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The nuclease activity of DNA2 promotes exonuclease 1–independent mismatch repair

Open AccessPublished:March 14, 2022DOI:https://doi.org/10.1016/j.jbc.2022.101831
      The DNA mismatch repair (MMR) system is a major DNA repair system that corrects DNA replication errors. In eukaryotes, the MMR system functions via mechanisms both dependent on and independent of exonuclease 1 (EXO1), an enzyme that has multiple roles in DNA metabolism. Although the mechanism of EXO1-dependent MMR is well understood, less is known about EXO1-independent MMR. Here, we provide genetic and biochemical evidence that the DNA2 nuclease/helicase has a role in EXO1-independent MMR. Biochemical reactions reconstituted with purified human proteins demonstrated that the nuclease activity of DNA2 promotes an EXO1-independent MMR reaction via a mismatch excision-independent mechanism that involves DNA polymerase δ. We show that DNA polymerase ε is not able to replace DNA polymerase δ in the DNA2-promoted MMR reaction. Unlike its nuclease activity, the helicase activity of DNA2 is dispensable for the ability of the protein to enhance the MMR reaction. Further examination established that DNA2 acts in the EXO1-independent MMR reaction by increasing the strand-displacement activity of DNA polymerase δ. These data reveal a mechanism for EXO1-independent mismatch repair.

      Abbreviations:

      EXO1 (exonuclease 1), MMR (DNA mismatch repair), Pol δ (DNA polymerase δ), Pol ε (DNA polymerase ε), PCNA (proliferating cell nuclear antigen), RFC (replication factor C), RPA (replication protein A)
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      • Zhang Y.
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      • Tian K.
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      • Constantin N.
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      Human mismatch repair: Reconstitution of a nick-directed bidirectional reaction.
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      ,
      • Modrich P.
      Mechanisms in eukaryotic mismatch repair.
      ).
      EXO1 deficiency in humans does not seem to cause significant cancer predisposition (
      • Peltomaki P.
      Lynch syndrome genes.
      ). Nevertheless, it is known that Exo1-/- mice are susceptible to the development of lymphomas (
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      • Tran H.T.
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      • Kadyrov F.A.
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      A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair.
      ). Furthermore, an EXO1-independent MMR reaction that occurred in a mammalian cell extract system without the formation of a gapped excision intermediate was observed (
      • Kadyrov F.A.
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      A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair.
      ). Together, these findings implicated the strand-displacement activity of Pol δ holoenzyme in EXO1-independent MMR.
      In this study, we investigated DNA2 in the context of MMR. DNA2 is an essential multifunctional protein that has nuclease, ATPase, and 5'→3′ helicase activities (
      • Budd M.E.
      • Campbell J.L.
      A yeast gene required for DNA replication encodes a protein with homology to DNA helicases.
      ,
      • Budd M.E.
      • Choe W.
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      The nuclease activity of the yeast DNA2 protein, which is related to the RecB-like nucleases, is essential in vivo.
      ,
      • Pinto C.
      • Kasaciunaite K.
      • Seidel R.
      • Cejka P.
      Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases.
      ). Previous research ascertained that DNA2 removes long flaps during Okazaki fragment maturation (
      • Budd M.E.
      • Campbell J.L.
      A yeast replicative helicase, Dna2 helicase, interacts with yeast FEN-1 nuclease in carrying out its essential function.
      ,
      • Burgers P.M.
      Polymerase dynamics at the eukaryotic DNA replication fork.
      ,
      • Jin Y.H.
      • Ayyagari R.
      • Resnick M.A.
      • Gordenin D.A.
      • Burgers P.M.
      Okazaki fragment maturation in yeast. II. Cooperation between the polymerase and 3'-5'-exonuclease activities of Pol delta in the creation of a ligatable nick.
      ), participates in the resection step of double-strand break repair (
      • Zhu Z.
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      • Lee S.E.
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      Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends.
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      • Niu H.
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      ,
      • Cejka P.
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      • Kowalczykowski S.C.
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      ), initiates the replication checkpoint (
      • Kumar S.
      • Burgers P.M.
      Lagging strand maturation factor Dna2 is a component of the replication checkpoint initiation machinery.
      ), and suppresses the expansions of GAA repeats (
      • Zhang Y.
      • Shishkin A.A.
      • Nishida Y.
      • Marcinkowski-Desmond D.
      • Saini N.
      • Volkov K.V.
      • Mirkin S.M.
      • Lobachev K.S.
      Genome-wide screen identifies pathways that govern GAA/TTC repeat fragility and expansions in dividing and nondividing yeast cells.
      ). We have found in vivo and in vitro evidence that DNA2 promotes EXO1-independent MMR. Our data have indicated that the nuclease activity of DNA2 enhances the strand-displacement activity of Pol δ holoenzyme in an EXO1-independent MMR reaction.

      Results

      A dna2 allele causes a defect in Exo1-independent MMR

      We started this work to investigate whether DNA2 has a role in MMR. Our initial genetic analysis in Saccharomyces cerevisiae demonstrated that introduction of a temperature-sensitive dna2 allele (
      • Budd M.E.
      • Campbell J.L.
      A yeast gene required for DNA replication encodes a protein with homology to DNA helicases.
      ), dna2-P504S, into a WT strain caused a 3-fold increase in the CAN1 mutation rate (Fig. 1). We then used the CAN1 mutation assay to examine how dna2-P504S interacted with exo1Δ and msh6Δ, two alleles that cause MMR defects. The data showed that there was a synergistic relationship between dna2-P504S and exo1Δ, but not between dna2-P504S and msh6Δ (Fig. 1). Next, we determined can1 mutation spectra in the WT, dna2-P504S, exo1Δ, dna2-P504S exo1Δ, msh2Δ, and msh2Δ dna2-P504S exo1Δ strains (Table 1). The can1 spectrum in the dna2-P504S strain was dominated by base–base substitutions, but some 1-nt deletions and mutational events that caused CAN1 loss were also observed. Comparison of the mutation spectra in the dna2-P504S, exo1Δ, and dna2-P504S exo1Δ showed that there was a synergistic interaction between dna2-P504S and exo1Δ for base substitutions and 1-nt deletions. Further analysis revealed that msh2Δ was epistatic to dna2-P504S exo1Δ for base substitutions. The observations that (i) msh2Δ was epistatic to dna2-P504S exo1Δ for base substitutions and that (ii) there was a synergistic relationship between dna2-P504S and exo1Δ for base substitutions supported the hypothesis that DNA2 is involved in an Msh2-dependent pathway that repairs base–base mismatches in an Exo1-independent manner. We also observed that there was a weak synergistic relationship between msh2Δ and dna2-P504S exo1Δ for 1-nt deletions. This is likely a result of participation of DNA2 in another genetic stabilization pathway.
      Figure thumbnail gr1
      Figure 1A synergistic interaction between dna2-P504S and exo1Δ for can1 mutations. Spontaneous CAN1 mutation rates were measured as described under . The data are presented as medians with 95% confidence intervals.
      Table 1msh2Δ is epistatic to dna2-P504S exo1Δ for base substitutions in CAN1
      GenotypeMutation rate (× 10−8)
      Base–base substitutions1-nt deletions1-nt insertionsCAN1 lossOther mutationsTotal
      WT (n=50)201.51.5<0.51.524 (21–34)
      dna2-P504S (n=50)428<281170 (50–90)
      exo1Δ (n=50)11014<2.8<2.814140 (130–190)
      dna2-P504S exo1Δ (n=80)28069292363460 (300–510)
      msh2Δ (n=82)43539090<11<11910 (900–1200)
      msh2Δ dna2-P504S exo1Δ (n=79)4256556161<151200 (1100–1700)
      pms1-Q723A (n=51)85361133140 (110–180)
      dna2-P504S pms1-Q723A (n=50)8011035520250 (230–320)
      n, a number of can1 mutants sequenced. The mutations were identified by DNA sequencing. 95% confidence intervals are in parentheses.
      A physical interaction between MutLα and PCNA is essential for MMR (
      • Genschel J.
      • Kadyrova L.Y.
      • Iyer R.R.
      • Dahal B.K.
      • Kadyrov F.A.
      • Modrich P.
      Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLalpha activation and function in mismatch repair.
      ). A mutation, pms1-Q723A, that results in an amino acid change in the PCNA-binding motif of MutLα causes a strong defect in EXO1-independent MMR (
      • Genschel J.
      • Kadyrova L.Y.
      • Iyer R.R.
      • Dahal B.K.
      • Kadyrov F.A.
      • Modrich P.
      Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLalpha activation and function in mismatch repair.
      ). We investigated how pms1-Q723A and dna2-P504S interacted with each other in the CAN1 mutation assay. Measurements of the mutation rates and analysis of the mutation spectra revealed that pms1-Q723A was epistatic to dna2-P504S for base substitutions in CAN1 (Table 1). This finding provided additional genetic evidence for a role of DNA2 in EXO1-independent MMR.

      DNA2 promotes an EXO1-independent MMR reaction in a reconstituted system via an excision-independent mechanism

      We next utilized biochemical approaches to investigate DNA2 in the context of MMR reactions. A previous study described a defined EXO1-independent human MMR reaction that occurs on heteroduplex DNAs in the presence of MutSα, MutLα, PCNA, RFC, RPA, and Pol δ (
      • Kadyrov F.A.
      • Genschel J.
      • Fang Y.
      • Penland E.
      • Edelmann W.
      • Modrich P.
      A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair.
      ). In this EXO1-independent MMR reaction, MutLα incises a heteroduplex DNA 5′ to the mismatch in a MutSα-, PCNA-, and RFC-dependent manner, and Pol δ holoenzyme utilizes the 5′ strand break to perform a strand-displacement DNA synthesis that removes the mismatch. We examined whether DNA2 affected the EXO1-independent MMR reaction on a 3' heteroduplex DNA. Human DNA2 for these and following experiments was produced in and purified from insect Sf9 cells (
      • Pinto C.
      • Kasaciunaite K.
      • Seidel R.
      • Cejka P.
      Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases.
      ) (Fig. S1). The data revealed that the purified DNA2 increased the level of MMR in the EXO1-independent reaction by ∼3 fold (Fig. 2A, lanes 2–3 and graph). In the next series of experiments, we analyzed whether the presence of MutSα, MutLα, and Pol δ was necessary for the DNA2-promoted MMR reaction. As expected, these experiments showed that the omission of MutSα, MutLα, or Pol δ abolished the DNA2-promoted MMR reaction (Fig. 2A, lanes 4–6 and graph). Thus, we concluded that the DNA2-promoted MMR reaction on a 3′ heteroduplex occurred in a MutSα-, MutLα-, and Pol δ-dependent manner.
      Figure thumbnail gr2
      Figure 2DNA2 promotes EXO1-independent MMR on a 3′ heteroduplex and physically interacts with MutSα. Reconstituted human MMR (A) and mismatch excision (B) reactions were carried out for 20 min at 37 °C as detailed under . The DNA substrate for the reactions contained a 21-nt gap 304-bp 3′ to an A-C mispair. When indicated, the reaction mixture contained MutSα (25 nM), MutLα (10 nM), DNA2 (15 nM), PCNA (30 nM), RFC (10 nM), RPA (10 nM), and Pol δ (10 nM). To score MMR, the reaction products were cleaved with BspEI and AlwNI. A, an outline of the MMR assay is shown on the left. In this 3′ MMR assay, a strand break-directed repair of the A-C mispair leads to restoration of a BspEI site. The gel image shows MMR products that were formed in the presence of indicated proteins. The data in the graph were obtained by quantification of gel images like the one shown and are presented as averages ±1 S.D. (n > 3). B, a sketch of the mismatch excision assay is depicted on the left. In this assay, a strand break-directed excision of the A-C mispair leads to the formation of a gap that renders the DNA resistant to cleavage with HindIII. A gel image on the right shows mismatch excision products that were generated in the presence indicated proteins. The excision values were calculated by subtracting percentages of the uncleaved/gapped species that were observed in reactions 2 and 3 from percentage of the uncleaved/gapped species that was observed in the control reaction containing the substrate DNA only (reaction 1). The excision data are presented as averages ±1 S.D. (n = 4). C, a coimmunoprecipitation assay () was employed to detect a protein–protein interaction between purified MutSα and DNA2. When indicated, 30-μl reaction mixtures contained MutSα (3.4 pmol) and DNA2 (3.4 pmol). 0.4% of the input and 25% of the eluted fractions were analyzed by immunoblotting. A representative image is shown. MMR, DNA mismatch pair; Pol δ, DNA polymerase; PCNA, proliferating cell nuclear antigen; RFC, replication factor C; RPA, replication protein A.
      EXO1 excises the DNA mismatch in the mismatch excision reaction (
      • Genschel J.
      • Modrich P.
      Mechanism of 5'-directed excision in human mismatch repair.
      ,
      • Constantin N.
      • Dzantiev L.
      • Kadyrov F.A.
      • Modrich P.
      Human mismatch repair: Reconstitution of a nick-directed bidirectional reaction.
      ,
      • Dzantiev L.
      • Constantin N.
      • Genschel J.
      • Iyer R.R.
      • Burgers P.M.
      • Modrich P.
      A defined human system that supports bidirectional mismatch-provoked excision.
      ). To better understand the mechanism of DNA2-promoted MMR, we performed experiments to analyze whether the addition of DNA2 to a reaction mixture containing MutSα, MutLα, PCNA, RFC, RPA, and a 3′ heteroduplex led to a mismatch excision (Fig. 2B). In agreement with a previous study (
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLalpha in human mismatch repair.
      ), a control experiment showed that a small level of mismatch excision took place in the reaction mixture containing MutSα, MutLα, PCNA, RFC, and RPA (Fig. 2B, lanes 1–2). This excision was a result of the activation of MutLα endonuclease by MutSα, PCNA, RFC, and the mismatch (
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLalpha in human mismatch repair.
      ). However, we found that the supplementation of the five-protein system with DNA2 did not trigger an increase in the level of mismatch excision (Fig. 2B, lanes 2–3). This finding indicated that the mechanism of the DNA2-promoted MMR reaction is different from that of the EXO1-dependent MMR reaction.
      Protein–protein interactions are involved in MMR reactions (
      • Iyer R.R.
      • Pluciennik A.
      • Burdett V.
      • Modrich P.L.
      DNA mismatch repair: Functions and mechanisms.
      ). We studied whether DNA2 physically interacted with the MMR factors MutSα and MutLα in a pull-down assay. The data showed that MutSα-containing agarose beads pulled down the purified DNA2 protein (Fig. 2C), but agarose beads containing MutLα did not (Fig. S2). Thus, these experiments revealed that DNA2 physically interacts with the mismatch recognition factor MutSα.

      DNA2 involves its nuclease activity to promote an EXO1-independent MMR reaction

      Human DNA2 has both helicase and nuclease activities (
      • Budd M.E.
      • Choe W.
      • Campbell J.L.
      The nuclease activity of the yeast DNA2 protein, which is related to the RecB-like nucleases, is essential in vivo.
      ,
      • Pinto C.
      • Kasaciunaite K.
      • Seidel R.
      • Cejka P.
      Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases.
      ,
      • Budd M.E.
      • Choe W.C.
      • Campbell J.L.
      DNA2 encodes a DNA helicase essential for replication of eukaryotic chromosomes.
      ). The DNA2-D277A variant lacks the nuclease activity and the DNA2-K654R mutant does not have the helicase activity (
      • Pinto C.
      • Kasaciunaite K.
      • Seidel R.
      • Cejka P.
      Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases.
      ). We investigated whether the DNA2-D277A and DNA2-K654R variants affected the EXO1-independent MMR reaction on a 3' heteroduplex DNA. We determined that the nuclease-deficient DNA2-D277A variant did not promote the 3′ gap-directed EXO1-independent MMR reaction (Fig. 3A and graph) but the helicase-deficient DNA2-K654R mutant protein did (Fig. 3B, lane 6 and graph). In agreement with the former result, we established the double mutant DNA2-D277A-K654R variant was not able to enhance the 3' gap-directed EXO1-independent MMR reaction. These findings indicated that DNA2 relies on its nuclease activity to promote the EXO1-independent MMR reaction on a 3′ heteroduplex DNA.
      Figure thumbnail gr3
      Figure 3The nuclease activity of DNA2 facilitates EXO1-independent MMR on a 3′ heteroduplex. MMR reactions were carried out and analyzed as described in . A, the effects of different concentrations of DNA2 and the nuclease-deficient DNA2-D277A on EXO1-independent MMR on a 3′ heteroduplex. B, the effects of the helicase-deficient DNA2-K654R and the nuclease- and helicase-deficient DNA2-D277A-K654R on EXO1-independent MMR that occurred on a 3′ heteroduplex. The data in the graphs are averages ±1 S.D. (n > 3). MMR, DNA mismatch pair.

      DNA2 increases the strand-displacement DNA synthesis activity of the Pol δ holoenzyme

      In addition to 3' heteroduplexes, the MMR system corrects mismatches on 5′ heteroduplexes. We therefore studied if DNA2 and its variants affected mismatch removal on a 5' heteroduplex in the presence of MutSα, MutLα, PCNA, RFC, and RPA. In line with our earlier data obtained using a 3′ heteroduplex DNA substrate (Fig. 3), we found that DNA2 and DNA2-K654R promoted mismatch removal on a 5' heteroduplex in the presence of MutSα, MutLα, PCNA, RFC, and RPA, but DNA2-D277A and DNA2-D277A-K654R did not (Fig. 4B, lanes 2–6, and graph). We next asked whether the DNA2-promoted mismatch removal reaction on the 5′ heteroduplex could occur in the absence of the mismatch recognition factor MutSα or MutLα endonuclease. The data showed that the omission of MutSα or MutLα from the reaction mixture did not abolish the DNA2-promoted mismatch removal reaction on a 5' heteroduplex (Fig. 4B, lanes 7–8, and graph). This suggested that DNA2 enhanced the MutSα-and MutLα-independent mismatch correction reaction by increasing the strand-displacement activity of Pol δ holoenzyme. Further support for this idea came from an experiment in which we ascertained that DNA2 strongly increased the strand displacement–based mismatch removal on the 5′ heteroduplex by a four-protein system consisting of Pol δ, PCNA, RFC, and RPA (Fig. 5A). In addition, we determined that human Pol ε, an enzyme that does not have a significant strand-displacement activity (
      • 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.
      ,
      • Ganai R.A.
      • Zhang X.P.
      • Heyer W.D.
      • Johansson E.
      Strand displacement synthesis by yeast DNA polymerase epsilon.
      ), was not able to replace Pol δ in the DNA2-promoted mismatch correction reaction on the 5′ heteroduplex (Fig. 5B).
      Figure thumbnail gr4
      Figure 4The nuclease activity of DNA2 enhances EXO1-independent MMR on a 5′ heteroduplex. MMR reactions were conducted as described in except that the DNA substrate was a 5′ heteroduplex (a 5' G-T DNA), which carried a nick 128 bp 5′ to a G-T mispair. To score MMR, the reaction products were cleaved with HindIII and ClaI. A, a graphical representation of the 5′ MMR assay. B, MMR products that were generated in the presence of indicated proteins. The data in the graph were obtained by quantification of gel images including the one shown and are averages ±1 S.D. (n > 3). MMR, DNA mismatch pair.
      Figure thumbnail gr5
      Figure 5DNA2 increases the strand-displacement activity of Pol δ holoenzyme. MMR reactions on a 5' G-T DNA and control reactions on a 5′ A-T DNA were performed as described in except that reactions in A and C were carried out for 10 min. A, the effect of DNA2 on the strand-displacement activity of Pol δ holoenzyme on a 5′ G-T DNA. B, the effect of replacement of Pol δ with Pol ε on the DNA2-promoted MMR on a 5′ G-T DNA. The data in A and B are averages ±1 S.D. (n > 3). C, a Southern hybridization analysis of strand-displacement products that were formed on 5' A-T and 5′ G-T DNAs in the presence of indicated proteins. Recovered products of the MMR reactions were cleaved with AccI, separated in denaturing agarose gels, transferred onto nylon membranes, and hybridized with a 32P-labeled probe (5'- ACTCTCAGGCAATGACCTGATAGCC-3′) that is complementary to the discontinuous strand of the 5' A-T and 5′ G-T DNAs. The indirectly labeled products were visualized using a Typhoon phosphorimager. The sketches outline the 5' A-T and 5′ G-T DNAs and indicate relative positions of the 32P-labeled probe. The arrow marks a location of smallest strand-displacement products that removed the G-T mismatch from the DNA. MMR, DNA mismatch pair; Pol δ, DNA polymerase; Pol ε, DNA polymerase ε.
      We also utilized a Southern hybridization with a 32P-labeled probe to visualize the DNA2-promoted strand-displacement DNA synthesis products that were separated on denaturing agarose gels. The experiments showed that DNA2 enhanced the strand-displacement DNA synthesis activity of Pol δ holoenzyme on the 5' heteroduplex and 5′ homoduplex with a similar efficiency (Fig. 5C, lanes 3 and 8), and that MutSα did not affect the DNA2-promoted strand-displacement DNA synthesis on either DNA (Fig. 5C, lanes 4 and 9).
      We next performed experiments to determine the size of DNA products formed by DNA2 in reconstituted strand-displacement DNA synthesis reactions that occurred in the presence or absence of MutSα. The substrate in these reactions was a 6.4-kb circular ssDNA that was annealed with 13 oligonucleotides, one of which had a base mismatch and was labeled at its 5′ end with 32P. The results showed that DNA2 formed 8 to 14 nt products in the strand-displacement DNA synthesis reactions in a MutSα-independent manner (Fig. S3). This finding suggests that when the Pol δ generated an 8 to 14 nt flap during the strand-displacement reaction, it was removed by DNA2.

      Discussion

      EXO1 is the only exonuclease that has been shown to excise the mismatch in eukaryotic MMR (
      • 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.
      ,
      • 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.
      ,
      • 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.
      ,
      • Genschel J.
      • Bazemore L.R.
      • Modrich P.
      Human exonuclease I is required for 5' and 3' mismatch repair.
      ). EXO1 preferentially acts on DNA replication errors formed by Pol α (
      • Liberti S.E.
      • Larrea A.A.
      • Kunkel T.A.
      Exonuclease 1 preferentially repairs mismatches generated by DNA polymerase alpha.
      ), but it is also involved in the correction of mismatches produced by Pol δ and Pol ε (
      • Liberti S.E.
      • Larrea A.A.
      • Kunkel T.A.
      Exonuclease 1 preferentially repairs mismatches generated by DNA polymerase alpha.
      ,
      • Hombauer H.
      • Campbell C.S.
      • Smith C.E.
      • Desai A.
      • Kolodner R.D.
      Visualization of eukaryotic DNA mismatch repair reveals distinct recognition and repair intermediates.
      ). Unlike the loss of MutSα or MutLα, the loss of EXO1 does not confer a strong mutator phenotype on yeast and mice (
      • 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.
      ,
      • 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.
      ,
      • 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.
      ). This finding indicates that EXO1-independent MMR removes the majority of DNA replication errors when EXO1 is not available. Although significant progress has been made in understanding EXO1-independent MMR (
      • Kadyrov F.A.
      • Genschel J.
      • Fang Y.
      • Penland E.
      • Edelmann W.
      • Modrich P.
      A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair.
      ,
      • Amin N.S.
      • Nguyen M.N.
      • Oh S.
      • Kolodner R.D.
      exo1-Dependent mutator mutations: model system for studying functional interactions in mismatch repair.
      ,
      • Stith C.M.
      • Sterling J.
      • Resnick M.A.
      • Gordenin D.A.
      • Burgers P.M.
      Flexibility of eukaryotic Okazaki fragment maturation through regulated strand displacement synthesis.
      ,
      • Johnson R.E.
      • Kovvali G.K.
      • Prakash L.
      • Prakash S.
      Requirement of the yeast RTH1 5' to 3' exonuclease for the stability of simple repetitive DNA.
      ,
      • Liu S.
      • Lu G.
      • Ali S.
      • Liu W.
      • Zheng L.
      • Dai H.
      • Li H.
      • Xu H.
      • Hua Y.
      • Zhou Y.
      • Ortega J.
      • Li G.M.
      • Kunkel T.A.
      • Shen B.
      Okazaki fragment maturation involves alpha-segment error editing by the mammalian FEN1/MutSalpha functional complex.
      ,
      • Kratz K.
      • Artola-Boran M.
      • Kobayashi-Era S.
      • Koh G.
      • Oliveira G.
      • Kobayashi S.
      • Oliveira A.
      • Zou X.
      • Richter J.
      • Tsuda M.
      • Sasanuma H.
      • Takeda S.
      • Loizou J.I.
      • Sartori A.A.
      • Nik-Zainal S.
      • et al.
      FANCD2-associated nuclease 1 partially compensates for the lack of Exonuclease 1 in mismatch repair.
      ,
      • Calil F.A.
      • Li B.Z.
      • Torres K.A.
      • Nguyen K.
      • Bowen N.
      • Putnam C.D.
      • Kolodner R.D.
      Rad27 and Exo1 function in different excision pathways for mismatch repair in Saccharomyces cerevisiae.
      ), this MMR pathway has remained enigmatic.
      Prior research revealed that the strand-displacement activity of Pol δ plays a role in EXO1-independent MMR (
      • Kadyrov F.A.
      • Genschel J.
      • Fang Y.
      • Penland E.
      • Edelmann W.
      • Modrich P.
      A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair.
      ,
      • Amin N.S.
      • Nguyen M.N.
      • Oh S.
      • Kolodner R.D.
      exo1-Dependent mutator mutations: model system for studying functional interactions in mismatch repair.
      ,
      • Stith C.M.
      • Sterling J.
      • Resnick M.A.
      • Gordenin D.A.
      • Burgers P.M.
      Flexibility of eukaryotic Okazaki fragment maturation through regulated strand displacement synthesis.
      ). Furthermore, the nucleases FEN1, Rad27, and FAN1 have been implicated in EXO1-independent MMR (
      • Johnson R.E.
      • Kovvali G.K.
      • Prakash L.
      • Prakash S.
      Requirement of the yeast RTH1 5' to 3' exonuclease for the stability of simple repetitive DNA.
      ,
      • Liu S.
      • Lu G.
      • Ali S.
      • Liu W.
      • Zheng L.
      • Dai H.
      • Li H.
      • Xu H.
      • Hua Y.
      • Zhou Y.
      • Ortega J.
      • Li G.M.
      • Kunkel T.A.
      • Shen B.
      Okazaki fragment maturation involves alpha-segment error editing by the mammalian FEN1/MutSalpha functional complex.
      ,
      • Kratz K.
      • Artola-Boran M.
      • Kobayashi-Era S.
      • Koh G.
      • Oliveira G.
      • Kobayashi S.
      • Oliveira A.
      • Zou X.
      • Richter J.
      • Tsuda M.
      • Sasanuma H.
      • Takeda S.
      • Loizou J.I.
      • Sartori A.A.
      • Nik-Zainal S.
      • et al.
      FANCD2-associated nuclease 1 partially compensates for the lack of Exonuclease 1 in mismatch repair.
      ,
      • Calil F.A.
      • Li B.Z.
      • Torres K.A.
      • Nguyen K.
      • Bowen N.
      • Putnam C.D.
      • Kolodner R.D.
      Rad27 and Exo1 function in different excision pathways for mismatch repair in Saccharomyces cerevisiae.
      ). To address the question of whether there is another player in EXO1-independent MMR, we investigated DNA2 nuclease/helicase, an essential eukaryotic protein that has multiple functions in DNA metabolism (
      • Budd M.E.
      • Campbell J.L.
      A yeast gene required for DNA replication encodes a protein with homology to DNA helicases.
      ,
      • Budd M.E.
      • Choe W.
      • Campbell J.L.
      The nuclease activity of the yeast DNA2 protein, which is related to the RecB-like nucleases, is essential in vivo.
      ,
      • Zhu Z.
      • Chung W.H.
      • Shim E.Y.
      • Lee S.E.
      • Ira G.
      Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends.
      ,
      • Niu H.
      • Chung W.H.
      • Zhu Z.
      • Kwon Y.
      • Zhao W.
      • Chi P.
      • Prakash R.
      • Seong C.
      • Liu D.
      • Lu L.
      • Ira G.
      • Sung P.
      Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae.
      ,
      • Cejka P.
      • Cannavo E.
      • Polaczek P.
      • Masuda-Sasa T.
      • Pokharel S.
      • Campbell J.L.
      • Kowalczykowski S.C.
      DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2.
      ). We have established that the replacement of DNA2 with dna2-P504S in a WT strain significantly increases the CAN1 mutation rate (Table 1). Furthermore, we have determined (i) that dna2-P504S interacts synergistically with exo1Δ for base substitutions, (ii) that msh2Δ is epistatic to dna2-P504S exo1Δ for base substitutions, and that (iii) pms1-Q723A is epistatic to dna2-P504S for base substitutions (Table 1). These data represent genetic evidence for DNA2 involvement in EXO1-independent MMR.
      Our biochemical experiments have shown that DNA2 promotes a defined EXO1-independent MMR reaction that relies on the strand-displacement activity of Pol δ holoenzyme (Figure 2, Figure 3, Figure 4). Earlier research revealed that DNA2 harbors both nuclease and helicase activities (
      • Budd M.E.
      • Campbell J.L.
      A yeast gene required for DNA replication encodes a protein with homology to DNA helicases.
      ,
      • Pinto C.
      • Kasaciunaite K.
      • Seidel R.
      • Cejka P.
      Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases.
      ) and that the helicase activity of DNA2 remains silent in the presence of the DNA2 nuclease activity (
      • Pinto C.
      • Kasaciunaite K.
      • Seidel R.
      • Cejka P.
      Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases.
      ). Our analysis of the helicase- and nuclease-deficient variants of DNA2 has established that it is the nuclease activity of DNA2 that enhances the EXO1-independent MMR reaction (Fig. 3). Subsequent experiments indicated that the DNA2 nuclease activity increases the efficiency of the EXO1-independent MMR reaction by enhancing strand displacement by the Pol δ holoenzyme (Fig. 5). This finding suggests that removal of ssDNA tails by DNA2 increases the strand-displacement DNA synthesis by Pol δ holoenzyme. Unlike Pol δ, Pol ε does not have a significant strand-displacement activity (
      • 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.
      ,
      • Ganai R.A.
      • Zhang X.P.
      • Heyer W.D.
      • Johansson E.
      Strand displacement synthesis by yeast DNA polymerase epsilon.
      ). Our observation that the replacement of Pol δ with Pol ε inactivates the DNA2-promoted MMR reaction (Fig. 5B) supports the conclusion that the strand-displacement activity of Pol δ holoenzyme drives mismatch removal in the DNA2-promoted MMR reaction.
      MutSα activates mismatch excision by EXO1 (
      • Genschel J.
      • Modrich P.
      Mechanism of 5'-directed excision in human mismatch repair.
      ). The functional MutSα–EXO1 interaction is likely to be driven by the physical contact between the two proteins (
      • Genschel J.
      • Modrich P.
      Mechanism of 5'-directed excision in human mismatch repair.
      ,
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      Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2.
      ). Although DNA2 and MutSα physically interact with each other (Fig. 2C), we have been unable to detect that MutSα increases the DNA2-promoted strand-displacement activity of Pol δ holoenzyme on a mismatch-containing DNA (Fig. 5, A and C). It might be that our system lacks a factor or a protein modification that enables MutSα to stimulate DNA2 in a mismatch-dependent manner.
      A basic feature of the eukaryotic MMR mechanism is that MutLα endonuclease incises the discontinuous daughter strand in a MutSα-, PCNA-, and RFC-dependent manner to afford mismatch correction (
      • Kadyrov F.A.
      • Dzantiev L.
      • Constantin N.
      • Modrich P.
      Endonucleolytic function of MutLalpha 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 MutLalpha is a mismatch repair endonuclease.
      ). The endonuclease function of MutLα provides a downstream factor with a window of opportunity to enter the DNA via a MutLα-generated strand break to remove the mismatch. Previous research uncovered a role for the strand-displacement activity of the Pol δ holoenzyme in mismatch removal in the absence of EXO1 (
      • 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.
      ,
      • Kadyrov F.A.
      • Genschel J.
      • Fang Y.
      • Penland E.
      • Edelmann W.
      • Modrich P.
      A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair.
      ,
      • Amin N.S.
      • Nguyen M.N.
      • Oh S.
      • Kolodner R.D.
      exo1-Dependent mutator mutations: model system for studying functional interactions in mismatch repair.
      ,
      • Stith C.M.
      • Sterling J.
      • Resnick M.A.
      • Gordenin D.A.
      • Burgers P.M.
      Flexibility of eukaryotic Okazaki fragment maturation through regulated strand displacement synthesis.
      ). We have now shown that DNA2 contributes to EXO1-independent mismatch removal by enhancing the strand-displacement activity of Pol δ.
      Of importance is an observation that like DNA2, the other two nucleases, Rad27/FEN1 and FAN1, that contribute to EXO1-independent MMR (
      • Johnson R.E.
      • Kovvali G.K.
      • Prakash L.
      • Prakash S.
      Requirement of the yeast RTH1 5' to 3' exonuclease for the stability of simple repetitive DNA.
      ,
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      • Ali S.
      • Liu W.
      • Zheng L.
      • Dai H.
      • Li H.
      • Xu H.
      • Hua Y.
      • Zhou Y.
      • Ortega J.
      • Li G.M.
      • Kunkel T.A.
      • Shen B.
      Okazaki fragment maturation involves alpha-segment error editing by the mammalian FEN1/MutSalpha functional complex.
      ,
      • Kratz K.
      • Artola-Boran M.
      • Kobayashi-Era S.
      • Koh G.
      • Oliveira G.
      • Kobayashi S.
      • Oliveira A.
      • Zou X.
      • Richter J.
      • Tsuda M.
      • Sasanuma H.
      • Takeda S.
      • Loizou J.I.
      • Sartori A.A.
      • Nik-Zainal S.
      • et al.
      FANCD2-associated nuclease 1 partially compensates for the lack of Exonuclease 1 in mismatch repair.
      ,
      • Calil F.A.
      • Li B.Z.
      • Torres K.A.
      • Nguyen K.
      • Bowen N.
      • Putnam C.D.
      • Kolodner R.D.
      Rad27 and Exo1 function in different excision pathways for mismatch repair in Saccharomyces cerevisiae.
      ) have 5′ flap endonuclease activities (
      • Harrington J.J.
      • Lieber M.R.
      The characterization of a mammalian DNA structure-specific endonuclease.
      ,
      • Li X.
      • Li J.
      • Harrington J.
      • Lieber M.R.
      • Burgers P.M.
      Lagging strand DNA synthesis at the eukaryotic replication fork involves binding and stimulation of FEN-1 by proliferating cell nuclear antigen.
      ,
      • Kratz K.
      • Schopf B.
      • Kaden S.
      • Sendoel A.
      • Eberhard R.
      • Lademann C.
      • Cannavo E.
      • Sartori A.A.
      • Hengartner M.O.
      • Jiricny J.
      Deficiency of FANCD2-associated nuclease KIAA1018/FAN1 sensitizes cells to interstrand crosslinking agents.
      ,
      • MacKay C.
      • Declais A.C.
      • Lundin C.
      • Agostinho A.
      • Deans A.J.
      • MacArtney T.J.
      • Hofmann K.
      • Gartner A.
      • West S.C.
      • Helleday T.
      • Lilley D.M.
      • Rouse J.
      Identification of KIAA1018/FAN1, a DNA repair nuclease recruited to DNA damage by monoubiquitinated FANCD2.
      ,
      • Smogorzewska A.
      • Desetty R.
      • Saito T.T.
      • Schlabach M.
      • Lach F.P.
      • Sowa M.E.
      • Clark A.B.
      • Kunkel T.A.
      • Harper J.W.
      • Colaiacovo M.P.
      • Elledge S.J.
      A genetic screen identifies FAN1, a Fanconi anemia-associated nuclease necessary for DNA interstrand crosslink repair.
      ,
      • Gueldener U.
      • Heinisch J.
      • Koehler G.J.
      • Voss D.
      • Hegemann J.H.
      A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast.
      ), and one of them (Rad27) enhances the strand-displacement DNA synthesis by a Pol δ holoenzyme (
      • Calil F.A.
      • Li B.Z.
      • Torres K.A.
      • Nguyen K.
      • Bowen N.
      • Putnam C.D.
      • Kolodner R.D.
      Rad27 and Exo1 function in different excision pathways for mismatch repair in Saccharomyces cerevisiae.
      ). This observation reinforces the view that the strand-displacement activity of the Pol δ holoenzyme plays a key role in EXO1-independent MMR. It will be important to perform quantitative analyses to determine the effects of EXO1, FEN1, Rad27, and FAN1 on the reconstituted DNA2-promoted MMR reaction to better understand the impact of DNA2 on MMR.

      Experimental procedures

      S. cerevisiae strains and measurements of the mutation rates

      Yeast strains used in this work were the haploid WT strain E134 (MATa ade5-1 lys2::InsE-A14 trp1-289 his7-2 leu2-3,112 ura3-52) (
      • 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.
      ) and its mutant derivatives. The gene knockouts were generated utilizing PCR-amplified disruption cassettes (
      • Gueldener U.
      • Heinisch J.
      • Koehler G.J.
      • Voss D.
      • Hegemann J.H.
      A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast.
      ) and the lithium/PEG-based transformation method (
      • Gietz R.D.
      • Woods R.A.
      Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.
      ). All gene disruptions were confirmed by PCR. The replacement of DNA2 with dna2-P504S allele was performed using the integration-excision method. The presence of the dna2-P504S mutation was confirmed by DNA sequencing.
      Spontaneous CAN1 mutation rates were measured using fluctuation tests that were carried out according to a previously described method (
      • Kadyrova L.Y.
      • Mertz T.M.
      • Zhang Y.
      • Northam M.R.
      • Sheng Z.
      • Lobachev K.S.
      • Shcherbakova P.V.
      • Kadyrov F.A.
      A reversible histone H3 acetylation cooperates with mismatch repair and replicative polymerases in maintaining genome stability.
      ). Briefly, single colonies obtained from 2 to 3 independent isolates of the same yeast genotype were used to start 12 to 24 cultures each in 3-ml YPD medium (1% yeast extract, 2% bactopeptone, 2% dextrose), supplemented with 60 mg/l adenine and 60 mg/l uracil that were grown at 23 °C. Dilutions of the cultures were plated on a synthetic complete medium to score the total number of cells and on a drop-out medium that lacked arginine and contained 60 mg/l L-canavanine to score the total number of can1 mutants. Colony counts were utilized to calculate the spontaneous CAN1 mutation rates with the Drake formula (
      • Drake J.W.
      A constant rate of spontaneous mutation in DNA-based microbes.
      ,
      • Shcherbakova P.V.
      • Kunkel T.A.
      Mutator phenotypes conferred by MLH1 overexpression and by heterozygosity for mlh1 mutations.
      ). Mutation rates are presented as median values with 95% confidence intervals.

      Human proteins

      Human MutLα, MutSα, PCNA, Pol δ, Pol ε, RFC, and RPA were isolated in near-homogeneous forms as previously described (
      • 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.
      ,
      • Kadyrov F.A.
      • Genschel J.
      • Fang Y.
      • Penland E.
      • Edelmann W.
      • Modrich P.
      A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair.
      ). Human DNA2, DNA2-D277A, DNA2-K654R, and DNA2-D277A-K654R that were flag-tagged at the C termini were each purified from insect Sf9 cells by chromatographies on α-Flag M2 beads (Sigma) and a MonoS column (GE HealthCare). Baculoviruses that carried the codon-optimized DNA2, DNA2-D277A, DNA2-K654R, and DNA2-D277A-K654R genes (
      • Pinto C.
      • Kasaciunaite K.
      • Seidel R.
      • Cejka P.
      Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases.
      ) were used for production and purification of DNA2 and its variants.

      MMR and mismatch excision reactions

      MMR reactions were carried out at 37 °C in 40-μl mixtures that contained 20 mM Hepes–NaOH (pH 7.4), 5 mM MgCl2, 3 mM ATP, 110 mM KCl, 25 μM dGTP, 25 μM dATP, 25 μM dTTP, 25 μM dCTP, 4 mM DTT, 0.2 mg/ml bovine serum albumin, 0.3 μg DNA (a 3′-gapped heteroduplex, a 5′-nicked heteroduplex, or a 5′-nicked homoduplex), and indicated human proteins. When human MutSα, MutLα, PCNA, RFC, RPA, Pol δ, and Pol ε were present in the reaction mixture, their concentrations were 25, 10, 30, 10, 10, 10, and 20 nM, respectively. Human DNA2 and its variants were included in the reaction mixtures at 0 to 30 nM as indicated. The 3′-gapped heteroduplex DNA (7.54 kb) contained a 21-nt gap that was 304 bp 3' to an A-C mispair, the 5′-nicked heteroduplex (6.44 kb) carried a nick that was 128 bp 5′ to a G-T mismatch (
      • Genschel J.
      • Bazemore L.R.
      • Modrich P.
      Human exonuclease I is required for 5' and 3' mismatch repair.
      ), and the 5' -nicked homoduplex DNA was identical to the 5′-nicked heteroduplex except that it lacked a mispair. Unless noted otherwise, MMR reactions were carried out for 20 min. Mismatch excision reactions were performed exactly as the MMR reactions except that the reaction mixtures lacked the four dNTPs. MMR and mismatch excision reactions were each terminated by the addition of a 30-μl mixture containing 0.31% SDS, 0.36 M NaCl, 12 mM EDTA, 0.3 μg/μl proteinase K, and 1.8 μg/μl glycogen, followed by incubation of the mixtures at 50 °C for 15 min. The mixtures were extracted with phenol/chloroform, and the DNAs from the supernatants were precipitated with isopropanol. MMR on the 3′-gapped heteroduplex was scored by cleavage of the recovered reaction products with BspEI and AlwNI. To score MMR on the 5′-nicked heteroduplex, the recovered reaction products were cleaved with HindIII and ClaI. To determine the level of mismatch excision on the 3′-gapped heteroduplex, the recovered reaction products were digested with HindIII and AlwNI. After cleavage with restriction endonucleases, the recovered reaction products were separated in 1.1% agarose gels in 1x TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.2) and stained with ethidium bromide. The images were obtained with a cooled charge-coupled device camera (Fotodyne) and the DNA species were quantified using an ImageJ software.

      Coimmunoprecipitation assays

      Coimmunoprecipitation assays were performed as previously described (
      • 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.
      ). Antibodies against human MSH2 (sc-376384, Santa Cruz Biotechnology), DNA2 (sc-393323, Santa Cruz Biotechnology), and MLH1 (sc-271978, Santa Cruz Biotechnology) were used in coimmunoprecipitation assays.

      Data availability

      All data are contained within the article.

      Supporting information

      This article contains supporting information (
      • Dzantiev L.
      • Constantin N.
      • Genschel J.
      • Iyer R.R.
      • Burgers P.M.
      • Modrich P.
      A defined human system that supports bidirectional mismatch-provoked excision.
      ).

      Conflicts of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We are grateful to Petr Cejka for a gift of the human codon-optimized DNA2 constructs and Tim Formosa for providing a dna2-1 plasmid.

      Author contributions

      L. Y. K., P. S., and F. A. K. methodology; L. Y. K., B. K. D., V. G., and F. A. K. investigation; L. Y. K., J. M. D., P. S., and F. A. K. resources; L. Y. K., B. K. D., V. G., and F. A. K. formal analysis; and L. Y. K. and F. A. K. wrote the writing–original draft.

      Funding and additional information

      Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R01GM132128 (to F. A. K.) and by the National Cancer Institute of the National Institutes of Health under Award Number R35CA241801 (to P. S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

      Supporting information

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