA mismatch repair.
      • Iyer R.R.
      • Pluciennik A.
      • Burdett V.
      • Modrich P.L.
      DNA mismatch repair: functions and mechanisms.
      ). The MMR system has several activities that are involved in genome metabolism (
      • Modrich P.
      Methyl-directed DNA mismatch correction.
      ,
      • Iyer R.R.
      • Pluciennik A.
      • Burdett V.
      • Modrich P.L.
      DNA mismatch repair: functions and mechanisms.
      • Jiricny J.
      The multifaceted mismatch-repair system.
      ,
      • Peña-Diaz J.
      • Jiricny J.
      Mammalian mismatch repair: error-free or error-prone?.
      ,
      • Kunkel T.A.
      • Erie D.A.
      Eukaryotic mismatch repair in relation to DNA replication.
      ,
      • Earley M.C.
      • Crouse G.F.
      The role of mismatch repair in the prevention of base pair mutations in Saccharomyces cerevisiae.
      ,
      • Russo M.T.
      • Blasi M.F.
      • Chiera F.
      • Fortini P.
      • Degan P.
      • Macpherson P.
      • Furuichi M.
      • Nakabeppu Y.
      • Karran P.
      • Aquilina G.
      • Bignami M.
      The oxidized deoxynucleoside triphosphate pool is a significant contributor to genetic instability in mismatch repair-deficient cells.
      ,
      • Shen Y.
      • Koh K.D.
      • Weiss B.
      • Storici F.
      Mispaired rNMPs in DNA are mutagenic and are targets of mismatch repair and RNases H.
      • Kadyrova L.Y.
      • Dahal B.K.
      • Kadyrov F.A.
      Evidence that the DNA mismatch repair system removes 1-nt Okazaki fragment flaps.
      ). The correction of DNA mismatches is the best understood activity of the MMR system. Eukaryotic MMR leads to the correction of errors caused by the replication fork DNA polymerases α, δ, and ϵ, and it also changes the outcome of homologous recombination (
      • Morrison A.
      • Johnson A.L.
      • Johnston L.H.
      • Sugino A.
      Pathway correcting DNA replication errors in Saccharomyces cerevisiae.
      • Morrison A.
      • Sugino A.
      The 3′ → 5′ exonucleases of both DNA polymerases δ and ϵ participate in correcting errors of DNA replication in Saccharomyces cerevisiae.
      ,
      • Tran H.T.
      • Keen J.D.
      • Kricker M.
      • Resnick M.A.
      • Gordenin D.A.
      Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants.
      ,
      • Tran H.T.
      • Gordenin D.A.
      • Resnick M.A.
      The 3′ → 5′ exonucleases of DNA polymerases delta and epsilon and the 5′ → 3′ exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae.
      ,
      • Greene C.N.
      • Jinks-Robertson S.
      Spontaneous frameshift mutations in Saccharomyces cerevisiae: accumulation during DNA replication and removal by proofreading and mismatch repair activities.
      ,
      • Burgers P.M.
      Polymerase dynamics at the eukaryotic DNA replication fork.
      • Stone J.E.
      • Petes T.D.
      Analysis of the proteins involved in the in vivo repair of base-base mismatches and four-base loops formed during meiotic recombination in the yeast Saccharomyces cerevisiae.
      ). A considerable number of proteins have been suggested to contribute to or regulate eukaryotic MMR (
      • Kunkel T.A.
      • Erie D.A.
      Eukaryotic mismatch repair in relation to DNA replication.
      ,
      • Kadyrova L.Y.
      • Kadyrov F.A.
      Endonuclease activities of MutLalpha and its homologs in DNA mismatch repair.
      ,
      • Kolodner R.D.
      A personal historical view of DNA mismatch repair with an emphasis on eukaryotic DNA mismatch repair.
      • Li F.
      • Ortega J.
      • Gu L.
      • Li G.M.
      Regulation of mismatch repair by histone code and posttranslational modifications in eukaryotic cells.
      ). Strong evidence indicates the primary mismatch recognition factor MutSα (MSH2-MSH6 heterodimer), MutLα (MLH1-PMS2 heterodimer in humans and Mlh1-Pms1 heterodimer in budding yeast) endonuclease, the replicative clamp proliferating cell nuclear antigen (PCNA), the clamp loader replication factor C (RFC), the exonuclease EXO1, the secondary mismatch recognition factor MutSβ (MSH2-MSH3 heterodimer), and the replicative DNA polymerase δ (Pol δ) play major roles in eukaryotic MMR (
      • Drummond J.T.
      • Li G.-M.
      • Longley M.J.
      • Modrich P.
      Isolation of an hMSH2·p160 heterodimer that restores mismatch repair to tumor cells.
      • Palombo F.
      • Gallinari P.
      • Iaccarino I.
      • Lettieri T.
      • Hughes M.
      • D'Arrigo A.
      • Truong O.
      • Hsuan J.J.
      • Jiricny J.
      GTBP, a 160-kilodalton protein essential for mismatch-binding activity in human cells.
      ,
      • Li G.-M.
      • Modrich P.
      Restoration of mismatch repair to nuclear extracts of H6 colorectal tumor cells by a heterodimer of human MutL homologs.
      ,
      • Szankasi P.
      • Smith G.R.
      A role for exonuclease I from S. pombe in mutation avoidance and mismatch correction.
      ,
      • Umar A.
      • Buermeyer A.B.
      • Simon J.A.
      • Thomas D.C.
      • Clark A.B.
      • Liskay R.M.
      • Kunkel T.A.
      Requirement for PCNA in DNA mismatch repair at a step preceding DNA resynthesis.
      ,
      • Johnson R.E.
      • Kovvali G.K.
      • Guzder S.N.
      • Amin N.S.
      • Holm C.
      • Habraken Y.
      • Sung P.
      • Prakash L.
      • Prakash S.
      Evidence for involvement of yeast proliferating cell nuclear antigen in DNA mismatch repair.
      ,
      • Longley M.J.
      • Pierce A.J.
      • Modrich P.
      DNA polymerase δ is required for human mismatch repair in vitro.
      ,
      • Tishkoff D.X.
      • Boerger A.L.
      • Bertrand P.
      • Filosi N.
      • Gaida G.M.
      • Kane M.F.
      • Kolodner R.D.
      Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2.
      ,
      • Palombo F.
      • Iaccarino I.
      • Nakajima E.
      • Ikejima M.
      • Shimada T.
      • Jiricny J.
      hMutSβ, a heterodimer of hMSH2 and hMSH3, binds to insertion/deletion loops in DNA.
      ,
      • Genschel J.
      • Littman S.J.
      • Drummond J.T.
      • Modrich P.
      Isolation of hMutSβ from human cells and comparison of the mismatch repair specificities of hMutSβ and hMutSα.
      ,
      • de Wind N.
      • Dekker M.
      • Claij N.
      • Jansen L.
      • van Klink Y.
      • Radman M.
      • Riggins G.
      • van der Valk M.
      • van't Wout K.
      • te Riele H.
      HNPCC-like cancer predisposition in mice through simultaneous loss of Msh3 and Msh6 mismatch-repair protein functions.
      ,
      • Genschel J.
      • Bazemore L.R.
      • Modrich P.
      Human exonuclease I is required for 5′ and 3′ mismatch repair.
      ,
      • Genschel J.
      • Modrich P.
      Mechanism of 5′-directed excision in human mismatch repair.
      ,
      • Wei K.
      • Clark A.B.
      • Wong E.
      • Kane M.F.
      • Mazur D.J.
      • Parris T.
      • Kolas N.K.
      • Russell R.
      • Hou Jr, H.
      • Kneitz B.
      • Yang G.
      • Kunkel T.A.
      • Kolodner R.D.
      • Cohen P.E.
      • Edelmann W.
      Inactivation of Exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility.
      ,
      • Dzantiev L.
      • Constantin N.
      • Genschel J.
      • Iyer R.R.
      • Burgers P.M.
      • Modrich P.
      A defined human system that supports bidirectional mismatch-provoked excision.
      ,
      • Constantin N.
      • Dzantiev L.
      • Kadyrov F.A.
      • Modrich P.
      Human mismatch repair: reconstitution of a nick-directed bidirectional reaction.
      ,
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLα in human mismatch repair.
      ,
      • Modrich P.
      Mechanisms in eukaryotic mismatch repair.
      • Kadyrov F.A.
      • Holmes S.F.
      • Arana M.E.
      • Lukianova O.A.
      • O'Donnell M.
      • Kunkel T.A.
      • Modrich P.
      Saccharomyces cerevisiae MutLα is a mismatch repair endonuclease.
      ).
      Several eukaryotic MMR reactions have been described (
      • Kadyrova L.Y.
      • Dahal B.K.
      • Kadyrov F.A.
      Evidence that the DNA mismatch repair system removes 1-nt Okazaki fragment flaps.
      ,
      • Constantin N.
      • Dzantiev L.
      • Kadyrov F.A.
      • Modrich P.
      Human mismatch repair: reconstitution of a nick-directed bidirectional reaction.
      ,
      • Zhang Y.
      • Yuan F.
      • Presnell S.R.
      • Tian K.
      • Gao Y.
      • Tomkinson A.E.
      • Gu L.
      • Li G.M.
      Reconstitution of 5′-directed human mismatch repair in a purified system.
      • Kadyrov F.A.
      • Genschel J.
      • Fang Y.
      • Penland E.
      • Edelmann W.
      • Modrich P.
      A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair.
      ,
      • Smith C.E.
      • Bowen N.
      • Graham 5th, W.J.
      • Goellner E.M.
      • Srivatsan A.
      • Kolodner R.D.
      Activation of Saccharomyces cerevisiae Mlh1-Pms1 endonuclease in a reconstituted mismatch repair system.
      • Rodriges Blanko E.
      • Kadyrova L.Y.
      • Kadyrov F.A.
      DNA mismatch repair interacts with CAF-1- and ASF1A-H3-H4-dependent histone (H3-H4)2 tetramer deposition.
      ). One of these reactions removes mismatches on both the 3′- and 5′-nicked DNA molecules in an excision-dependent manner and is probably necessary for the majority of MMR events in wild-type cells (
      • Constantin N.
      • Dzantiev L.
      • Kadyrov F.A.
      • Modrich P.
      Human mismatch repair: reconstitution of a nick-directed bidirectional reaction.
      ). This MMR reaction depends on the activities of MutSα, MutLα, PCNA, RFC, EXO1, RPA, and Pol δ (
      • Genschel J.
      • Bazemore L.R.
      • Modrich P.
      Human exonuclease I is required for 5′ and 3′ mismatch repair.
      ,
      • Genschel J.
      • Modrich P.
      Mechanism of 5′-directed excision in human mismatch repair.
      ,
      • Dzantiev L.
      • Constantin N.
      • Genschel J.
      • Iyer R.R.
      • Burgers P.M.
      • Modrich P.
      A defined human system that supports bidirectional mismatch-provoked excision.
      ,
      • Constantin N.
      • Dzantiev L.
      • Kadyrov F.A.
      • Modrich P.
      Human mismatch repair: reconstitution of a nick-directed bidirectional reaction.
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLα in human mismatch repair.
      ,
      • Kadyrov F.A.
      • Genschel J.
      • Fang Y.
      • Penland E.
      • Edelmann W.
      • Modrich P.
      A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair.
      ) and is initiated by recognition of the mismatch by MutSα (
      • Drummond J.T.
      • Li G.-M.
      • Longley M.J.
      • Modrich P.
      Isolation of an hMSH2·p160 heterodimer that restores mismatch repair to tumor cells.
      ,
      • Palombo F.
      • Gallinari P.
      • Iaccarino I.
      • Lettieri T.
      • Hughes M.
      • D'Arrigo A.
      • Truong O.
      • Hsuan J.J.
      • Jiricny J.
      GTBP, a 160-kilodalton protein essential for mismatch-binding activity in human cells.
      ). After recognizing the mismatch, MutSα cooperates with RFC-loaded PCNA to activate MutLα endonuclease (
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLα in human mismatch repair.
      ,
      • Kadyrov F.A.
      • Genschel J.
      • Fang Y.
      • Penland E.
      • Edelmann W.
      • Modrich P.
      A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair.
      ,
      • Pluciennik A.
      • Dzantiev L.
      • Iyer R.R.
      • Constantin N.
      • Kadyrov F.A.
      • Modrich P.
      PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair.
      ). The activated MutLα endonuclease incises the discontinuous daughter strand. An incision that is generated by MutLα endonuclease 5′ to the mismatch is used by EXO1 to enter the DNA and excise the mismatch in a 5′ → 3′-directed reaction modulated by MutSα and RPA (
      • Genschel J.
      • Modrich P.
      Mechanism of 5′-directed excision in human mismatch repair.
      ,
      • Dzantiev L.
      • Constantin N.
      • Genschel J.
      • Iyer R.R.
      • Burgers P.M.
      • Modrich P.
      A defined human system that supports bidirectional mismatch-provoked excision.
      ,
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLα in human mismatch repair.
      ,
      • Shao H.
      • Baitinger C.
      • Soderblom E.J.
      • Burdett V.
      • Modrich P.
      Hydrolytic function of Exo1 in mammalian mismatch repair.
      ). A gap generated by EXO1 action is filled in by Pol δ holoenzyme. Although Pol ϵ holoenzyme can also perform gap filling in a reconstituted excision-dependent MMR reaction, the specific activity of this enzyme is much lower than that of Pol δ holoenzyme (
      • Rodriges Blanko E.
      • Kadyrova L.Y.
      • Kadyrov F.A.
      DNA mismatch repair interacts with CAF-1- and ASF1A-H3-H4-dependent histone (H3-H4)2 tetramer deposition.
      ). The mutator phenotype of an exo1Δ mutant is weaker compared with the mutator phenotype of an msh2Δ or mlh1Δ mutant, indicating that the MMR system can remove DNA polymerase errors in EXO1-deficient cells (
      • Tran H.T.
      • Gordenin D.A.
      • Resnick M.A.
      The 3′ → 5′ exonucleases of DNA polymerases delta and epsilon and the 5′ → 3′ exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae.
      ,
      • Tishkoff D.X.
      • Boerger A.L.
      • Bertrand P.
      • Filosi N.
      • Gaida G.M.
      • Kane M.F.
      • Kolodner R.D.
      Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2.
      ,
      • Wei K.
      • Clark A.B.
      • Wong E.
      • Kane M.F.
      • Mazur D.J.
      • Parris T.
      • Kolas N.K.
      • Russell R.
      • Hou Jr, H.
      • Kneitz B.
      • Yang G.
      • Kunkel T.A.
      • Kolodner R.D.
      • Cohen P.E.
      • Edelmann W.
      Inactivation of Exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility.
      ). In agreement with this idea, an excision-independent MMR reaction that involves MutSα, MutLα, PCNA, RFC, RPA, and Pol δ has been described (
      • Kadyrov F.A.
      • Genschel J.
      • Fang Y.
      • Penland E.
      • Edelmann W.
      • Modrich P.
      A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair.
      ). Like the excision-dependent MMR reaction, the excision-independent MMR reaction requires the MutLα endonuclease activity for incision of the discontinuous daughter strand. After MutLα endonuclease incises the discontinuous daughter strand, the Pol δ holoenzyme uses a MutLα-generated 3′ end that is 5′ to the mismatch to perform strand displacement DNA synthesis that removes the mismatch. The role of the Pol δ holoenzyme in the excision-independent MMR reaction may be unique because the replacement of the Pol δ holoenzyme with the Pol ϵ holoenzyme abolishes the excision-independent MMR reaction (
      • Rodriges Blanko E.
      • Kadyrova L.Y.
      • Kadyrov F.A.
      DNA mismatch repair interacts with CAF-1- and ASF1A-H3-H4-dependent histone (H3-H4)2 tetramer deposition.
      ).
      One of the activities of the MMR system is required for the cytotoxic response of MGMT (O6-methyl guanine methyl transferase)-deficient mammalian cells and yeast mgt1Δ rad52Δ cells to Sn1-type methylating agents (
      • Branch P.
      • Aquilina G.
      • Bignami M.
      • Karran P.
      Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage.
      ,
      • Cejka P.
      • Mojas N.
      • Gillet L.
      • Schär P.
      • Jiricny J.
      Homologous recombination rescues mismatch-repair-dependent cytotoxicity of S(N)1-type methylating agents in S. cerevisiae.
      ). This activity of the MMR system depends on MutSα and MutLα and is necessary for several therapies against MGMT-deficient cancers (
      • Iyer R.R.
      • Pluciennik A.
      • Burdett V.
      • Modrich P.L.
      DNA mismatch repair: functions and mechanisms.
      ). The treatment involves dacarbazine, procarbazine, or temozolomide, each of which is an Sn1-type methylating drug that triggers death of MGMT-deficient cancer cells by activating the MMR system. O6-Methylguanine (O6-mG) is the cytotoxic product of treatment of the cell with the Sn1-type methylating agent (
      • Loveless A.
      Possible relevance of O-6 alkylation of deoxyguanosine to the mutagenicity and carcinogenicity of nitrosamines and nitrosamides.
      ). Normally, O6-mG lesions are removed by MGMT that protects the cell from killing by the Sn1-type methylating agent (
      • Glassner B.J.
      • Weeda G.
      • Allan J.M.
      • Broekhof J.L.
      • Carls N.H.
      • Donker I.
      • Engelward B.P.
      • Hampson R.J.
      • Hersmus R.
      • Hickman M.J.
      • Roth R.B.
      • Warren H.B.
      • Wu M.M.
      • Hoeijmakers J.H.
      • Samson L.D.
      DNA repair methyltransferase (Mgmt) knockout mice are sensitive to the lethal effects of chemotherapeutic alkylating agents.
      ). However, a significant number of cancers are deficient in MGMT due to methylation of the MGMT promoter (
      • Esteller M.
      • Garcia-Foncillas J.
      • Andion E.
      • Goodman S.N.
      • Hidalgo O.F.
      • Vanaclocha V.
      • Baylin S.B.
      • Herman J.G.
      Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents.
      ). MGMT-deficient cancer cells treated with the Sn1-type methylating agent accumulate O6-mG-T mispairs that are recognized by MutSα (
      • Duckett D.R.
      • Drummond J.T.
      • Murchie A.I.H.
      • Reardon J.T.
      • Sancar A.
      • Lilley D.M.J.
      • Modrich P.
      Human MutSα recognizes damaged DNA base pairs containing O6-methylguanine, O4-methylthymine or the cisplatin-d(GpG) adduct.
      ). Upon recognition of an O6-mG-T mispair, MutSα initiates its repair. If the O6-mG is on the discontinuous strand, it gets repaired (
      • York S.J.
      • Modrich P.
      Mismatch repair-dependent iterative excision at irreparable O6-methylguanine lesions in human nuclear extracts.
      ). In contrast, if the O6-mG is on the continuous strand, it triggers futile cycles of MMR (
      • York S.J.
      • Modrich P.
      Mismatch repair-dependent iterative excision at irreparable O6-methylguanine lesions in human nuclear extracts.
      ). The futile cycles of MMR lead to the formation of persistent strand breaks (
      • Mojas N.
      • Lopes M.
      • Jiricny J.
      Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA.
      ), which are converted in the next S phase into double strand breaks that cause cell cycle arrest followed by cell death (
      • Zhukovskaya N.
      • Branch P.
      • Aquilina G.
      • Karran P.
      DNA replication arrest and tolerance to DNA methylation damage.
      ,
      • Stojic L.
      • Mojas N.
      • Cejka P.
      • Di Pietro M.
      • Ferrari S.
      • Marra G.
      • Jiricny J.
      Mismatch repair-dependent G2 checkpoint induced by low doses of SN1 type methylating agents requires the ATR kinase.
      ). Consistent with this, double strand break repair defects sensitize eukaryotic cells to the killing effects of Sn1-type methylating agents (
      • Cejka P.
      • Mojas N.
      • Gillet L.
      • Schär P.
      • Jiricny J.
      Homologous recombination rescues mismatch-repair-dependent cytotoxicity of S(N)1-type methylating agents in S. cerevisiae.
      ,
      • Tsaryk R.
      • Fabian K.
      • Thacker J.
      • Kaina B.
      Xrcc2 deficiency sensitizes cells to apoptosis by MNNG and the alkylating anticancer drugs temozolomide, fotemustine and mafosfamide.
      ).
      The heterotrimeric CAF-1 is the major histone chaperone for the assembly of nucleosomes onto the newly replicated DNA (
      • Smith S.
      • Stillman B.
      Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro.
      • Kaufman P.D.
      • Kobayashi R.
      • Kessler N.
      • Stillman B.
      The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication.
      ,
      • Verreault A.
      • Kaufman P.D.
      • Kobayashi R.
      • Stillman B.
      Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4.
      ,
      • Kaufman P.D.
      • Kobayashi R.
      • Stillman B.
      Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I.
      • Hoek M.
      • Stillman B.
      Chromatin assembly factor 1 is essential and couples chromatin assembly to DNA replication in vivo.
      ). CAF-1 loads histone (H3-H4)2 tetramers onto DNA, producing tetrasomes (
      • Smith S.
      • Stillman B.
      Stepwise assembly of chromatin during DNA replication in vitro.
      ,
      • Kadyrova L.Y.
      • Blanko E.R.
      • Kadyrov F.A.
      CAF-I-dependent control of degradation of the discontinuous strands during mismatch repair.
      ). Each tetrasome is then converted into a nucleosome by the addition of two histone H2A-H2B dimers (
      • Smith S.
      • Stillman B.
      Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro.
      ,
      • Kadyrova L.Y.
      • Rodriges Blanko E.
      • Kadyrov F.A.
      Human CAF-1-dependent nucleosome assembly in a defined system.
      ). CAF-1 interacts physically with PCNA, and this interaction is necessary for the action of CAF-1 on the newly replicated DNA (
      • Shibahara K.
      • Stillman B.
      Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin.
      ,
      • Rolef Ben-Shahar T.
      • Castillo A.G.
      • Osborne M.J.
      • Borden K.L.
      • Kornblatt J.
      • Verreault A.
      Two fundamentally distinct PCNA interaction peptides contribute to chromatin assembly factor 1 function.
      ). Recent research has indicated that postreplicative MMR coincides with CAF-1-dependent nucleosome assembly and that the two processes interact with each other (
      • Rodriges Blanko E.
      • Kadyrova L.Y.
      • Kadyrov F.A.
      DNA mismatch repair interacts with CAF-1- and ASF1A-H3-H4-dependent histone (H3-H4)2 tetramer deposition.
      ,
      • Kadyrova L.Y.
      • Blanko E.R.
      • Kadyrov F.A.
      CAF-I-dependent control of degradation of the discontinuous strands during mismatch repair.
      ,
      • Schöpf B.
      • Bregenhorn S.
      • Quivy J.P.
      • Kadyrov F.A.
      • Almouzni G.
      • Jiricny J.
      Interplay between mismatch repair and chromatin assembly.
      ). Furthermore, recent findings are consistent with the idea that the eukaryotic MMR system degrades irreparable O6-mG-T mispair-containing DNA when it is being packaged into nucleosomes by the CAF-1-dependent mechanism (
      • Mojas N.
      • Lopes M.
      • Jiricny J.
      Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA.
      ,
      • Kadyrova L.Y.
      • Blanko E.R.
      • Kadyrov F.A.
      CAF-I-dependent control of degradation of the discontinuous strands during mismatch repair.
      ,
      • Schöpf B.
      • Bregenhorn S.
      • Quivy J.P.
      • Kadyrov F.A.
      • Almouzni G.
      • Jiricny J.
      Interplay between mismatch repair and chromatin assembly.
      ). We show here that CAF-1 suppresses the activity of the MMR system in the cytotoxic response to Sn1-type methylating agents.

      Results

      CAF-1 Suppressed the Activity of the MMR System in the Cytotoxic Response of Yeast mgt1Δ rad52Δ Cells to MNNG

      The cytotoxic response to Sn1-type methylating agents occurs in the chromatin environment (
      • Branch P.
      • Aquilina G.
      • Bignami M.
      • Karran P.
      Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage.
      ,
      • York S.J.
      • Modrich P.
      Mismatch repair-dependent iterative excision at irreparable O6-methylguanine lesions in human nuclear extracts.
      ,
      • Mojas N.
      • Lopes M.
      • Jiricny J.
      Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA.
      ,
      • Erdeniz N.
      • Nguyen M.
      • Deschênes S.M.
      • Liskay R.M.
      Mutations affecting a putative MutLα endonuclease motif impact multiple mismatch repair functions.
      ). However, it has remained unknown whether the chromatin environment affects the cytotoxic response to Sn1-type methylating agents. It has also been unknown whether histone chaperones, proteins that are involved in the control of chromatin environment, influence the cytotoxic response to Sn1-type methylating agents. We wanted to study whether the major replicative histone chaperone CAF-1 impacts the cytotoxic response to an Sn1-type methylating agent. Previous research showed that MGMT-deficient mammalian cells and Saccharomyces cerevisiae mgt1Δ rad52Δ that lack Mgt (the yeast ortholog of MGMT (
      • Xiao W.
      • Derfler B.
      • Chen J.
      • Samson L.
      Primary sequence and biological functions of a Saccharomyces cerevisiae O6-methylguanine/O4-methylthymine DNA repair methyltransferase gene.
      )) and the recombination mediator Rad52 (
      • Sung P.
      Function of yeast Rad52 protein as a mediator between replication protein A and the Rad51 recombinase.
      ) are efficiently killed by the prototypic Sn1-type methylating agent MNNG in a manner that involves the MMR system (
      • Branch P.
      • Aquilina G.
      • Bignami M.
      • Karran P.
      Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage.
      ,
      • Cejka P.
      • Mojas N.
      • Gillet L.
      • Schär P.
      • Jiricny J.
      Homologous recombination rescues mismatch-repair-dependent cytotoxicity of S(N)1-type methylating agents in S. cerevisiae.
      ,
      • Mojas N.
      • Lopes M.
      • Jiricny J.
      Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA.
      ). Thus, we utilized the yeast mgt1Δ rad52Δ cells to determine whether CAF-1 impacts the cytotoxic response to MNNG. In budding yeast, CAC1 encodes the largest subunit of CAF-1 (
      • Kaufman P.D.
      • Kobayashi R.
      • Stillman B.
      Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I.
      ). Accordingly, we investigated whether loss of the CAC1 gene increased the sensitivity of the yeast mgt1Δ rad52Δ cells to killing by MNNG. The use of an MNNG cytotoxicity assay permitted us to establish that the cac1Δ mgt1Δ rad52Δ cells were more sensitive to treatment with MNNG than the mgt1Δ rad52Δ cells (Fig. 1A). A more detailed analysis revealed that ∼1.7% of the mgt1Δ rad52Δ cells and only ∼0.2% of the cac1Δ mgt1Δ rad52Δ cells survived the treatment with MNNG (Fig. 1C). Thus, the surviving fraction of the MNNG-treated cac1Δ mgt1Δ rad52Δ cells was ∼9 times smaller than that of the MNNG-treated mgt1Δ rad52Δ cells. We then conducted experiments to determine whether MNNG killed the cac1Δ mgt1Δ rad52Δ cells via an MMR system-dependent mechanism (Fig. 1C). The results showed that the deletion of the MMR system gene MLH1 rescued the sensitivity of the cac1Δ mgt1Δ rad52Δ cells to the cytotoxic effect of MNNG. The experiments also demonstrated that the mlh1Δ cac1Δ mgt1Δ rad52Δ cells were as resistant to the MNNG treatment as the mlh1Δ mgt1Δ rad52Δ cells. Based on these findings, we concluded that loss of CAC1 sensitizes the yeast mgt1Δ rad52Δ cells to MMR system-dependent killing by MNNG.
      Figure thumbnail gr1
      FIGURE 1.CAF-1 suppresses the activity of the MMR system in the cytotoxic response of yeast mgt1Δ rad52Δ cells to MNNG. Cytotoxicity assays were carried out as detailed under “Experimental Procedures.” A and B, cytotoxic responses of cac1Δ mgt1Δ rad52Δ and cac2Δ mgt1Δ rad52Δ cells to treatment with 1 μm MNNG. 10-Fold serial dilutions of yeast cultures that were treated or not treated with 1 μm MNNG were spotted on the YPDAU plates. C, quantitative analysis of cytotoxic responses of cac1Δ mgt1Δ rad52Δ and cac2Δ mgt1Δ rad52Δ cells to treatment with 1 μm MNNG. D, quantitative analysis of cytotoxic response of cac1Δ mgt1Δ rad52Δ to treatment with 30 μg/ml bleomycin (BLE). The data in C and D are averages ± 1 S.D. (error bars) (n ≥ 3). E and F, cytotoxic responses of cac1Δ mgt1Δ rad52Δ and cac2Δ mgt1Δ rad52Δ cells to treatments with 0.5 μg/ml camptothecin and 10 mm hydroxyurea. 10-Fold serial dilutions of yeast cultures were spotted on the YPDAU plates, YPDAU plate with 0.5 μg/ml camptothecin, and YPDAU plate with 10 mm hydroxyurea. G, cytotoxic responses of hht2-hhf2Δ mgt1Δ rad52Δ, hir2Δ mgt1Δ rad52Δ, and rtt106Δ mgt1Δ rad52Δ cells to treatment with 1 μm MNNG. 10-Fold serial dilutions of yeast cultures that were treated or not with 1 μm MNNG were spotted on the YPDAU plates.
      The cytotoxicity of Sn1-type methylators is mediated by replication- and MMR system-dependent double strand breaks that these agents form. A number of other DNA-damaging agents generate strand breaks that kill cells. Among them are camptothecin, hydroxyurea, and bleomycin. Camptothecin induces replication-dependent double strand breaks by stabilizing topoisomerase I-DNA covalent complexes (
      • Hsiang Y.H.
      • Lihou M.G.
      • Liu L.F.
      Arrest of replication forks by drug-stabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by camptothecin.
      ), hydroxyurea causes double strand breaks by depleting the dNTP pools (
      • Krakoff I.H.
      • Brown N.C.
      • Reichard P.
      Inhibition of ribonucleoside diphosphate reductase by hydroxyurea.
      ), and bleomycin creates double strand and single strand breaks by attacking DNA (
      • Povirk L.F.
      DNA damage and mutagenesis by radiomimetic DNA-cleaving agents: bleomycin, neocarzinostatin and other enediynes.
      ). Unlike the Sn1-type methylating agent, camptothecin, hydroxyurea, and bleomycin kill cells via MMR system-independent mechanisms. Nevertheless, there is a significant similarity between one of these drugs, camptothecin, and MNNG in that double strand breaks caused by these two agents are formed during DNA replication. The results of our genetic experiments (Fig. 1, A and C) raised the possibility that loss of CAC1 sensitized the yeast mgt1Δ rad52Δ cells to the cytotoxic effects of DNA-damaging agents that generate DNA breaks. To address this possibility, we studied the effect of CAC1 absence on the sensitivity of the mgt1Δ rad52Δ cells to camptothecin, hydroxyurea, and bleomycin (Fig. 1, D and E). An analysis of the data showed that the cac1Δ mgt1Δ rad52Δ cells and the mgt1Δ rad52Δ cells had the same sensitivities to bleomycin, camptothecin, and hydroxyurea. Thus, CAC1 absence did not affect the sensitivity of the yeast mgt1Δ rad52Δ cells to bleomycin, camptothecin, and hydroxyurea, drugs that kill cells via MMR system-independent mechanisms. Because MNNG kills the yeast mgt1Δ rad52Δ cells by activating an MMR system-dependent mechanism and bleomycin, camptothecin, and hydroxyurea kill the cells via other mechanisms, the results of our genetic experiments (Fig. 1, A and C–E) indicated that Cac1 increases the survival of the MNNG-treated mgt1Δ rad52Δ cells by being involved in a process that suppresses the cytotoxic activity of the MMR system.
      CAC2 codes for the second subunit of yeast CAF-1 (
      • Kaufman P.D.
      • Kobayashi R.
      • Stillman B.
      Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I.
      ). We studied whether deletion of CAC2 sensitized the mgt1Δ rad52Δ cells to MNNG (Fig. 1B). The data showed that the cac2Δ mgt1Δ rad52Δ cells were more sensitive to MNNG than the mgt1Δ rad52Δ cells (Fig. 1, B and C). A comparison of the sensitivities of the cac2Δ msh2Δ mgt1Δ rad52Δ, msh2Δ mgt1Δ rad52Δ, cac2Δ mgt1Δ rad52Δ, and mgt1Δ rad52Δ cells (Fig. 1C) revealed that MNNG killed the cac2Δ mgt1Δ rad52Δ cells via an MMR system-dependent mechanism. As expected, the cac2Δ mgt1Δ rad52Δ cells were as sensitive to camptothecin and hydroxyurea as the mgt1Δ rad52Δ cells (Fig. 1F). Thus, the results of these and previous experiments (Fig. 1, A–F) demonstrated that CAF-1 increases the survival of MNNG-treated mgt1Δ rad52Δ cells by suppressing the cytotoxic activity of the MMR system.

      Loss of HIR2, RTT106, or HHT2-HHF2 Did Not Change the Sensitivity of Yeast mgt1Δ rad52Δ Cells to MNNG

      Histone H3-H4 chaperone HIR (Hir1-Hir2-Hir3-Hpc2 complex) plays a major role in replication-independent nucleosome assembly (
      • Green E.M.
      • Antczak A.J.
      • Bailey A.O.
      • Franco A.A.
      • Wu K.J.
      • Yates 3rd, J.R.
      • Kaufman P.D.
      Replication-independent histone deposition by the HIR complex and Asf1.
      ), and histone H3-H4 chaperone Rtt106 participates in replication-coupled nucleosome assembly (
      • Burgess R.J.
      • Zhang Z.
      Histone chaperones in nucleosome assembly and human disease.
      ). We analyzed whether the absence of HIR2 or RTT106 had an effect on the sensitivity of the mgt1Δ rad52Δ cells to MNNG. The experiments showed that lack of HIR2 or RTT106 did not change the sensitivity of the mgt1Δ rad52Δ cells to MNNG (Fig. 1G).
      The molecular activity of CAF-1 is to load histone H3-H4 tetramers onto newly replicated DNA (
      • Verreault A.
      • Kaufman P.D.
      • Kobayashi R.
      • Stillman B.
      Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4.
      ,
      • Smith S.
      • Stillman B.
      Stepwise assembly of chromatin during DNA replication in vitro.
      ,
      • Kadyrova L.Y.
      • Rodriges Blanko E.
      • Kadyrov F.A.
      Human CAF-1-dependent nucleosome assembly in a defined system.
      ). We wanted to explore whether decreasing histone H3-H4 gene dosage affected the survival of MNNG-treated mgt1Δ rad52Δ cells. The yeast genome contains two histone H3-H4 gene loci, HHT1-HHF1 and HHT2-HHF2. Compared with HHT1-HHF1, HHT2-HHF2 produces about 5–7 times more mRNA (
      • Cross S.L.
      • Smith M.M.
      Comparison of the structure and cell cycle expression of mRNAs encoded by two histone H3-H4 loci in Saccharomyces cerevisiae.
      ). Either locus is sufficient to maintain the existence of the yeast cell. We measured the sensitivity of hht2-hhf2Δ mgt1Δ rad52Δ cells to MNNG and found it to be no different from that of the mgt1Δ rad52Δ cells (Fig. 1G).

      CAF-1-dependent Packaging of Irreparable O6-mG-T Mispair-containing DNA into Nucleosomes Suppressed Its Degradation by the MMR System

      MMR system-dependent degradation of irreparable O6-mG-T mispair-containing DNA is involved in the cytotoxic response to the Sn1-type methylating drug (
      • York S.J.
      • Modrich P.
      Mismatch repair-dependent iterative excision at irreparable O6-methylguanine lesions in human nuclear extracts.
      ,
      • Mojas N.
      • Lopes M.
      • Jiricny J.
      Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA.
      ,
      • Erdeniz N.
      • Nguyen M.
      • Deschênes S.M.
      • Liskay R.M.
      Mutations affecting a putative MutLα endonuclease motif impact multiple mismatch repair functions.
      ). Our genetic experiments indicated that CAF-1 activity suppresses degradation of irreparable O6-mG-T mispair-containing DNA by the MMR system (Fig. 1). To find evidence that CAF-1-dependent incorporation of irreparable O6-mG-T mispair-containing DNA into nucleosomes suppresses its degradation by the MMR system, we performed biochemical experiments that are summarized in FIGURE 2., FIGURE 3., FIGURE 4., FIGURE 5., FIGURE 6., FIGURE 7.; DNA substrates that we utilized in these experiments were 3′-nicked O6-mG-T (3O6-mG-T), 3′-nicked G-T (3G-T), and 3′-nicked A-T (3A-T) DNAs. The substrates were made using a plasmid, pAH1A, as a starting material and differed from each other by 1–2 bases (
      • York S.J.
      • Modrich P.
      Mismatch repair-dependent iterative excision at irreparable O6-methylguanine lesions in human nuclear extracts.
      ). The 3′-nicked O6-mG-T DNA contained a single O6-mG-T mispair, the 3′-nicked G-T DNA carried a single G-T mispair, and the 3′-nicked A-T DNA lacked a mispair. The 3′-nicked O6-mG-T DNA was irreparable by the MMR system because the O6-mG was on the continuous strand. In these biochemical experiments, we used a cytosolic extract prepared from human embryonic kidney cell line 293T that lacked MutLα and MGMT (
      • Trojan J.
      • Zeuzem S.
      • Randolph A.
      • Hemmerle C.
      • Brieger A.
      • Raedle J.
      • Plotz G.
      • Jiricny J.
      • Marra G.
      Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system.
      ,
      • Cejka P.
      • Stojic L.
      • Mojas N.
      • Russell A.M.
      • Heinimann K.
      • Cannavó E.
      • di Pietro M.
      • Marra G.
      • Jiricny J.
      Methylation-induced G2/M arrest requires a full complement of the mismatch repair protein hMLH1.
      ) and had a reduced level of CAF-1 (Fig. 2A) (
      • Kaufman P.D.
      • Kobayashi R.
      • Kessler N.
      • Stillman B.
      The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication.
      ). Although the 293T cytosolic extract lacks the MMR system due to the absence of MutLα (
      • Trojan J.
      • Zeuzem S.
      • Randolph A.
      • Hemmerle C.
      • Brieger A.
      • Raedle J.
      • Plotz G.
      • Jiricny J.
      • Marra G.
      Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system.
      ), supplementation of the extract with purified MutLα reconstitutes the MMR system (
      • Cejka P.
      • Stojic L.
      • Mojas N.
      • Russell A.M.
      • Heinimann K.
      • Cannavó E.
      • di Pietro M.
      • Marra G.
      • Jiricny J.
      Methylation-induced G2/M arrest requires a full complement of the mismatch repair protein hMLH1.
      ). In agreement with previous studies (
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLα in human mismatch repair.
      ,
      • York S.J.
      • Modrich P.
      Mismatch repair-dependent iterative excision at irreparable O6-methylguanine lesions in human nuclear extracts.
      ,
      • Kadyrova L.Y.
      • Blanko E.R.
      • Kadyrov F.A.
      CAF-I-dependent control of degradation of the discontinuous strands during mismatch repair.
      ,
      • Trojan J.
      • Zeuzem S.
      • Randolph A.
      • Hemmerle C.
      • Brieger A.
      • Raedle J.
      • Plotz G.
      • Jiricny J.
      • Marra G.
      Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system.
      ,
      • Cejka P.
      • Stojic L.
      • Mojas N.
      • Russell A.M.
      • Heinimann K.
      • Cannavó E.
      • di Pietro M.
      • Marra G.
      • Jiricny J.
      Methylation-induced G2/M arrest requires a full complement of the mismatch repair protein hMLH1.
      • Holmes Jr., J.
      • Clark S.
      • Modrich P.
      Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines.
      ), our control experiments showed that 1) the reconstituted MMR system failed to repair an O6-mG-T mispair on a nicked DNA (3′-nicked O6-mG-T DNA) but repaired a G-T mispair on a similar nicked DNA (the 3′-nicked G-T DNA) (Fig. 3), 2) supplementation of the 293T cytosolic extract with purified MutLα-E705K endonuclease mutant (
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLα in human mismatch repair.
      ) did not lead to reconstitution of the MMR system (Fig. 3), 3) the omission of exogenous dNTPs and the addition of the DNA polymerase inhibitor aphidicolin led to a nearly complete inhibition of the mismatch correction activity of the reconstituted MMR system (Fig. 3), 4) the 293T cytosolic extract had a weak nucleosome assembly activity (Fig. 2B) due to the reduced level of CAF-1 (Fig. 2A).
      Figure thumbnail gr2
      FIGURE 2.Nucleosome assembly reactions reconstituted with an extract and purified CAF-1. The experiments were conducted and analyzed as described under “Experimental Procedures.” A, analysis of CAF-1 in 293T cytosolic extract (293T CE) and 293T nuclear extract (293T NE) by Western blotting with antibodies against the CAF-1 p150 subunit. Quantification of the data showed that 293T cytosolic extract (150 μg) contains 0.077 ± 0.009 pmol of CAF-1. B, analysis of nucleosome assembly in reaction mixtures containing the indicated components. The nucleosome assembly products were visualized with the 32P-labeled probe c154, which is complementary to the continuous strand. A diagram on the left indicates the relative positions of the 32P-labeled probe (a bar with an asterisk), the strand break, and a mismatch.
      Figure thumbnail gr3
      FIGURE 3.MMR system reconstituted with an extract and purified MutLα does not correct a mispair that contains O6-mG on the continuous strand. The experiments were carried out and analyzed as described under “Experimental Procedures.” Each reaction mixture contained a DNA substrate (162 fmol) and the other indicated components and was incubated for 10 min at 37 °C. No exogenous dNTPs were present in the reaction mixtures that included aphidicolin (0.1 mm). To score MMR, the recovered DNAs were digested with XhoI and BanI, separated on native agarose gels, and visualized with ethidium bromide staining. A, outline of the assay. The assay is based on the knowledge that the repair of the mismatched T on the 3′-nicked G-T or the 3′-nicked O6-mG-T DNA restores the XhoI site. B, DNA species present in the indicated reaction mixtures. C, amounts of MMR products formed in the indicated reaction mixtures. The data are averages ± 1 S.D. (error bars) (n = 3) and were obtained by quantification of images including the one shown in B.
      Figure thumbnail gr4
      FIGURE 4.MMR system reconstituted with an extract and purified MutLα degrades the discontinuous strand of an irreparable O6-mG-T mispair-containing DNA. The experiments were carried out as described in the legend to . The recovered DNAs were digested with ClaI, separated on alkaline agarose gels, transferred onto nylon membranes, and hybridized with the 32P-labeled probes ts154 and ts1, which are complementary to the discontinuous strands. A and B, DNA species visualized with the ts154 probe and the ts1 probe, respectively. Each of the diagrams outlines relative positions of the probe (a bar with an asterisk), the unique ClaI site, the strand break, and a mismatch. The distance between the ClaI site and the mismatch is 145 bp.
      Figure thumbnail gr7
      FIGURE 7.CAF-1-dependent suppression of the formation of a gap in an irreparable O6-mG-T mispair-containing DNA in an extract system. The experiments were performed and analyzed as described under “Experimental Procedures.” Each reaction mixture contained a DNA substrate (41 fmol) and other indicated components and was incubated for 60 min at 37 °C. The recovered DNA products were digested with AhdI, separated on native agarose gels, transferred onto nylon membranes, and hybridized with the 32P-labeled probes c154 and ts154. A, outline of the assay. B and C, DNA species visualized with the 32P-labeled probes c154 and ts154, respectively. The c154 probe is complementary to the continuous strand, and the ts154 probe is complementary to the discontinuous strand. Each diagram depicts the relative positions of the 32P-labeled probe (a bar with an asterisk), AhdI sites, the strand break, and a mismatch. D, quantification of gapped DNA formed in MutLα endonuclease-dependent reactions in the indicated reaction mixtures. E, effects of the different amounts of purified CAF-1 on the formation of the gapped product of the 3′-nicked O6-mG-T DNA in 293T cytosolic extract-containing reaction mixture. The reaction mixtures contained 293T cytosolic extract (150 μg), MutLα (3.2 pmol), and CAF-1 (0, 0.3, 0.6, or 2.4 pmol). The data in D and E were obtained with the 32P-labeled probe c154 and are averages ± 1 S.D. (error bars) (n = 3).
      Figure thumbnail gr6
      FIGURE 6.CAF-1 suppresses persistent degradation of an irreparable O6-mG-T mispair-containing DNA in an extract system in a concentration-dependent manner. The experiments were carried out and analyzed as detailed under “Experimental Procedures.” Each reaction mixture contained a DNA substrate (41 fmol) and the other indicated components and was incubated for 60 min at 37 °C. The recovered DNAs were digested with ClaI, separated on an alkaline agarose gel, transferred onto a nylon membrane, and hybridized with the 32P-labeled probe ts154, which is complementary to the discontinuous strand. A, DNA species visualized in one of the experiments. The diagram outlines relative positions of the probe (a bar with an asterisk), unique ClaI site, strand break, and mismatch. B, formation of the ∼130-nt product of degradation of the 3′-nicked O6-mG-T DNA as a function of the amount of purified CAF-1. The data were obtained by quantification of images, including the one in A, and are averages ± 1 S.D. (error bars) (n = 3).
      Figure thumbnail gr5
      FIGURE 5.CAF-1-dependent suppression of persistent MutLα endonuclease-dependent degradation of an irreparable O6-mG-T mispair-containing DNA in an extract system. The experiments were carried out and analyzed as described under “Experimental Procedures.” The reaction mixtures included the indicated components and were incubated for 60 min (A and C–F) or 10–120 min (B) at 37 °C. Each reaction mixture analyzed in A–E contained 162 fmol of a DNA substrate, and each reaction mixture analyzed in F contained 41 fmol of a DNA substrate. Exogenous dNTPs were not included in the reaction mixtures that contained aphidicolin (0.1 mm). The recovered DNAs were digested with ClaI, separated on alkaline agarose gels, transferred onto nylon membranes, and hybridized with the 32P-labeled probes ts154, ts182, ts1, and c154. The ts154, ts182, and ts1 probes are complementary to the discontinuous strands, and the c154 probe is complementary to the continuous strands. A, DNA species visualized with the 32P-labeled probe ts154. B, effect of the presence of purified CAF-1 (2.4 pmol) on the formation of the ∼130-nt product of degradation of the discontinuous strand of the 3′-nicked O6-mG-T DNA. The data were obtained by quantification of images, including those shown in A and A and are averages ± 1 S.D. (error bars) (n ≥ 2). C–E, DNA species visualized with the 32P-labeled probes ts182, ts1, and c154, respectively. Each of the diagrams outlines relative positions of the probe (a bar with an asterisk), the unique ClaI site, the strand break, and a mismatch. F, quantification of ∼130-nt degradation products formed in the indicated reaction mixtures. The data were obtained using the ts154 probe and are averages ± 1 S.D. (n = 3).
      We next utilized Southern hybridization to detect MMR system-dependent degradation of the irreparable O6-mG-T mispair-containing DNA that was reconstituted with the 293T cytosolic extract in the absence or presence of the purified CAF-1 (FIGURE 4., FIGURE 5., FIGURE 6., FIGURE 7.). The initial experiments in this series analyzed degradation products that were separated on denaturing agarose gels (FIGURE 4., FIGURE 5., FIGURE 6.). The data showed that the reconstituted MMR system degraded the discontinuous strand of an irreparable O6-mG-T mispair-containing DNA (the 3′-nicked O6-mG-T DNA) leading to the formation of a ∼130-nt product (Figs. 4A and 5A, lane 10). Importantly, the ∼130-nt product was not observed in the reaction mixture that contained endonuclease-deficient MutLα-E705K instead of MutLα (Figs. 4A and 5A, lane 13) demonstrating that the endonuclease activity of MutLα is required for the degradation of the 3′-nicked O6-mG-T DNA. Additional experiments revealed that the ∼130-nt product or a similar product was not formed from the 3′-nicked A-T DNA in the reaction mixture that contained the reconstituted MMR system (Figs. 4A and 5A, lane 17). The results of a time course analysis demonstrated that the ∼130-nt product of degradation of the discontinuous strand of the 3′-nicked O6-mG-T DNA was observed in the reaction mixture that was incubated for 10–120 min (Fig. 5B). This observation indicated that the reconstituted MMR system caused persistent degradation of the discontinuous strand of the 3′-nicked O6-mG-T DNA. In contrast, the products of degradation of the discontinuous strand of the 3′-nicked G-T DNA that were present in the reaction mixture incubated for 10 min (Fig. 4A, lane 3) were not detected in the same reaction mixture that was incubated for 60 min (Fig. 5A, lane 3). This finding is consistent with the view that the product of degradation of the discontinuous strand of the 3′-nicked G-T DNA is an intermediate of the MMR reaction (
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLα in human mismatch repair.
      ).
      The ∼130-nt product was identified by Southern hybridization with a 32P-labeled probe that was complementary to a discontinuous strand sequence located 2 nt downstream from the mismatched T (Figs. 4A and 5A). However, Southern hybridization with a 32P-labeled probe that was complementary to a discontinuous strand sequence located 2 nt upstream from the mismatched T did not detect the ∼130-nt product (Fig. 5C). This finding indicated that the 5′ end of the ∼130-nt product was located at or near the mismatch. To determine whether a different part of the 3′-nicked O6-mG-T DNA contained a MutLα endonuclease-dependent strand break, we carried out Southern hybridizations with two other 32P-labeled probes (Fig. 5, D and E). One of the probes was complementary to a discontinuous strand sequence that was downstream from the preexisting strand break (Fig. 5D) and the other to a continuous strand sequence (Fig. 5E). These two Southern hybridizations did not identify any additional MutLα endonuclease-dependent strand break in the 3′-nicked O6-mG-T DNA.
      The addition of purified CAF-1 to the 293T extract- and MutLα-containing reaction mixture led to packaging of the 3′-nicked O6-mG-T DNA into nucleosomes (Fig. 2B, lane 9). The presence of purified CAF-1 in the 293T extract- and MutLα-containing reaction mixture also caused a significant decrease in the yield of the ∼130-nt product of degradation of the irreparable O6-mG-containing DNA (Fig. 5, A and B). The effect of CAF-1 on the yield of the ∼130-nt product was especially pronounced in the reaction mixture in which the concentration of the 3′-nicked O6-mG-T DNA was decreased 4 times (Fig. 5F). In this case, the addition of purified CAF-1 reduced the yield of the ∼130-nt degradation product 5-fold. In the above experiments, we included 2.4 pmol of purified CAF-1 in the 293T extract- and MutLα-containing reaction mixtures. Further experiments showed that the addition of 0.3 pmol of purified CAF-1 was sufficient to cause a significant decrease in the yield of the ∼130-nt product (Fig. 6, A and B). The simplest interpretation of these results is that CAF-1-dependent packaging of irreparable O6-mG-T mispair-containing DNA into nucleosomes reduces its degradation by the MMR system.
      We also utilized Southern hybridization to analyze the degradation products that were separated on native agarose gels (Fig. 7). The data revealed that in the 293T extract- and MutLα-containing reaction mixture, a significant fraction of the 3′-nicked O6-mG-T DNA was converted into a gapped product (Fig. 7). A gap was formed in the discontinuous but not continuous strand of the 3′-nicked O6-mG-T DNA (Fig. 7, B and C, lane 7). The same or a similar gapped product was largely absent in the 293T extract- and MutLα-E705K-containing mixture (Fig. 7B, lane 10). Although some of the 3′-nicked G-T DNA products in the 293T extract- and MutLα-containing reaction mixture were also gapped (Fig. 7B, lane 2), their yield was ∼2.5 times lower than that of the gapped product of the 3′-nicked O6-mG-T DNA (Fig. 7D). As expected, 3′-nicked A-T DNA products that were formed in the 293T extract- and MutLα-containing reaction mixture lacked gaps (Fig. 7, B (lane 12) and C). Supplementation of the 293T extract- and MutLα-containing reaction mixture with 0.6 or 2.4 pmol of purified CAF-1 caused a significant reduction in the yield of the gapped product of the 3′-nicked O6-mG-T DNA (Fig. 7, B–E). This finding supports the view that CAF-1-dependent packaging of irreparable O6-mG-T mispair-containing DNA into nucleosomes suppresses its degradation by the MMR system.

      Degradation of an Irreparable O6-mG-T Mispair-containing DNA by the Activated MutLα Endonuclease in a Defined System

      Our biochemical experiments (FIGURE 4., FIGURE 5., FIGURE 6., FIGURE 7.) have implicated MutLα endonuclease activity in MMR system-dependent degradation of the discontinuous strand of irreparable O6-mG-T mispair-containing DNA. However, these experiments did not provide a clear view of how MutLα endonuclease activity is involved in this process. To better understand the involvement of MutLα endonuclease activity in MMR system-dependent degradation of the discontinuous strand of irreparable O6-mG-T mispair-containing DNA, we carried out additional experiments summarized in Fig. 8. In these experiments, we studied degradation of the discontinuous strand of the 3′-nicked O6-mG-T DNA in a defined system (
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLα in human mismatch repair.
      ). Purified proteins that were included in the defined system were MutLα endonuclease, the mismatch recognition factor MutSα, the PCNA clamp, the RFC clamp loader, and the single-stranded DNA-binding protein RPA. It can be seen that the discontinuous strand of the 3′-nicked O6-mG-T DNA was degraded in the defined system in a MutLα endonuclease concentration-dependent manner (Fig. 8, A (lanes 13–17) and B). The level of degradation of the discontinuous strand of the 3′-nicked O6-mG-T DNA was 5–6 times higher than the degradation level of the discontinuous strand of the control 3′-nicked A-T DNA (Fig. 8B). No degradation of the discontinuous strand of the 3′-nicked O6-mG-T DNA was observed in the defined system in which the endonuclease-deficient MutLα-E705K substituted for MutLα (Fig. 8, A (lanes 6, 12, and 18) and C). This information indicated that MutLα provided the endonuclease activity that degraded the discontinuous strand of an irreparable O6-mG-T mispair-containing DNA in the presence of MutSα, PCNA, RFC, and RPA. Importantly, the ∼130-nt fragment that was detected in the cell extract system (Fig. 5A, lane 10) was not a preferred product of the endonuclease reaction in the defined system (Fig. 8A, lanes 15–17). This is an indication that the defined system lacks one or more factors that are involved in the formation of the ∼130-nt fragment in the extract system. While degrading the discontinuous strand, MutLα endonuclease did not incise the continuous strand of the 3′-nicked O6-mG-T DNA (Fig. 8E, lanes 13–17). An inspection of the data in Fig. 8A showed that MutLα endonuclease incised the discontinuous strand of the 3′-nicked O6-mG-T DNA at random sites, displaying no significant site or sequence specificity. These findings suggested that MutLα endonuclease contributes to MMR system-dependent degradation of irreparable O6-mG-T mispair-containing DNA by introducing strand breaks at random sites on the discontinuous strand. In addition, these findings suggested that processing of the MutLα endonuclease-incised discontinuous strand of the 3′-nicked O6-mG-T DNA in the extract system led to the formation of the ∼130-nt product (Fig. 4A, lane 10). In agreement with this idea, the pattern of degradation of the discontinuous strand of the 3′-nicked O6-mG-T DNA in the extract system that was supplemented with the DNA polymerase inhibitor aphidicolin (Fig. 4A, lane 14) is similar to the pattern of degradation of the discontinuous strand of the 3′-nicked O6-mG-T DNA in the defined system (Fig. 8A, lanes 13–17).
      Figure thumbnail gr8
      FIGURE 8.MutLα endonuclease-dependent degradation of an irreparable O6-mG-T mispair-containing DNA in a defined system. The experiments were performed and analyzed as described under “Experimental Procedures.” Each reaction mixture contained a DNA substrate (81 fmol) and the indicated proteins and was incubated for 10 min at 37 °C. When MutSα, PCNA, RFC, and RPA were present in the reaction mixtures, their concentrations were 40, 24, 4, and 40 nm, respectively. The recovered DNAs that either were not digested (A and C) or were digested (E) with ClaI were resolved on alkaline agarose gels and hybridized with the 32P-labeled probes ts154, ts1, and c29. The ts154 and ts1 probes are complementary to the discontinuous strands, and the c29 probe is complementary to the continuous strands. A and C, DNA species visualized with the 32P-labeled probes ts154 and ts1, respectively. B, degradation of the discontinuous strands of the 3′-nicked DNA substrates as a function of MutLα endonuclease concentration. The data were obtained by quantification of images generated with the 32P-labeled probe ts154. One of the images is shown in A. D, degradation of the discontinuous strands of the 3′-nicked DNA substrates in the presence of the MutLα-E705K mutant. The data were obtained by quantification of images generated with the 32P-labeled probe ts1. One of the images is shown in C. The data in B and D are averages ± 1 S.D. (error bars) (n = 3). E, DNA species visualized with the 32P-labeled probe c29. Each of the diagrams depicts the relative positions of the 32P-labeled probe (a bar with an asterisk), the strand break, and a mismatch.

      Discussion

      Mammalian MGMT-deficient and yeast mgt1Δ rad52Δ cells are efficiently killed by low doses of Sn1-type methylating agents (
      • Branch P.
      • Aquilina G.
      • Bignami M.
      • Karran P.
      Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage.
      ,
      • Cejka P.
      • Mojas N.
      • Gillet L.
      • Schär P.
      • Jiricny J.
      Homologous recombination rescues mismatch-repair-dependent cytotoxicity of S(N)1-type methylating agents in S. cerevisiae.
      ,
      • Cejka P.
      • Stojic L.
      • Mojas N.
      • Russell A.M.
      • Heinimann K.
      • Cannavó E.
      • di Pietro M.
      • Marra G.
      • Jiricny J.
      Methylation-induced G2/M arrest requires a full complement of the mismatch repair protein hMLH1.
      ,
      • Kat A.
      • Thilly W.G.
      • Fang W.H.
      • Longley M.J.
      • Li G.M.
      • Modrich P.
      An alkylation-tolerant, mutator human cell line is deficient in strand-specific mismatch repair.
      ,
      • de Wind N.
      • Dekker M.
      • Berns A.
      • Radman M.
      • te Riele H.
      Inactivation of the mouse Msh2 gene results in mismatch repair deficiency, methylation tolerance, hyperrecombination, and predisposition to cancer.
      • Umar A.
      • Koi M.
      • Risinger J.I.
      • Glaab W.E.
      • Tindall K.R.
      • Kolodner R.D.
      • Boland C.R.
      • Barrett J.C.
      • Kunkel T.A.
      Correction of hypermutability, N-methyl-N′-nitro-N-nitrosoguanidine resistance, and defective DNA mismatch repair by introducing chromosome 2 into human tumor cells with mutations in MSH2 and MSH6.
      ). Several anticancer therapies exploit the marked sensitivity of MGMT-deficient cells to Sn1-type methylating agents. The marked sensitivity of mammalian MGMT-deficient and yeast mgt1Δ rad52Δ cells to Sn1-type methylating agents is a result of the MMR system-dependent cytotoxic response (
      • Branch P.
      • Aquilina G.
      • Bignami M.
      • Karran P.
      Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage.
      ,
      • Cejka P.
      • Mojas N.
      • Gillet L.
      • Schär P.
      • Jiricny J.
      Homologous recombination rescues mismatch-repair-dependent cytotoxicity of S(N)1-type methylating agents in S. cerevisiae.
      ,
      • Cejka P.
      • Stojic L.
      • Mojas N.
      • Russell A.M.
      • Heinimann K.
      • Cannavó E.
      • di Pietro M.
      • Marra G.
      • Jiricny J.
      Methylation-induced G2/M arrest requires a full complement of the mismatch repair protein hMLH1.
      ,
      • Kat A.
      • Thilly W.G.
      • Fang W.H.
      • Longley M.J.
      • Li G.M.
      • Modrich P.
      An alkylation-tolerant, mutator human cell line is deficient in strand-specific mismatch repair.
      ,
      • de Wind N.
      • Dekker M.
      • Berns A.
      • Radman M.
      • te Riele H.
      Inactivation of the mouse Msh2 gene results in mismatch repair deficiency, methylation tolerance, hyperrecombination, and predisposition to cancer.
      • Umar A.
      • Koi M.
      • Risinger J.I.
      • Glaab W.E.
      • Tindall K.R.
      • Kolodner R.D.
      • Boland C.R.
      • Barrett J.C.
      • Kunkel T.A.
      Correction of hypermutability, N-methyl-N′-nitro-N-nitrosoguanidine resistance, and defective DNA mismatch repair by introducing chromosome 2 into human tumor cells with mutations in MSH2 and MSH6.
      ). Previous research showed that the cytotoxic response to the Sn1-type methylating drug involves MMR system-dependent degradation of irreparable O6-mG-containing DNA that leads to the formation of lethal double strand breaks (
      • Cejka P.
      • Mojas N.
      • Gillet L.
      • Schär P.
      • Jiricny J.
      Homologous recombination rescues mismatch-repair-dependent cytotoxicity of S(N)1-type methylating agents in S. cerevisiae.
      ,
      • Mojas N.
      • Lopes M.
      • Jiricny J.
      Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA.
      ,
      • Tsaryk R.
      • Fabian K.
      • Thacker J.
      • Kaina B.
      Xrcc2 deficiency sensitizes cells to apoptosis by MNNG and the alkylating anticancer drugs temozolomide, fotemustine and mafosfamide.
      ). The MMR system starts to degrade irreparable O6-mG-containing nascent DNA behind the replication fork (
      • Mojas N.
      • Lopes M.
      • Jiricny J.
      Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA.
      ). At the same time, this DNA is incorporated into nucleosomes in the CAF-1-orchestrated process (
      • Smith S.
      • Stillman B.
      Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro.
      • Kaufman P.D.
      • Kobayashi R.
      • Kessler N.
      • Stillman B.
      The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication.
      ,
      • Verreault A.
      • Kaufman P.D.
      • Kobayashi R.
      • Stillman B.
      Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4.
      ,
      • Kaufman P.D.
      • Kobayashi R.
      • Stillman B.
      Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I.
      • Hoek M.
      • Stillman B.
      Chromatin assembly factor 1 is essential and couples chromatin assembly to DNA replication in vivo.
      ). It was previously unknown whether concomitant CAF-1-dependent nucleosome assembly affects degradation of irreparable O6-mG-containing DNA by the MMR system. We have found that CAF-1 suppresses the activity of the MMR system in the cytotoxic response of yeast mgt1Δ rad52Δ cells to MNNG (Fig. 1). We have also found that in an MGMT-deficient extract system, CAF-1-dependent incorporation of an irreparable O6-mG-T mispair-containing DNA into nucleosomes (Fig. 2B) correlates with a substantial decrease in degradation of this DNA by a MutLα endonuclease-dependent mechanism (FIGURE 5., FIGURE 6., FIGURE 7.). These findings imply that CAF-1-dependent packaging of irreparable O6-mG-T mispair-containing DNA into nucleosomes suppresses its degradation by the MMR system, therefore defending the cell against killing by the Sn1-type methylating drug. Consistent with this, we have established that loss of CAF-1 does not affect the sensitivity of yeast mgt1Δ rad52Δ cells to bleomycin, camptothecin, and hydroxyurea, DNA-damaging drugs that kill cells in an MMR system-independent manner (Fig. 1). It is known that 1–2 double strand breaks are sufficient to kill the yeast rad52 cell (
      • Resnick M.A.
      • Martin P.
      The repair of double-strand breaks in the nuclear DNA of Saccharomyces cerevisiae and its genetic control.
      ). Therefore, the fact that the MNNG treatment kills the cac1Δ mgt1Δ rad52Δ and cac2Δ mgt1Δ rad52Δ cells more efficiently than the mgt1Δ rad52Δ cells indicates that CAF-1 loss increases the fraction of MNNG-treated cells that experience at least 1–2 double strand breaks.
      In addition to CAF-1, S. cerevisiae cells contain several other histone chaperones, including HIR and Rtt106 (
      • Burgess R.J.
      • Zhang Z.
      Histone chaperones in nucleosome assembly and human disease.
      ). Our results indicated that loss of HIR2 or RTT106 does not increase the sensitivity of mgt1Δ rad52Δ cells to MNNG (Fig. 1G). Thus, these results imply that neither of these histone chaperones has a non-redundant function that provides a protection for mgt1Δ rad52Δ cells from the cytotoxic activity of the MMR system. We determined that decreasing histone H3-H4 gene dosage by deletion of the HHT2-HHF2 locus in the mgt1Δ rad52Δ cells does not change their sensitivity to MNNG (Fig. 1G). A previous report described that hht2-hhf2Δ does not affect the level of the chromatin H3-H4 histones but decreases the level of the soluble H3-H4 histones 2-fold (
      • Liang D.
      • Burkhart S.L.
      • Singh R.K.
      • Kabbaj M.H.
      • Gunjan A.
      Histone dosage regulates DNA damage sensitivity in a checkpoint-independent manner by the homologous recombination pathway.
      ). Thus, it appears that a small decrease in the level of the soluble histones H3-H4 does not affect the cytotoxic activity of the MMR system.
      An earlier study has implicated MutLα endonuclease activity in the cytotoxic response to the Sn1-type methylating drug in the yeast and mammalian cells (
      • Erdeniz N.
      • Nguyen M.
      • Deschênes S.M.
      • Liskay R.M.
      Mutations affecting a putative MutLα endonuclease motif impact multiple mismatch repair functions.
      ). We have now shown that activated MutLα endonuclease degrades the discontinuous strand of an irreparable O6-mG-T mispair-containing DNA in an extract system and in a purified system (FIGURE 4., FIGURE 5., FIGURE 6., FIGURE 7., FIGURE 8.). This information implies that MutLα endonuclease-dependent degradation of the discontinuous strand of irreparable O6-mG-T mispair-containing nuclear DNA is involved in the cytotoxic response to the Sn1-type methylating drug. MutLα endonuclease degrades the 3′-nicked O6-mG-T and G-T DNA substrates in the presence of MutSα, PCNA, RFC, and RPA in a very similar way (Fig. 8A, lanes 3–5 and lanes 15–17), suggesting that the same mechanism activates MutLα endonuclease in MMR and in the cytotoxic response to the Sn1-type methylating drug. The mechanism of activation of MutLα endonuclease in MMR requires the presence of MutSα and a mismatch (
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLα in human mismatch repair.
      ,
      • Kadyrov F.A.
      • Holmes S.F.
      • Arana M.E.
      • Lukianova O.A.
      • O'Donnell M.
      • Kunkel T.A.
      • Modrich P.
      Saccharomyces cerevisiae MutLα is a mismatch repair endonuclease.
      ,
      • Smith C.E.
      • Bowen N.
      • Graham 5th, W.J.
      • Goellner E.M.
      • Srivatsan A.
      • Kolodner R.D.
      Activation of Saccharomyces cerevisiae Mlh1-Pms1 endonuclease in a reconstituted mismatch repair system.
      ) (Fig. 8). The O6-mG-T mispair is one of many mispairs recognized by MutSα (
      • Drummond J.T.
      • Li G.-M.
      • Longley M.J.
      • Modrich P.
      Isolation of an hMSH2·p160 heterodimer that restores mismatch repair to tumor cells.
      ). A crystallographic study determined that MutSα has the same structure in the MutSα-G-T DNA and MutSα-O6-mG-T DNA crystals (
      • Warren J.J.
      • Pohlhaus T.J.
      • Changela A.
      • Iyer R.R.
      • Modrich P.L.
      • Beese L.S.
      Structure of the human MutSα DNA lesion recognition complex.
      ). This information and the results of our analysis of the defined reactions (Fig. 8) suggest that adoption of the same structure by MutSα on the G-T mispair and on the irreparable O6-mG-T mispair permits the protein to convey the same activating signal to MutLα endonuclease during MMR and the cytotoxic response to the Sn1-type methylating drug. Although the major product of persistent MutLα endonuclease-dependent degradation of the 3′-nicked O6-mG-T DNA in the extract system has a size of ∼130 nt (Figs. 4A and 5A (lane 10) and 6A (lane 8)), the products of MutLα endonuclease-dependent degradation of the 3′-nicked O6-mG-T DNA in the extract system that was supplemented with aphidicolin have sizes in the range of 100–2,000 nt (Fig. 4, A and B, lane 14). Because aphidicolin is an inhibitor of the biosynthetic activities of Pol δ and Pol ϵ, these findings suggest that DNA synthesis and ligation that occur on the MutLα-degraded DNA in the extract system in the absence of aphidicolin are necessary to remove the majority of the 100–2,000-nt products.
      It is important to note that a previous work described that MutLα-dependent processing of the 3′-nicked O6-mG-T DNA in the HCT116BBR nuclear extract-containing system leads to the formation of a ∼130-nt product (
      • York S.J.
      • Modrich P.
      Mismatch repair-dependent iterative excision at irreparable O6-methylguanine lesions in human nuclear extracts.
      ) that does not appear to be different from the one that we have detected in our extract system (FIGURE 4., FIGURE 5., FIGURE 6., FIGURE 7.). This observation implies that reactions that occur in different extract systems generate the same product of degradation of the 3′-nicked O6-mG-T DNA. We have observed that MutLα endonuclease-dependent degradation of the 3′-nicked O6-mG-T DNA in our extract system leads to the formation of a gapped product (Fig. 7). The gap was generated in the discontinuous strand in the presence of dNTPs and was located downstream from the mispaired T. Thus, the MMR system-dependent processing of the 3′-nicked O6-mG-T DNA in the cell extract generates two kinds of DNA products; one of them carries the gap (Fig. 7), and the other contains the ∼130-nt fragment (Figs. 5A and 6A). It is likely that the gap is formed when the excision step is not blocked by the O6-mG, whereas the ∼130-nt fragment is formed when the excision step is blocked by the lesion. The presence of a gap in the irreparable DNA is in good agreement with a previous study that documented that gaps are formed behind replication forks in response to treatment of both mammalian MGMT-deficient cells and yeast mgt1Δ rad52Δ cells with MNNG (
      • Mojas N.
      • Lopes M.
      • Jiricny J.
      Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA.
      ). The fact that the gap was generated downstream from the mismatched T (Fig. 7) suggests that O6-mG is a stronger block for the DNA polymerization reaction than for the excision reaction.
      The Sn1-type methylating drug temozolomide is used for treatment of glioblastoma patients. However, recurrent glioblastomas often arise during post-treatment period (
      • Cahill D.P.
      • Levine K.K.
      • Betensky R.A.
      • Codd P.J.
      • Romany C.A.
      • Reavie L.B.
      • Batchelor T.T.
      • Futreal P.A.
      • Stratton M.R.
      • Curry W.T.
      • Iafrate A.J.
      • Louis D.N.
      Loss of the mismatch repair protein MSH6 in human glioblastomas is associated with tumor progression during temozolomide treatment.
      ). This indicates that an approach that increases the sensitivity of MGMT-deficient tumors to treatment with the Sn1-type methylating drug might suppress recurrent cancers. More research is needed to determine whether defective replication-coupled nucleosome assembly increases the sensitivity of MGMT-deficient cancer cells to treatment with the Sn1-type methylating drug.

      Experimental Procedures

      Yeast Strains

      S. cerevisiae strains that were used in this study were derivatives of the wild-type haploid strain E134 (MATα ade5-1 lys2::InsE-A14 trp1-289 his7-2 leu2-3,112 ura3-52) (
      • Tran H.T.
      • Keen J.D.
      • Kricker M.
      • Resnick M.A.
      • Gordenin D.A.
      Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants.
      ). The lithium acetate/PEG4000/DMSO transformation method and PCR-amplified disruption cassettes were used to generate the strains.

      MNNG and Bleomycin Cytotoxicity Assays

      Liquid YPDAU medium (1% yeast extract, 2% Bacto-peptone, 2% dextrose, 60 mg/liter adenine, 60 mg/liter uracil), YPDAU plates, 1 mm stock solutions of MNNG (Wako Chemicals USA) in DMSO, a 2 mg/ml stock solution of bleomycin (Santa Cruz Biotechnology) in DMSO, and DMSO were used in the assays. Yeast cultures were grown to saturation in liquid YPDAU for ∼20 h at 30 °C. The saturated cultures were diluted 10-fold in fresh medium and grown for 4 h at 30 °C. The cultures were then diluted with fresh medium to A600 = 1.3, and aliquots of the diluted cultures were treated with 1 μm MNNG, 0.1% DMSO (vehicle control in the MNNG toxicity assay), 30 μg/ml bleomycin, and 1.5% DMSO (vehicle control in the bleomycin cytotoxicity assay) for 2 h at 30 °C. After treatment, the cultures were diluted, and appropriate dilutions of the cultures were spread on YPDAU plates. The plates were incubated for 3–4 days at 30 °C, and colonies were counted. A somewhat different MNNG cytotoxicity assay was also used in this study. In this assay, 10-fold serial dilutions of the treated cultures were made and spotted on YPDAU plates. The plates were incubated for 2 days at 30 °C and photographed.

      Camptothecin and Hydroxyurea Cytotoxicity Assays

      Yeast cultures were grown to saturation as described above and diluted to A600 = 1.4 with sterile water. 10-fold serial dilutions of the cultures were prepared and spotted on YPDAU plates, YPDAU plates containing 0.5 μg/ml camptothecin (Enzo Life Science), and YPDAU plates containing 10 mm hydroxyurea (US Biological). After incubation for 2 days at 30 °C, the plates were photographed.

      Cell Extract and Recombinant Proteins

      293T cells were grown as an attached culture in DMEM/high glucose medium that was supplemented with 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.29 μg/ml l-glutamine. Cytosolic and nuclear extracts from proliferating 293T cells were prepared according to a described procedure (
      • Kadyrova L.Y.
      • Blanko E.R.
      • Kadyrov F.A.
      CAF-I-dependent control of degradation of the discontinuous strands during mismatch repair.
      ). Recombinant human CAF-1, MutSα, MutLα, MutLα-E705K, PCNA, RFC, and RPA were isolated in nearly homogeneous forms as described previously (
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLα in human mismatch repair.
      ,
      • Kadyrova L.Y.
      • Blanko E.R.
      • Kadyrov F.A.
      CAF-I-dependent control of degradation of the discontinuous strands during mismatch repair.
      ).

      Western Blotting

      Samples of the purified CAF-1, 293T cytosolic extract (15 μg), and 293T nuclear extract (15 μg) were separated on a denaturing SDS gel and transferred onto a PVDF membrane. After the protein transfer step, the membrane was incubated with α-CAF-1 p150 antibodies (catalog no. sc-10772, lot E2004, rabbit polyclonal IgG, Santa Cruz Biotechnology, Inc.) and then with ECL HRP-conjugated secondary antibodies (catalog no. NA934V, lot 389592, donkey antibody, GE Healthcare). Immune complexes were visualized utilizing ECL2 Western blotting substrate (Thermo Fisher Scientific) and a CCD camera. Amounts of CAF-1 in the samples of the 293T cytosolic and nuclear extracts were measured by quantification of the data with the ImageJ software.

      DNA Substrates, Oligonucleotides, and 32P-Labeled Hybridization Probes

      3′-Nicked O6-mG-T, 3′-nicked G-T, and 3′-nicked A-T DNAs were essentially prepared as described previously (
      • York S.J.
      • Modrich P.
      Mismatch repair-dependent iterative excision at irreparable O6-methylguanine lesions in human nuclear extracts.
      ), except that after DNA ligation, unligated material was degraded by exonuclease III (New England Biolabs), and the remaining DNA was purified by chromatography on BND-cellulose (Sigma). The O6-mG-containing oligonucleotide that was used to prepare the 3′-nicked O6-mG-T DNA was synthesized by Midland Certified Reagent Co. All other oligonucleotides used in this study were synthesized by IDT. To prepare a 32P-labeled hybridization probe, the oligonucleotide was labeled at the 5′ end with 32P by T4 polynucleotide kinase in the presence of [γ-32P]ATP. The sequences of oligonucleotides used to construct the DNA substrates and prepare 32P-labeled hybridization probes are shown in Table 1.
      TABLE 1Oligonucleotides that were used to prepare the DNA substrates and 32P-labeled hybridization probes
      Oligonucleotide
      aThe first three oligonucleotides were used to prepare the 3′-nicked DNA substrates, and the remaining oligonucleotides were used to prepare 32P-labeled hybridization probes.
      Oligonucleotide sequence
      AH1A A-T5′-GCTACCGTCCTCGAAGCTTCCGCATCGGAGTCGACG-3′
      AH1A G-T5′-GCTACCGTCCTCGAGGCTTCCGCATCGGAGTCGACG-3′
      AH1A O6-mG-T5′-GCTACCGTCCTCGAO6mGGCTTCCGCATCGGAGTCGACG-3′
      ts15′-CTCTTCCTTTTTCAATTGGGATCC-3′
      ts1545′-TCGAGTCTCTCGCTACCGTCCTCG-3′
      ts1825′-TTCCGCATCGGAGTCGACGGCTTC-3′
      ts11315′-GACAGTTACCAATGCTTAATCAGTG-3′
      c295′-CTGAGGATCCCAATTGAAAAAGGA-3′
      c1545′-CGAGGACGGTAGCGAGAGACTCGA-3′
      a aThe first three oligonucleotides were used to prepare the 3′-nicked DNA substrates, and the remaining oligonucleotides were used to prepare 32P-labeled hybridization probes.

      Nucleosome Assembly Reactions in 293T Cytosolic Extract-containing Mixtures

      The nucleosome assembly reactions were carried out at 37 °C in 40-μl mixtures that contained 20 mm HEPES-NaOH (pH 7.4), 100 mm KCl, 8 mm MgCl2, 2 mm DTT, 0.2 mg/ml BSA, 0.1 mm each of the four dNTPs, 3 mm ATP, 20 mm creatine phosphate, 0.02 mg/ml creatine phosphokinase, 1% glycerol (v/v), 75 μg of 293T cytosolic extract, purified CAF-1 (0 or 1.2 pmol), MutLα (0 or 1.6 pmol), and 81 fmol (0.1 μg) of a 3′-nicked DNA (the 3′-nicked O6-mG-T DNA, the 3′-nicked G-T DNA, or the 3′-nicked A-T DNA). After 60 min of incubation, a 35-μl fraction of each reaction mixture was mixed with a 5-μl mixture containing 20 mm HEPES-NaOH (pH 7.4), 64 mm CaCl2, 8 units/μl micrococcal nuclease, and 0.02 mg/ml RNase A and incubated for 20 min at 21–23 °C. Each reaction mixture was then supplemented with a 10-μl solution containing 0.5% SDS, 150 mm EDTA, 40% glycerol, and 2 mg/ml Proteinase K, followed by incubation of the mixture for 20 min at 50 °C. Proteinase K in the mixtures was inactivated by the addition of PMSF to a final concentration of 0.8 mm. The nucleosome assembly products were separated on native 1.7% agarose gels, transferred onto nitrocellulose membranes, and hybridized with 32P-labeled probe c154. Indirectly labeled DNA species were visualized with a Typhoon biomolecular imager (GE Healthcare) and quantified with ImageQuant software.

      Mismatch-provoked Reactions in an Extract System

      The mismatch-provoked and control reactions in the extract system were carried out at 37 °C in 80-μl mixtures containing 20 mm HEPES-NaOH (pH 7.4), 100 mm KCl, 8 mm MgCl2, 2 mm DTT, 0.2 mg/ml BSA, 0.1 mm each of the four dNTPs, 3 mm ATP, 20 mm creatine phosphate, 0.02 mg/ml creatine phosphokinase, 1% glycerol (v/v), 150 μg of 293T cell extract, MutLα (0 or 3.2 pmol), MutLα-E705K (0 or 3.2 pmol), purified CAF-1 (0, 0.3, 0.6, or 2.4 pmol), and 41 or 162 fmol of a 3′-nicked DNA (the 3′-nicked O6-mG-T DNA, the 3′-nicked G-T DNA, or the 3′-nicked A-T DNA). The reaction mixtures were incubated for 10–120 min. Reactions in each mixture were stopped by the addition of a 60-μl solution containing 0.35% SDS, 0.4 m NaCl, 13 mm EDTA, 0.33 mg/ml Proteinase K, and 1 mg/ml glycogen, and the resulting mixtures were incubated for 20 min at 50 °C, followed by their extraction with phenol/chloroform mixture. DNAs present in the supernatants were recovered by isopropyl alcohol precipitation. To detect whether the repair of O6-mG-T or G-T mispairs occurred in a reaction mixture, a fraction of the recovered DNA was cleaved with BanI and XhoI and separated on a 1.2% agarose gel, followed by staining of the gel with ethidium bromide and quantification of DNA species with the ImageJ software. To detect whether a 3′-nicked DNA was degraded in the reaction, a fraction of the recovered DNA was cleaved with ClaI, separated on an alkaline 1.4% agarose gel, transferred onto a nylon membrane, and hybridized with a 32P-labeled probe. To detect whether gapped DNA was produced in a reaction mixture, a fraction of the recovered DNA was digested with AhdI, resolved on a native 1.4% agarose gel, transferred onto a nylon membrane, and hybridized with an indicated 32P-labeled probe. Indirectly labeled DNA species were visualized and quantified as described above.

      Cleavage of DNA by Activated MutLα Endonuclease in a Defined System

      The reactions were performed in 40-μl mixtures that contained 20 mm HEPES-NaOH (pH 7.4), 120 mm KCl, 5 mm MgCl2, 3 mm ATP, 2 mm DTT, 0.2 mg/ml BSA, 2% glycerol (v/v), and 2 nm (81 fmol) of a 3′-nicked DNA (the 3′-nicked O6-mG-T DNA, the 3′-nicked G-T DNA, or the 3′-nicked A-T DNA). When indicated, MutSα (40 nm), MutLα (2, 6, or 20 nm), MutLα-E705K (20 nm), PCNA (24 nm), RFC (4 nm), and RPA (40 nm) were included in the reaction mixtures. The reaction mixtures were incubated for 10 min at 37 °C, and each reaction was terminated by the addition of a 30-μl mixture containing 0.35% SDS, 0.4 m NaCl, 13 mm EDTA, 0.33 mg/ml Proteinase K, and 2 mg/ml glycogen, followed by incubation of the mixtures for 15 min at 50 °C. The mixtures were then extracted by phenol/chloroform, and the DNAs were recovered by isopropyl alcohol precipitation. Recovered DNAs that were digested or not with ClaI were separated on alkaline 1.4% denaturing agarose gels, transferred onto nylon membranes, and hybridized with 32P-labeled probes. Indirectly labeled DNA species were visualized and quantified as described above.

      Author Contributions

      L. K., B. D., and F. K. performed experiments and analyzed data. F. K. and L. K. designed experiments, contributed reagents, prepared the figures, and wrote the paper.

      Acknowledgments

      We thank Farid F. Kadyrov for critical reading of the manuscript.

      References

        • Modrich P.
        Methyl-directed DNA mismatch correction.
        J. Biol. Chem. 1989; 264: 6597-6600
        • Kunkel T.A.
        • Erie D.A.
        DNA mismatch repair.
        Annu. Rev. Biochem. 2005; 74: 681-710
        • Iyer R.R.
        • Pluciennik A.
        • Burdett V.
        • Modrich P.L.
        DNA mismatch repair: functions and mechanisms.
        Chem. Rev. 2006; 106: 302-323
        • Jiricny J.
        The multifaceted mismatch-repair system.
        Nat. Rev. Mol. Cell Biol. 2006; 7: 335-346
        • Peña-Diaz J.
        • Jiricny J.
        Mammalian mismatch repair: error-free or error-prone?.
        Trends Biochem. Sci. 2012; 37: 206-214
        • Kunkel T.A.
        • Erie D.A.
        Eukaryotic mismatch repair in relation to DNA replication.
        Annu. Rev. Genet. 2015; 49: 291-313
        • Earley M.C.
        • Crouse G.F.
        The role of mismatch repair in the prevention of base pair mutations in Saccharomyces cerevisiae.
        Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 15487-15491
        • Russo M.T.
        • Blasi M.F.
        • Chiera F.
        • Fortini P.
        • Degan P.
        • Macpherson P.
        • Furuichi M.
        • Nakabeppu Y.
        • Karran P.
        • Aquilina G.
        • Bignami M.
        The oxidized deoxynucleoside triphosphate pool is a significant contributor to genetic instability in mismatch repair-deficient cells.
        Mol. Cell. Biol. 2004; 24: 465-474
        • Shen Y.
        • Koh K.D.
        • Weiss B.
        • Storici F.
        Mispaired rNMPs in DNA are mutagenic and are targets of mismatch repair and RNases H.
        Nat. Struct. Mol. Biol. 2011; 19: 98-104
        • Kadyrova L.Y.
        • Dahal B.K.
        • Kadyrov F.A.
        Evidence that the DNA mismatch repair system removes 1-nt Okazaki fragment flaps.
        J. Biol. Chem. 2015; 290: 24051-24065
        • Morrison A.
        • Johnson A.L.
        • Johnston L.H.
        • Sugino A.
        Pathway correcting DNA replication errors in Saccharomyces cerevisiae.
        EMBO J. 1993; 12: 1467-1473
        • Morrison A.
        • Sugino A.
        The 3′ → 5′ exonucleases of both DNA polymerases δ and ϵ participate in correcting errors of DNA replication in Saccharomyces cerevisiae.
        Mol. Gen. Genet. 1994; 242: 289-296
        • Tran H.T.
        • Keen J.D.
        • Kricker M.
        • Resnick M.A.
        • Gordenin D.A.
        Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants.
        Mol. Cell. Biol. 1997; 17: 2859-2865
        • Tran H.T.
        • Gordenin D.A.
        • Resnick M.A.
        The 3′ → 5′ exonucleases of DNA polymerases delta and epsilon and the 5′ → 3′ exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae.
        Mol. Cell. Biol. 1999; 19: 2000-2007
        • Greene C.N.
        • Jinks-Robertson S.
        Spontaneous frameshift mutations in Saccharomyces cerevisiae: accumulation during DNA replication and removal by proofreading and mismatch repair activities.
        Genetics. 2001; 159: 65-75
        • Burgers P.M.
        Polymerase dynamics at the eukaryotic DNA replication fork.
        J. Biol. Chem. 2009; 284: 4041-4045
        • Stone J.E.
        • Petes T.D.
        Analysis of the proteins involved in the in vivo repair of base-base mismatches and four-base loops formed during meiotic recombination in the yeast Saccharomyces cerevisiae.
        Genetics. 2006; 173: 1223-1239
        • Kadyrova L.Y.
        • Kadyrov F.A.
        Endonuclease activities of MutLalpha and its homologs in DNA mismatch repair.
        DNA Repair. 2016; 38: 42-49
        • Kolodner R.D.
        A personal historical view of DNA mismatch repair with an emphasis on eukaryotic DNA mismatch repair.
        DNA Repair. 2016; 38: 3-13
        • Li F.
        • Ortega J.
        • Gu L.
        • Li G.M.
        Regulation of mismatch repair by histone code and posttranslational modifications in eukaryotic cells.
        DNA Repair. 2016; 38: 68-74
        • Drummond J.T.
        • Li G.-M.
        • Longley M.J.
        • Modrich P.
        Isolation of an hMSH2·p160 heterodimer that restores mismatch repair to tumor cells.
        Science. 1995; 268: 1909-1912
        • Palombo F.
        • Gallinari P.
        • Iaccarino I.
        • Lettieri T.
        • Hughes M.
        • D'Arrigo A.
        • Truong O.
        • Hsuan J.J.
        • Jiricny J.
        GTBP, a 160-kilodalton protein essential for mismatch-binding activity in human cells.
        Science. 1995; 268: 1912-1914
        • Li G.-M.
        • Modrich P.
        Restoration of mismatch repair to nuclear extracts of H6 colorectal tumor cells by a heterodimer of human MutL homologs.
        Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 1950-1954
        • Szankasi P.
        • Smith G.R.
        A role for exonuclease I from S. pombe in mutation avoidance and mismatch correction.
        Science. 1995; 267: 1166-1169
        • Umar A.
        • Buermeyer A.B.
        • Simon J.A.
        • Thomas D.C.
        • Clark A.B.
        • Liskay R.M.
        • Kunkel T.A.
        Requirement for PCNA in DNA mismatch repair at a step preceding DNA resynthesis.
        Cell. 1996; 87: 65-73
        • Johnson R.E.
        • Kovvali G.K.
        • Guzder S.N.
        • Amin N.S.
        • Holm C.
        • Habraken Y.
        • Sung P.
        • Prakash L.
        • Prakash S.
        Evidence for involvement of yeast proliferating cell nuclear antigen in DNA mismatch repair.
        J. Biol. Chem. 1996; 271: 27987-27990
        • Longley M.J.
        • Pierce A.J.
        • Modrich P.
        DNA polymerase δ is required for human mismatch repair in vitro.
        J. Biol. Chem. 1997; 272: 10917-10921
        • Tishkoff D.X.
        • Boerger A.L.
        • Bertrand P.
        • Filosi N.
        • Gaida G.M.
        • Kane M.F.
        • Kolodner R.D.
        Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2.
        Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 7487-7492
        • Palombo F.
        • Iaccarino I.
        • Nakajima E.
        • Ikejima M.
        • Shimada T.
        • Jiricny J.
        hMutSβ, a heterodimer of hMSH2 and hMSH3, binds to insertion/deletion loops in DNA.
        Curr. Biol. 1996; 6: 1181-1184
        • Genschel J.
        • Littman S.J.
        • Drummond J.T.
        • Modrich P.
        Isolation of hMutSβ from human cells and comparison of the mismatch repair specificities of hMutSβ and hMutSα.
        J. Biol. Chem. 1998; 273: 19895-19901
        • de Wind N.
        • Dekker M.
        • Claij N.
        • Jansen L.
        • van Klink Y.
        • Radman M.
        • Riggins G.
        • van der Valk M.
        • van't Wout K.
        • te Riele H.
        HNPCC-like cancer predisposition in mice through simultaneous loss of Msh3 and Msh6 mismatch-repair protein functions.
        Nat. Genet. 1999; 23: 359-362
        • Genschel J.
        • Bazemore L.R.
        • Modrich P.
        Human exonuclease I is required for 5′ and 3′ mismatch repair.
        J. Biol. Chem. 2002; 277: 13302-13311
        • Genschel J.
        • Modrich P.
        Mechanism of 5′-directed excision in human mismatch repair.
        Mol. Cell. 2003; 12: 1077-1086
        • Wei K.
        • Clark A.B.
        • Wong E.
        • Kane M.F.
        • Mazur D.J.
        • Parris T.
        • Kolas N.K.
        • Russell R.
        • Hou Jr, H.
        • Kneitz B.
        • Yang G.
        • Kunkel T.A.
        • Kolodner R.D.
        • Cohen P.E.
        • Edelmann W.
        Inactivation of Exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility.
        Genes Dev. 2003; 17: 603-614
        • Dzantiev L.
        • Constantin N.
        • Genschel J.
        • Iyer R.R.
        • Burgers P.M.
        • Modrich P.
        A defined human system that supports bidirectional mismatch-provoked excision.
        Mol. Cell. 2004; 15: 31-41
        • Constantin N.
        • Dzantiev L.
        • Kadyrov F.A.
        • Modrich P.
        Human mismatch repair: reconstitution of a nick-directed bidirectional reaction.
        J. Biol. Chem. 2005; 280: 39752-39761
        • Kadyrov F.A.
        • Dzantiev L.
        • Constantin N.
        • Modrich P.
        Endonucleolytic function of MutLα in human mismatch repair.
        Cell. 2006; 126: 297-308
        • Modrich P.
        Mechanisms in eukaryotic mismatch repair.
        J. Biol. Chem. 2006; 281: 30305-30309
        • Kadyrov F.A.
        • Holmes S.F.
        • Arana M.E.
        • Lukianova O.A.
        • O'Donnell M.
        • Kunkel T.A.
        • Modrich P.
        Saccharomyces cerevisiae MutLα is a mismatch repair endonuclease.
        J. Biol. Chem. 2007; 282: 37181-37190
        • Zhang Y.
        • Yuan F.
        • Presnell S.R.
        • Tian K.
        • Gao Y.
        • Tomkinson A.E.
        • Gu L.
        • Li G.M.
        Reconstitution of 5′-directed human mismatch repair in a purified system.
        Cell. 2005; 122: 693-705
        • Kadyrov F.A.
        • Genschel J.
        • Fang Y.
        • Penland E.
        • Edelmann W.
        • Modrich P.
        A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8495-8500
        • Smith C.E.
        • Bowen N.
        • Graham 5th, W.J.
        • Goellner E.M.
        • Srivatsan A.
        • Kolodner R.D.
        Activation of Saccharomyces cerevisiae Mlh1-Pms1 endonuclease in a reconstituted mismatch repair system.
        J. Biol. Chem. 2015; 290: 21580-21590
        • Rodriges Blanko E.
        • Kadyrova L.Y.
        • Kadyrov F.A.
        DNA mismatch repair interacts with CAF-1- and ASF1A-H3-H4-dependent histone (H3-H4)2 tetramer deposition.
        J. Biol. Chem. 2016; 291: 9203-9217
        • Pluciennik A.
        • Dzantiev L.
        • Iyer R.R.
        • Constantin N.
        • Kadyrov F.A.
        • Modrich P.
        PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 16066-16071
        • Shao H.
        • Baitinger C.
        • Soderblom E.J.
        • Burdett V.
        • Modrich P.
        Hydrolytic function of Exo1 in mammalian mismatch repair.
        Nucleic Acids Res. 2014; 42: 7104-7112
        • Branch P.
        • Aquilina G.
        • Bignami M.
        • Karran P.
        Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage.
        Nature. 1993; 362: 652-654
        • Cejka P.
        • Mojas N.
        • Gillet L.
        • Schär P.
        • Jiricny J.
        Homologous recombination rescues mismatch-repair-dependent cytotoxicity of S(N)1-type methylating agents in S. cerevisiae.
        Curr. Biol. 2005; 15: 1395-1400
        • Loveless A.
        Possible relevance of O-6 alkylation of deoxyguanosine to the mutagenicity and carcinogenicity of nitrosamines and nitrosamides.
        Nature. 1969; 223: 206-207
        • Glassner B.J.
        • Weeda G.
        • Allan J.M.
        • Broekhof J.L.
        • Carls N.H.
        • Donker I.
        • Engelward B.P.
        • Hampson R.J.
        • Hersmus R.
        • Hickman M.J.
        • Roth R.B.
        • Warren H.B.
        • Wu M.M.
        • Hoeijmakers J.H.
        • Samson L.D.
        DNA repair methyltransferase (Mgmt) knockout mice are sensitive to the lethal effects of chemotherapeutic alkylating agents.
        Mutagenesis. 1999; 14: 339-347
        • Esteller M.
        • Garcia-Foncillas J.
        • Andion E.
        • Goodman S.N.
        • Hidalgo O.F.
        • Vanaclocha V.
        • Baylin S.B.
        • Herman J.G.
        Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents.
        N. Engl. J. Med. 2000; 343: 1350-1354
        • Duckett D.R.
        • Drummond J.T.
        • Murchie A.I.H.
        • Reardon J.T.
        • Sancar A.
        • Lilley D.M.J.
        • Modrich P.
        Human MutSα recognizes damaged DNA base pairs containing O6-methylguanine, O4-methylthymine or the cisplatin-d(GpG) adduct.
        Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 6443-6447
        • York S.J.
        • Modrich P.
        Mismatch repair-dependent iterative excision at irreparable O6-methylguanine lesions in human nuclear extracts.
        J. Biol. Chem. 2006; 281: 22674-22683
        • Mojas N.
        • Lopes M.
        • Jiricny J.
        Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA.
        Genes Dev. 2007; 21: 3342-3355
        • Zhukovskaya N.
        • Branch P.
        • Aquilina G.
        • Karran P.
        DNA replication arrest and tolerance to DNA methylation damage.
        Carcinogenesis. 1994; 15: 2189-2194
        • Stojic L.
        • Mojas N.
        • Cejka P.
        • Di Pietro M.
        • Ferrari S.
        • Marra G.
        • Jiricny J.
        Mismatch repair-dependent G2 checkpoint induced by low doses of SN1 type methylating agents requires the ATR kinase.
        Genes Dev. 2004; 18: 1331-1344
        • Tsaryk R.
        • Fabian K.
        • Thacker J.
        • Kaina B.
        Xrcc2 deficiency sensitizes cells to apoptosis by MNNG and the alkylating anticancer drugs temozolomide, fotemustine and mafosfamide.
        Cancer Lett. 2006; 239: 305-313
        • Smith S.
        • Stillman B.
        Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro.
        Cell. 1989; 58: 15-25
        • Kaufman P.D.
        • Kobayashi R.
        • Kessler N.
        • Stillman B.
        The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication.
        Cell. 1995; 81: 1105-1114
        • Verreault A.
        • Kaufman P.D.
        • Kobayashi R.
        • Stillman B.
        Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4.
        Cell. 1996; 87: 95-104
        • Kaufman P.D.
        • Kobayashi R.
        • Stillman B.
        Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I.
        Genes Dev. 1997; 11: 345-357
        • Hoek M.
        • Stillman B.
        Chromatin assembly factor 1 is essential and couples chromatin assembly to DNA replication in vivo.
        Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12183-12188
        • Smith S.
        • Stillman B.
        Stepwise assembly of chromatin during DNA replication in vitro.
        EMBO J. 1991; 10: 971-980
        • Kadyrova L.Y.
        • Blanko E.R.
        • Kadyrov F.A.
        CAF-I-dependent control of degradation of the discontinuous strands during mismatch repair.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 2753-2758
        • Kadyrova L.Y.
        • Rodriges Blanko E.
        • Kadyrov F.A.
        Human CAF-1-dependent nucleosome assembly in a defined system.
        Cell Cycle. 2013; 12: 3286-3297
        • Shibahara K.
        • Stillman B.
        Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin.
        Cell. 1999; 96: 575-585
        • Rolef Ben-Shahar T.
        • Castillo A.G.
        • Osborne M.J.
        • Borden K.L.
        • Kornblatt J.
        • Verreault A.
        Two fundamentally distinct PCNA interaction peptides contribute to chromatin assembly factor 1 function.
        Mol. Cell. Biol. 2009; 29: 6353-6365
        • Schöpf B.
        • Bregenhorn S.
        • Quivy J.P.
        • Kadyrov F.A.
        • Almouzni G.
        • Jiricny J.
        Interplay between mismatch repair and chromatin assembly.
        Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 1895-1900
        • Erdeniz N.
        • Nguyen M.
        • Deschênes S.M.
        • Liskay R.M.
        Mutations affecting a putative MutLα endonuclease motif impact multiple mismatch repair functions.
        DNA Repair. 2007; 6: 1463-1470
        • Xiao W.
        • Derfler B.
        • Chen J.
        • Samson L.
        Primary sequence and biological functions of a Saccharomyces cerevisiae O6-methylguanine/O4-methylthymine DNA repair methyltransferase gene.
        EMBO J. 1991; 10: 2179-2186
        • Sung P.
        Function of yeast Rad52 protein as a mediator between replication protein A and the Rad51 recombinase.
        J. Biol. Chem. 1997; 272: 28194-28197
        • Hsiang Y.H.
        • Lihou M.G.
        • Liu L.F.
        Arrest of replication forks by drug-stabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by camptothecin.
        Cancer Res. 1989; 49: 5077-5082
        • Krakoff I.H.
        • Brown N.C.
        • Reichard P.
        Inhibition of ribonucleoside diphosphate reductase by hydroxyurea.
        Cancer Res. 1968; 28: 1559-1565
        • Povirk L.F.
        DNA damage and mutagenesis by radiomimetic DNA-cleaving agents: bleomycin, neocarzinostatin and other enediynes.
        Mutat. Res. 1996; 355: 71-89
        • Green E.M.
        • Antczak A.J.
        • Bailey A.O.
        • Franco A.A.
        • Wu K.J.
        • Yates 3rd, J.R.
        • Kaufman P.D.
        Replication-independent histone deposition by the HIR complex and Asf1.
        Curr. Biol. 2005; 15: 2044-2049
        • Burgess R.J.
        • Zhang Z.
        Histone chaperones in nucleosome assembly and human disease.
        Nat. Struct. Mol. Biol. 2013; 20: 14-22
        • Cross S.L.
        • Smith M.M.
        Comparison of the structure and cell cycle expression of mRNAs encoded by two histone H3-H4 loci in Saccharomyces cerevisiae.
        Mol. Cell. Biol. 1988; 8: 945-954
        • Trojan J.
        • Zeuzem S.
        • Randolph A.
        • Hemmerle C.
        • Brieger A.
        • Raedle J.
        • Plotz G.
        • Jiricny J.
        • Marra G.
        Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system.
        Gastroenterology. 2002; 122: 211-219
        • Cejka P.
        • Stojic L.
        • Mojas N.
        • Russell A.M.
        • Heinimann K.
        • Cannavó E.
        • di Pietro M.
        • Marra G.
        • Jiricny J.
        Methylation-induced G2/M arrest requires a full complement of the mismatch repair protein hMLH1.
        EMBO J. 2003; 22: 2245-2254
        • Holmes Jr., J.
        • Clark S.
        • Modrich P.
        Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines.
        Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 5837-5841
        • Kat A.
        • Thilly W.G.
        • Fang W.H.
        • Longley M.J.
        • Li G.M.
        • Modrich P.
        An alkylation-tolerant, mutator human cell line is deficient in strand-specific mismatch repair.
        Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 6424-6428
        • de Wind N.
        • Dekker M.
        • Berns A.
        • Radman M.
        • te Riele H.
        Inactivation of the mouse Msh2 gene results in mismatch repair deficiency, methylation tolerance, hyperrecombination, and predisposition to cancer.
        Cell. 1995; 82: 321-330
        • Umar A.
        • Koi M.
        • Risinger J.I.
        • Glaab W.E.
        • Tindall K.R.
        • Kolodner R.D.
        • Boland C.R.
        • Barrett J.C.
        • Kunkel T.A.
        Correction of hypermutability, N-methyl-N′-nitro-N-nitrosoguanidine resistance, and defective DNA mismatch repair by introducing chromosome 2 into human tumor cells with mutations in MSH2 and MSH6.
        Cancer Res. 1997; 57: 3949-3955
        • Resnick M.A.
        • Martin P.
        The repair of double-strand breaks in the nuclear DNA of Saccharomyces cerevisiae and its genetic control.
        Mol. Gen. Genet. 1976; 143: 119-129
        • Liang D.
        • Burkhart S.L.
        • Singh R.K.
        • Kabbaj M.H.
        • Gunjan A.
        Histone dosage regulates DNA damage sensitivity in a checkpoint-independent manner by the homologous recombination pathway.
        Nucleic Acids Res. 2012; 40: 9604-9620
        • Warren J.J.
        • Pohlhaus T.J.
        • Changela A.
        • Iyer R.R.
        • Modrich P.L.
        • Beese L.S.
        Structure of the human MutSα DNA lesion recognition complex.
        Mol. Cell. 2007; 26: 579-592
        • Cahill D.P.
        • Levine K.K.
        • Betensky R.A.
        • Codd P.J.
        • Romany C.A.
        • Reavie L.B.
        • Batchelor T.T.
        • Futreal P.A.
        • Stratton M.R.
        • Curry W.T.
        • Iafrate A.J.
        • Louis D.N.
        Loss of the mismatch repair protein MSH6 in human glioblastomas is associated with tumor progression during temozolomide treatment.
        Clin. Cancer Res. 2007; 13: 2038-2045