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Roles of Residues Arg-61 and Gln-38 of Human DNA Polymerase η in Bypass of Deoxyguanosine and 7,8-Dihydro-8-oxo-2′-deoxyguanosine*

  • Yan Su
    Affiliations
    From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Amritraj Patra
    Affiliations
    From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Joel M. Harp
    Affiliations
    From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Martin Egli
    Affiliations
    From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • F. Peter Guengerich
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University School of Medicine, 638B Robinson Research Bldg., 2200 Pierce Ave., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-4349;
    Affiliations
    From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants R01 ES010375 (to F. P. G. and M. E.) and P01 CA160032 (to M. E.). The authors declare that they have no conflicts of interest with the contents of this article.
Open AccessPublished:May 06, 2015DOI:https://doi.org/10.1074/jbc.M115.653691
      Like the other Y-family DNA polymerases, human DNA polymerase η (hpol η) has relatively low fidelity and is able to tolerate damage during DNA synthesis, including 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG), one of the most abundant DNA lesions in the genome. Crystal structures show that Arg-61 and Gln-38 are located near the active site and may play important roles in the fidelity and efficiency of hpol η. Site-directed mutagenesis was used to replace these side chains either alone or together, and the wild type or mutant proteins were purified and tested by replicating DNA past deoxyguanosine (G) or 8-oxoG. The catalytic activity of hpol η was dramatically disrupted by the R61M and Q38A/R61A mutations, as opposed to the R61A and Q38A single mutants. Crystal structures of hpol η mutant ternary complexes reveal that polarized water molecules can mimic and partially compensate for the missing side chains of Arg-61 and Gln-38 in the Q38A/R61A mutant. The combined data indicate that the positioning and positive charge of Arg-61 synergistically contribute to the nucleotidyl transfer reaction, with additional influence exerted by Gln-38. In addition, gel filtration chromatography separated multimeric and monomeric forms of wild type and mutant hpol η, indicating the possibility that hpol η forms multimers in vivo.

      Introduction

      Y-family DNA polymerases are characterized by their low fidelity and ability to tolerate a wide range of lesions during DNA synthesis. Due to their relatively large active sites and loss of 3′–5′ exonuclease domains compared with replicative DNA polymerases, they readily introduce mutations into the genome. These enzymes are able to insert dNTPs opposite and beyond DNA lesions, therefore rescuing blocked replication forks and releasing the arrested cell cycle (
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      Human DNA polymerase η (hpol η)
      The abbreviations used are: hpol η
      human DNA polymerase η
      FAM
      6-carboxyfluorescein
      8-oxoG
      7,8-dihydro-8-oxo-2′-deoxyguanosine
      PDB
      Protein Data Bank
      dCMPNPP
      2′-deoxycytidine-5′-[(α,β)-imido] triphosphate.
      belongs to the DNA polymerase Y family (
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      An overview of Y-family DNA polymerases and a case study of human DNA polymerase η.
      ). It plays a crucial role in bypassing cyclobutane pyrimidine dimers, which arise as a result of UV exposure. Defects in hpol η bypass of cyclobutane pyrimidine dimers in xeroderma pigmentosum variant patients results in increased rates of skin and internal cancers (
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      Rad54 dissociates homologous recombination intermediates by branch migration.
      ). hpol η also contributes to somatic hypermutation in B cells by introducing mutations in specific DNA stretches embedded within antigen-binding sites, thereby causing structural changes and potentially elevating the antigen-binding ability (
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      DNA polymerase η is the sole contributor of A/T modifications during immunoglobulin gene hypermutation in the mouse.
      ).
      hpol η and its yeast homolog Rad30 also play roles in the bypass of 7,8-dihydro-8-oxoguanine (8-oxoG) (
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      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ), one of the most common DNA lesions introduced by oxidative stress. 8-oxoG is generated at a rate of >1000 molecules/cell/day and prefers the syn conformation, which facilitates formation of a Hoogsteen pair with dATP and introduces mutations into the genome (
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      ). hpol η replicates past 8-oxoG mainly by inserting dCTP, scaffolding the dCTP:8-oxoG pair in the Watson-Crick geometry, although the dATP misincorporation rate is 280-fold higher in the bypass of 8-oxoG compared with unmodified G (
      • Patra A.
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      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ).
      Crystal structures of the catalytic core of hpol η (amino acids 1–432) and its yeast homolog with unmodified or lesion-containing oligonucleotides have been published (
      • Zhao Y.
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      • Gregory M.T.
      • Hua Y.J.
      • Hanaoka F.
      • Yang W.
      Structural basis of human DNA polymerase η-mediated chemoresistance to cisplatin.
      ,
      • Gregory M.T.
      • Park G.Y.
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      • Lee Y.S.
      • Yang W.
      • Lippard S.J.
      Structural and mechanistic studies of polymerase η bypass of phenanthriplatin DNA damage.
      ,
      • Zhao Y.
      • Gregory M.T.
      • Biertümpfel C.
      • Hua Y.J.
      • Hanaoka F.
      • Yang W.
      Mechanism of somatic hypermutation at the WA motif by human DNA polymerase η.
      ,
      • Silverstein T.D.
      • Jain R.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Structural basis for error-free replication of oxidatively damaged DNA by yeast DNA polymerase η.
      ,
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ,
      • Nakamura T.
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      Watching DNA polymerase η make a phosphodiester bond.
      ,
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      Structure and mechanism of human DNA polymerase η.
      ,
      • Silverstein T.D.
      • Johnson R.E.
      • Jain R.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Structural basis for the suppression of skin cancers by DNA polymerase η.
      ,
      • Patra A.
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      • Egli M.
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      Structural and kinetic analysis of nucleoside triphosphate incorporation opposite an abasic site by human translesion DNA polymerase η.
      ,
      • Ummat A.
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      • Jain R.
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      • Johnson R.E.
      • Prakash L.
      • Prakash S.
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      Human DNA polymerase η is pre-aligned for dNTP binding and catalysis.
      ,
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      Mechanism of replication blocking and bypass of Y-family polymerase η by bulky acetylaminofluorene DNA adducts.
      ). These studies provide a better understanding of the catalytic mechanism and have yielded valuable information regarding the design of potential drugs against hpol η in the hope of blocking tumor proliferation. The residue Arg-61, located in a short loop region, is highly conserved. Its dynamic movement contributes to the catalytic activity of the enzyme. X-ray crystal structures reveal that after binding the DNA substrate in the absence of an incoming dNTP, the positively charged guanidino moiety of Arg-61 is projected toward the nucleobase of the 5′-overhanging, single-stranded region of template DNA of the template-primer duplex (
      • Ummat A.
      • Silverstein T.D.
      • Jain R.
      • Buku A.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Human DNA polymerase η is pre-aligned for dNTP binding and catalysis.
      ). However, following binding of dNTP and divalent metal ions, the side chain of Arg-61 undergoes a rotation and interacts with the α-phosphate of the incoming dNTP. Interestingly, the side chain of Arg-61 also moves during phosphodiester bond formation to allow enough space for a third divalent metal ion, which interacts with both the α-phosphate and the oxygen, bridging the α- and β-phosphates. Thus, Arg-61 can adopt different conformations during each stage of the catalytic reaction cycle. In addition, the reported structures have also indicated that another highly conserved residue near the active site, Gln-38, may play an important role in stabilizing the template base in the nucleotidyl transfer reaction (
      • Zhao Y.
      • Biertümpfel C.
      • Gregory M.T.
      • Hua Y.J.
      • Hanaoka F.
      • Yang W.
      Structural basis of human DNA polymerase η-mediated chemoresistance to cisplatin.
      ,
      • Gregory M.T.
      • Park G.Y.
      • Johnstone T.C.
      • Lee Y.S.
      • Yang W.
      • Lippard S.J.
      Structural and mechanistic studies of polymerase η bypass of phenanthriplatin DNA damage.
      ,
      • Zhao Y.
      • Gregory M.T.
      • Biertümpfel C.
      • Hua Y.J.
      • Hanaoka F.
      • Yang W.
      Mechanism of somatic hypermutation at the WA motif by human DNA polymerase η.
      ,
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ,
      • Nakamura T.
      • Zhao Y.
      • Yamagata Y.
      • Hua Y.J.
      • Yang W.
      Watching DNA polymerase η make a phosphodiester bond.
      ,
      • Biertümpfel C.
      • Zhao Y.
      • Kondo Y.
      • Ramón-Maiques S.
      • Gregory M.
      • Lee J.Y.
      • Masutani C.
      • Lehmann A.R.
      • Hanaoka F.
      • Yang W.
      Structure and mechanism of human DNA polymerase η.
      ,
      • Patra A.
      • Zhang Q.
      • Lei L.
      • Su Y.
      • Egli M.
      • Guengerich F.P.
      Structural and kinetic analysis of nucleoside triphosphate incorporation opposite an abasic site by human translesion DNA polymerase η.
      ).
      Site-directed mutagenesis was used in this study to further investigate the roles of Arg-61 and Gln-38 in the catalytic activity. Wild-type and mutant forms of hpol η were analyzed and crystallized with unmodified DNA or DNA containing 8-oxoG. The combined results of the structural and biochemical analysis indicate that the positioning and positive charge of the Arg-61 synergistically contribute to the nucleotidyl transfer reaction in addition to the hydrogen bonding effect of Gln-38 with the template base. When both Arg-61 and Gln-38 were replaced by Ala, polarized water molecules could mimic and partially compensate for the missing wild-type side chains.

      Experimental Procedures

      Materials

      All oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA), and some were purified by HPLC. dNTPs were from New England Biolabs (Ipswich, MA). PreScission protease was from GE Healthcare. Polyethylene glycol monomethyl ether 2000 (for crystallization) was from Hampton Research (Aliso Viejo, CA).

      Site-directed Mutagenesis, Protein Expression, and Purification

      Wild-type hpol η (residues 1–432, in a pET28a plasmid) was a gift from Dr. Wei Yang (NIDDK, National Institutes of Health, Bethesda, MD). Site-directed mutagenesis was used to make point mutations by the following primers: Q38A, 5′-CATGCGCCGTTGTGGCGTATAAAAGCTGG-3′ and 5′-CCAGCTTTTATACGCCACAACGGCGCATG-3′; Q38L, 5′-CCATGCGCCGTTGTGCTGTATAAAAGCTGGAAAGG-3′ and 5′-CCTTTCCAGCTTTTATACAGCACAACGGCGCATGG-3′; R61A, 5′-CGTTTGGCGTCACCGCCAGCATGTGGGCCGATG-3′ and 5′-CATCGGCCCACATGCTGGCGGTGACGCCAAACG-3′; R61K, 5′-GTTTGGCGTCACCAAGAGCATGTGGGCCGATGAC-3′ and 5′-GTCATCGGCCCACATGCTCTTGGTGACGCCAAAC-3′; R61M, 5′-GTTTGGCGTCACCATGAGCATGTGGGCCGATGAC-3′ and 5′-GTCATCGGCCCACATGCTCATGGTGACGCCAAAC-3′. Wild-type and mutant hpol η derivatives were expressed in Escherichia coli BL21 (DE3) pLysS cells, followed by purification through a HisTrap HP column (5 ml; GE Healthcare). After removal of the N-terminal His tag by PreScission protease (GE Healthcare), the proteins were purified through a Mono S column (GE Healthcare), eluted with the buffer containing 20 mm sodium MES (pH 6.0), 3 mm dithiothreitol (DTT), 0.1 mm EDTA, 10% glycerol (v/v), and ∼350 mm KCl (using a gradient of 250–500 mm KCl). The proteins were used for activity assays after flash freezing in liquid nitrogen and storage at −80 °C. Proteins were further purified through a Superdex-75 10/300 GL column (GE Healthcare) in 20 mm Tris-HCl buffer (pH 7.5) containing 450 mm KCl and 3 mm DTT (
      • Biertümpfel C.
      • Zhao Y.
      • Kondo Y.
      • Ramón-Maiques S.
      • Gregory M.
      • Lee J.Y.
      • Masutani C.
      • Lehmann A.R.
      • Hanaoka F.
      • Yang W.
      Structure and mechanism of human DNA polymerase η.
      ) to obtain crystals. In addition, the R61M mutant was analyzed with a Superdex-200 10/300 GL column (GE Healthcare) with 20 mm Tris-HCl (pH 7.5) containing 450 mm KCl and 3 mm DTT as well as bovine thyroglobulin (Sigma-Aldrich) and bovine serum albumin (BSA).

      Annealing and Extension Assays

      Primer and template DNA were annealed (1:1 molar ratio) by heating to 95 °C for 5 min and slow cooling to room temperature: (a) 5′-6-carboxyfluorescein (FAM)-CGGGCTCGTAAGCGTCAT-3′ and 3′-GCCCGAGCATTCGCAGTAGTACT-5′; (b) 5′-FAM-CGGGCTCGTAAGCGTCAT-3′ and 3′-GCCCGAGCATTCGCAGTA(8-oxoG)TACT-5′; (c) 5′-FAM-CGGGCTCGTAAGCGTCATC-3′ and 3′-GCCCGAGCATTCGCAGTA(8-oxoG)TACT-5′; (d) 5′-FAM-CGGGCTCGTAAGCGTCATA-3′ and 3′-GCCCGAGCATTCGCAGTA(8-oxoG)TACT-5′; (e) 5′-AGCGTCAT-3′ and 3′-TCGCAGTAGTAC-5′; (f) 5′-AGCGTCAT-3′ and 3′-TCGCAGTA(8-oxoG)TAC-5′; (g) 5′-FAM-CGGGCTCGTAAGCGTCATC-3′ and 3′-GCCCGAGCATTCGCAGTAGTACT-5′. DNA complexes a–d were used for extension assays; a–c and g were applied in steady-state kinetic assays; a and b were used in pre-steady-state kinetic assays; e and f were used for crystallization. For the extension assays, wild-type hpol η or mutant proteins (25 nm) were incubated with 5 μm FAM-labeled primer·template DNA complex in 40 mm Tris-HCl buffer (pH 7.5), 10 mm DTT, 0.1 mg/ml BSA, 5% glycerol (v/v), 5 mm MgCl2, 100 mm KCl, and 200 μm each dNTP. The reactions were conducted for 0, 2, 5, and 15 min at 37 °C. After stopping the reactions, the products were separated using 18% denaturing polyacrylamide gels (w/v) and visualized using a Typhoon system (GE Healthcare).

      Steady-state Kinetics

      Reactions were conducted by incubating hpol η (2–500 nm), 5 μm FAM-labeled primer·template DNA complex, 40 mm Tris-HCl (pH 7.5) containing 10 mm DTT, 0.1 mg/ml BSA, 5% glycerol (v/v), 5 mm MgCl2, 100 mm KCl, and various amounts of dCTP or dATP at 37 °C for 5 min. After quenching the reactions, products were applied on 18% denaturing polyacrylamide gels (w/v), visualized with a Typhoon system (GE Healthcare), and quantified using ImageJ software (National Institutes of Health). The data were fit to the (hyperbolic) Michaelis-Menten equation with Prism software (GraphPad, La Jolla, CA) (
      • Zhao Y.
      • Biertümpfel C.
      • Gregory M.T.
      • Hua Y.J.
      • Hanaoka F.
      • Yang W.
      Structural basis of human DNA polymerase η-mediated chemoresistance to cisplatin.
      ,
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ).

      Pre-steady-state Kinetics

      FAM-labeled primer·template DNA complex (1 μm) was mixed with wild-type (100 nm) or mutant (500 nm) hpol η, 40 mm Tris-HCl (pH 7.5), 10 mm DTT, 0.1 mg/ml BSA, 5% glycerol (v/v), and 100 mm KCl (Solution A). Solution B contained 1 mm dCTP and 10 mm MgCl2. The two solutions were mixed rapidly with a KinTek RP-3 instrument (KinTek Corp., Austin, TX) at 25 °C for 0.005–5 s (the final reaction concentration for wild-type hpol η was 50 nm, and that for mutant hpol η was 250 nm) and stopped by the addition of 0.5 m EDTA. The products were separated with 18% denaturing polyacrylamide gels (w/v) and visualized with a Typhoon system (GE Healthcare). After quantification using ImageJ software, the results were fit to a burst equation using GraphPad Prism: y = A(1 - e kpt) + kssE0t, where A is the burst amplitude, representing the apparent concentration of the active form of the enzyme, kp is the burst rate, kss is the steady-state rate, and E0 is the total enzyme concentration (
      • Patra A.
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      • Zhang Q.
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      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ,
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      • Zhang Q.
      • Lei L.
      • Su Y.
      • Egli M.
      • Guengerich F.P.
      Structural and kinetic analysis of nucleoside triphosphate incorporation opposite an abasic site by human translesion DNA polymerase η.
      ,
      • Sassa A.
      • Beard W.A.
      • Shock D.D.
      • Wilson S.H.
      Steady-state, pre-steady-state, and single-turnover kinetic measurement for DNA glycosylase activity.
      ).

      Crystallizations

      The hanging drop vapor diffusion technique was used to obtain crystals. DNA complex and protein (1.2:1 molar ratio) were first incubated at room temperature for 10 min, followed by the addition of 0.5 μl of 1 m CaCl2 and concentration by centrifugation through a filter (10 kDa, Millipore). After the addition of 0.5 μl of 100 mm dNTP (New England Biolabs), the solution (1 μl) was mixed with an equal volume of reservoir solution (100 mm sodium MES (pH 6.0), 5 mm CaCl2, and 15–22% (w/v) polyethylene glycol monomethyl ether 2000 (Hampton Research, Aliso Viejo, CA)). The mixture was equilibrated against 500 μl of the reservoir solution at 18 °C. Crystals were typically observed after overnight incubation (
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ,
      • Biertümpfel C.
      • Zhao Y.
      • Kondo Y.
      • Ramón-Maiques S.
      • Gregory M.
      • Lee J.Y.
      • Masutani C.
      • Lehmann A.R.
      • Hanaoka F.
      • Yang W.
      Structure and mechanism of human DNA polymerase η.
      ).

      X-ray Diffraction Data Collection and Structure Determination

      Crystals were mounted in nylon loops and swiped through a mixture of reservoir solution plus 25% glycerol (v/v), followed by flash-cooling in liquid nitrogen. Diffraction data were collected on the Sector 21-ID-G beamline at the Advanced Photon Source (Life Sciences Collaborative Access Team, Argonne National Laboratory, Argonne, IL). All collected data were integrated and scaled using HKL2000 (
      • Otwinowski Z.
      • Minor W.
      Processing of x-ray diffraction data collected in oscillation mode.
      ). Structures were determined by molecular replacement phasing using the program Phaser MR (
      • McCoy A.J.
      • Grosse-Kunstleve R.W.
      • Adams P.D.
      • Winn M.D.
      • Storoni L.C.
      • Read R.J.
      Phaser crystallographic software.
      ). Coordinates with Protein Data Bank (PDB) code 4O3N were used as the search model for R61M·G·dCTP and Q38A/R61A·G·dCTP, those with PDB code 4O3P were used for R61M·8-oxoG·dCTP and Q38A/R61A·8-oxoG·dCTP, and those with PDB code 4O3O were used for R61M·8-oxoG·dATP. PHENIX (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • Richardson J.S.
      • Terwilliger T.C.
      • Zwart P.H.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ) was applied for refinements, and ARP/ω ARP classic (
      • Langer G.G.
      • Hazledine S.
      • Wiegels T.
      • Carolan C.
      • Lamzin V.S.
      Visual automated macromolecular model building.
      ,
      • Murshudov G.N.
      • Skubák P.
      • Lebedev A.A.
      • Pannu N.S.
      • Steiner R.A.
      • Nicholls R.A.
      • Winn M.D.
      • Long F.
      • Vagin A.A.
      REFMAC5 for the refinement of macromolecular crystal structures.
      ) and COOT (
      • Emsley P.
      • Lohkamp B.
      • Scott W.G.
      • Cowtan K.
      Features and development of Coot.
      ) were used for model building. Structural illustrations were generated with the program UCSF Chimera (
      • Pettersen E.F.
      • Goddard T.D.
      • Huang C.C.
      • Couch G.S.
      • Greenblatt D.M.
      • Meng E.C.
      • Ferrin T.E.
      UCSF Chimera: a visualization system for exploratory research and analysis.
      ).

      Results

      Primer Extension past G or 8-oxoG by Wild-type or Mutant hpol η in the Presence of All Four dNTPs

      The catalytic core (residues 1–432) of hpol η was used in this study because its activity is similar to that of the full-length protein in vitro (
      • Zhao Y.
      • Biertümpfel C.
      • Gregory M.T.
      • Hua Y.J.
      • Hanaoka F.
      • Yang W.
      Structural basis of human DNA polymerase η-mediated chemoresistance to cisplatin.
      ). In order to understand the mechanism of dNTP incorporation by hpol η, point mutations were introduced at Arg-61 and Gln-38. In the extension assays, wild-type hpol η bypassed both unmodified template G and 8-oxoG and fully extended the primer in 15 min, whereas mutants R61M and Q38A/R61A partially elongated the primer by inserting at most two nucleotides, indicating that these mutations strongly attenuate the activity of hpol η. In comparison, the mutations Q38A, Q38L, R61A, and R61K only slightly reduced the catalytic activity (Fig. 1, A and B).
      Figure thumbnail gr1
      FIGURE 1Primer extension assays for bypass of G or 8-oxoG by wild-type or mutant hpol η. Wild-type or mutant hpol η (25 nm), FAM-labeled primer·template DNA complex (5 μm), and a saturating concentration of all four dNTPs (200 μm each dNTP) were incubated at 37 °C for 0, 2, 5, and 15 min. The products were separated on denaturing polyacrylamide gels. In A, X represents G; in B, X represents 8-oxoG.

      Steady-state Kinetics of dCTP or dATP Incorporation Opposite G or 8-oxoG by Wild-type and Mutant hpol η

      All hpol η mutants used in our study had attenuated efficiency for dCTP insertion opposite unmodified G compared with the wild-type enzyme. The Q38L, R61M, and Q38A/R61A mutants showed 22-, 19-, and 16-fold attenuation in enzyme efficiency, respectively, whereas Q38A, R61A, and R61K showed only small decreases. Even when these mutations were introduced, hpol η still maintained relatively high fidelity opposite G, and the dATP misincorporation frequency for each hpol η was <4% (Fig. 2A and Table 1). For dCTP incorporation opposite 8-oxoG (Fig. 2B and Table 2), the lowest catalytic efficiencies (kcat/Km) were observed for the Q38L, R61M, and Q38A/R61A mutants (i.e. 4.9, 1.4, and 2.2 μm−1 min−1 respectively, compared with 31 μm−1 min−1 for wild-type hpol η). In the study of steady-state kinetics of dATP misinsertion opposite 8-oxoG, a second insertion band was always observed due to the next base T, 3′ to 8-oxoG on the template strand (Fig. 2C). To obtain catalytic efficiencies specifically for the insertion step opposite 8-oxoG, the two insertion bands were both included in the quantitation. We reported previously that the dATP insertion frequency was 280-fold higher for wild-type hpol η replicating past 8-oxoG than that for replicating past G (
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ). For each mutant, dATP was more frequently inserted opposite 8-oxoG than G. Interestingly, all mutants except R61K showed higher fidelity (inserting dCTP instead of dATP) than wild-type hpol η in bypassing 8-oxoG. The R61K mutant, consistent with a previous report (
      • Zhao Y.
      • Gregory M.T.
      • Biertümpfel C.
      • Hua Y.J.
      • Hanaoka F.
      • Yang W.
      Mechanism of somatic hypermutation at the WA motif by human DNA polymerase η.
      ), had a higher misincorporation frequency (0.60, compared with 0.28 for wild-type hpol η) (Fig. 2B and Table 2).
      Figure thumbnail gr2
      FIGURE 2Enzyme efficiencies for dNTP incorporation opposite G or 8-oxoG by wild-type or mutant hpol η. Enzyme efficiencies (kcat/Km) were obtained from steady-state kinetic studies by incubating 2–500 nm hpol η, 5 μm FAM-labeled primer·template DNA complex, and varying concentrations of dNTPs at 37 °C for 5 min. After separating the products on denaturing polyacrylamide gels, data were fit to a (hyperbolic) Michaelis-Menten equation using non-linear regression (Prism). Each experiment was conducted at least twice, and error bars indicate S.D. A, dCTP and dATP incorporation opposite G; B, dCTP and dATP incorporation opposite 8-oxoG; C, an example (R61A mutant) of second band insertion during a steady-state-kinetic study for dATP incorporation opposite 8-oxoG.
      TABLE 1Steady-state kinetics of incorporation of dCTP and dATP opposite G by wild-type and mutant hpol η
      hpol ηdNTPKmkcatkcat/Kmf
      Misinsertion frequency: f = (kcat/Km) dATP /(kcat/Km)dCTP.
      μmmin−1μm−1 min−1
      WT
      Steady-state kinetic data of dNTP incorporation opposite G by WT hpol η were from Ref. 30. The experimental conditions for WT and hpol η mutants were identical.
      dCTP1.3 ± 0.280 ± 362 ± 10
      dATP92 ± 236.0 ± 0.60.065 ± 0.0180.0010
      Q38AdCTP5.2 ± 0.988 ± 517 ± 3
      dATP103 ± 1314 ± 10.14 ± 0.020.0082
      Q38LdCTP20 ± 555 ± 52.8 ± 0.7
      dATP148 ± 3615 ± 10.10 ± 0.030.036
      R61AdCTP1.5 ± 0.221 ± 114 ± 2
      dATP49 ± 100.92 ± 0.090.019 ± 0.0040.0014
      R61KdCTP8.7 ± 1.772 ± 38.3 ± 1.7
      dATP73 ± 108.0 ± 0.40.11 ± 0.020.013
      R61MdCTP1.3 ± 0.14.2 ± 0.13.2 ± 0.3
      dATP47 ± 130.14 ± 0.010.0030 ± 0.00090.00094
      Q38A/R61AdCTP6.5 ± 2.226 ± 24.0 ± 1.4
      dATP78 ± 152.1 ± 0.10.027 ± 0.0050.0068
      a Misinsertion frequency: f = (kcat/Km) dATP /(kcat/Km)dCTP.
      b Steady-state kinetic data of dNTP incorporation opposite G by WT hpol η were from Ref.
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      . The experimental conditions for WT and hpol η mutants were identical.
      TABLE 2Steady-state kinetics of incorporation of dCTP and dATP opposite 8-oxoG by wild-type and mutant hpol η
      hpol ηdNTPKmkcatkcat/Kmf
      Misinsertion frequency: f = (kcat/Km)dATP/(kcat/Km)dCTP.
      μmmin−1μm−1min−1
      WT
      Steady-state kinetic data of dNTP incorporation opposite 8-oxoG by WT hpol η were from Ref. 30. The experimental conditions were the same for WT and mutant forms of hpol η.
      dCTP2.3 ± 0.272 ± 231 ± 3
      dATP7.6 ± 0.966 ± 28.7 ± 1.10.28
      Q38AdCTP9.1 ± 1.893 ± 810 ± 2
      dATP14 ± 331 ± 22.2 ± 0.50.22
      Q38LdCTP13 ± 464 ± 74.9 ± 1.6
      dATP34 ± 422 ± 10.65 ± 0.080.13
      R61AdCTP1.3 ± 0.218 ± 114 ± 2
      dATP11 ± 117 ± 0.41.5 ± 0.10.11
      R61KdCTP8.4 ± 1.380 ± 49.5 ± 1.5
      dATP12 ± 268 ± 25.7 ± 1.00.60
      R61MdCTP1.7 ± 0.32.3 ± 0.11.4 ± 0.2
      dATP15 ± 21.7 ± 0.10.11 ± 0.020.079
      Q38A/R61AdCTP7.7 ± 1.317 ± 12.2 ± 0.4
      dATP29 ± 86.9 ± 0.50.24 ± 0.070.11
      a Misinsertion frequency: f = (kcat/Km)dATP/(kcat/Km)dCTP.
      b Steady-state kinetic data of dNTP incorporation opposite 8-oxoG by WT hpol η were from Ref.
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      . The experimental conditions were the same for WT and mutant forms of hpol η.

      Pre-steady-state Kinetics of dCTP Incorporation Opposite G or 8-oxoG by Wild-type and Mutant hpol η

      In the pre-steady-state kinetic analysis of dCTP insertion opposite G, the burst rates for R61M and Q38A/R61A were 2.6- and 5.9-fold lower than that for wild-type hpol η (Fig. 3 and Table 3). A similar trend was observed for bypass of 8-oxoG, with 90 s−1 for wild-type, 35 s−1 for R61M, and 53 s−1 for Q38A/R61A. In the bypass of G, the burst amplitude of wild type was nearly 100% of the estimated enzyme concentration, but the burst amplitudes for R61M and Q38A/R61A were only 21 and 16%, respectively, suggesting the presence of either inactive enzymes or multiple conformations in mutant·DNA·dNTP ternary complexes (
      • Furge L.L.
      • Guengerich F.P.
      Explanation of pre-steady-state kinetics and decreased burst amplitude of HIV-1 reverse transcriptase at sites of modified DNA bases with an additional, nonproductive enzyme-DNA-nucleotide complex.
      ). For the bypass of 8-oxoG, the burst amplitudes were ∼14% for each hpol η mutant and ∼70% for wild-type hpol η. Notably, for each individual enzyme, the burst amplitude for incorporation opposite G was always larger than that for 8-oxoG, suggesting that non-productive forms are more prominent in the presence of 8-oxoG (
      • Furge L.L.
      • Guengerich F.P.
      Explanation of pre-steady-state kinetics and decreased burst amplitude of HIV-1 reverse transcriptase at sites of modified DNA bases with an additional, nonproductive enzyme-DNA-nucleotide complex.
      ) (Table 3).
      Figure thumbnail gr3
      FIGURE 3Pre-steady-state kinetic analysis of dCTP incorporation opposite G or 8-oxoG by wild-type or mutant hpol η. A mixture of 1 μm FAM-labeled DNA complex and 100 nm wild-type or 500 nm mutant hpol η was mixed with an equal volume of solution containing 1 mm dCTP and 10 mm MgCl2 for 0.005–5 s at 25 °C (wild-type hpol η was 50 nm; mutant was 250 nm in the reactions), followed by the addition of quenching solution (0.5 m EDTA) and separation of products on denaturing polyacrylamide gels. The quantified data were fit to a burst equation, y = A(1 − ekpt) + kssE0t. Each experiment was conducted twice, and error bars represent S.D. A, wild-type hpol η (WT) incorporation of dCTP opposite G; B, wild-type hpol η (WT) incorporation of dCTP opposite 8-oxoG; C, mutant R61M incorporation of dCTP opposite G; D, mutant R61M incorporation of dCTP opposite 8-oxoG; E, mutant Q38A/R61A incorporation of dCTP opposite G; F, mutant Q38A/R61A incorporation of dCTP opposite 8-oxoG; G, comparison of the burst rates (kp) for dCTP incorporation opposite G and 8-oxoG.
      TABLE 3Burst kinetics of incorporation of dCTP opposite G and 8-oxoG
      hpol η
      The final enzyme concentrations were 50 nm WT hpol η and 250 nm R61M and Q38A/R61A.
      TemplatekpkssBurst amplitude
      s−1s−1nm
      WTG124 ± 110.32 ± 0.0549 ± 1
      8-oxoG90 ± 110.26 ± 0.0635 ± 1
      R61MG47 ± 60.064 ± 0.00452 ± 2
      8-oxoG35 ± 50.060 ± 0.00334 ± 2
      Q38A/R61AG21 ± 30.078 ± 0.00440 ± 2
      8-oxoG53 ± 110.095 ± 0.00435 ± 2
      a The final enzyme concentrations were 50 nm WT hpol η and 250 nm R61M and Q38A/R61A.

      Post-G or -8-oxoG Synthesis by Wild-type or Mutant hpol η

      The ternary crystal structure shows that 8-oxoG formed a Watson-Crick pair with an incoming dCTP analog in the presence of wild-type hpol η (PDB code 4O3P) (
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ). The question can be raised as to whether the presence of an 8-oxoG:C pair affects primer extension by wild-type or mutant hpol η. In the presence of all four dNTPs, wild-type hpol η fully extended the primer beyond the 8-oxoG:C pair in 15 min, and the Q38A, Q38L, R61A, and R61K mutants elongated the primer at slightly slower rates, but only part of the primer was extended (by one nucleotide) by the R61M and Q38A/R61A mutants (Fig. 4A).
      Figure thumbnail gr4
      FIGURE 4Extension of primer beyond an 8-oxoG:C or 8-oxoG:A pair by wild-type or mutant hpol η. A FAM-labeled 19-mer primer and 23-mer template were annealed, with 8-oxoG located at position 19 from the 3′-end in the template strand. The DNA complex (5 μm) was incubated with a 25 nm concentration of each enzyme and all four dNTPs (200 μm each dNTP) at 37 °C for 0, 2, 5, and 15 min, followed by separation of products on denaturing polyacrylamide gels. A, C was at the 3′-end of the primer and opposite 8-oxoG. B, A was at the 3′-end of the primer and opposite 8-oxoG.
      To further investigate primer extension post-8-oxoG, steady-state kinetic analysis was applied for the next base (dATP) insertion beyond an 8-oxoG:C pair, as well as a G:C pair as a control. Consistent with the observation in Fig. 4A, the catalytic efficiencies (kcat/Km) for dATP insertion post-8-oxoG were much lower for R61M (1.8 μm−1 min−1) or Q38A/R61A (1.2 μm−1 min−1) compared with wild-type hpol η (47 μm−1 min−1). Interestingly, both wild-type hpol η and the two mutants had similar kcat/Km values for dATP incorporation post-G and -8-oxoG, indicating that 8-oxoG does not affect next base insertion by hpol η, even with mutations at Arg-61 or Gln-38 (Table 4).
      TABLE 4Steady-state kinetics of next base (dATP) insertion post-unmodified G or 8-oxoG by wild-type and mutant hpol η
      hpol ηTemplateKmkcatkcat/Km
      μmmin−1μm−1 min−1
      WTPost-G2.0 ± 0.585 ± 743 ± 11
      Post-8-oxoG1.8 ± 0.284 ± 247 ± 5
      R61MPost-G2.5 ± 0.26.0 ± 0.12.4 ± 0.2
      Post-8-oxoG1.9 ± 0.33.5 ± 0.11.8 ± 0.3
      Q38A/R61APost-G15 ± 324 ± 11.6 ± 0.3
      Post-8-oxoG17 ± 220 ± 11.2 ± 0.2
      Because wild-type or mutant hpol η may incorporate dATP opposite 8-oxoG, extension assays beyond the 8-oxoG:A pair were also studied. The extension pattern beyond an 8-oxoG:A pair was very similar to that beyond an 8-oxoG:C pair for wild-type hpol η or either mutant, indicating that the mismatched 8-oxoG:A pair does not dramatically affect subsequent dNTP insertion (Fig. 4B).

      X-ray Crystallography of Mutant hpol η Inserting dCTP Opposite G

      In order to further understand the roles of Arg-61 and Gln-38 in bypassing unmodified G, crystal structures of R61M·G·dCTP (R61M mutant incorporating dCTP opposite unmodified template G) and Q38A/R61A·G·dCTP were obtained in the presence of Ca2+, which does not support nucleotidyl transfer reactions (Table 5). Electron density for the active site of each ternary complex is shown in Fig. 5. Similar to the structure of the wild-type hpol η·G·dCTP complex, hpol η mutants R61M and Q38A/R61A provided a scaffold for the incoming dCTP and template G to form a Watson-Crick base pair (Fig. 6). The distances were 3.8 and 4.1 Å from the 3′-OH of T of the primer strand to Pα of the incoming dCTP in the R61M·G·dCTP and Q38A/R61A·G·dCTP complexes respectively, compared with 3.3 or 3.5 Å (two conformations) for wild-type hpol η (
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ).
      TABLE 5Crystal data, data collection parameters, and structure refinement statistics
      ComplexR61M 8-oxoG·dCTPR61M 8-oxoG·dATPR61M G·dCTPQ38A/R61A 8-oxoG·dCTPQ38A/R61A G·dCTP
      Data collection
      Wavelength (Å)0.978560.978560.978560.978560.97856
      Space groupP61P61P61P61P61
      Resolution (Å)50.00-1.78 (1.81-1.78)
      R-merge: R linear = SUM(ABS(I − 〈I〉))/SUM(I).
      50.00-1.95 (1.98-1.95)50.00-2.00 (2.03-2.00)50.00-1.89 (1.92-1.89)50.00-2.06 (2.10-2.06)
      Unit cell a = b, c (Å)98.73, 82.4598.97, 82.1898.71, 81.9598.77, 81.9799.24, 82.38
      Unique reflections43,702 (2,144)33,388 (1,642)30,423 (1,496)36,455 (1,805)28,389 (1,229)
      Completeness (%)99.7 (98.6)99.9 (99.6)98.7 (98.1)99.9 (100)99.2 (85.6)
      I/σ(I)13.1 (1.87)21.0 (1.65)12.3 (2.23)14.7 (2.21)10.2 (1.29)
      Wilson B-factor (Å2)19.527.520.323.826.2
      R-merge
      Data shown in parentheses are from the highest resolution shell.
      0.094 (0.793)0.080 (0.775)0.121 (0.813)0.087 (0.793)0.149 (0.964)
      Redundancy5.5 (4.4)5.5 (4.4)5.8 (5.7)5.7 (5.7)5.5 (3.9)
      Refinement
      R-work0.172 (0.282)0.168 (0.242)0.176 (0.235)0.169 (0.245)0.174 (0.253)
      R-free0.213 (0.347)0.218 (0.310)0.219 (0.262)0.216 (0.311)0.227 (0.328)
      No. of atoms
      Protein/DNA3260/4093236/3843294/3673235 /3633293/415
      dNTP/Ca2+28/230/228/228/228/2
      H2O/glycerol507/12404/12411/0406/12302/12
      Protein residues422423424424427
      B-factor (Å)
      Average23.630.624.628.329.7
      Protein/DNA22.4/25.829.2/36.623.4/29.226.8/33.528.9/33.5
      dNTP/Ca2+13.5/11.531.7/28.813.0/14.118.6/16.720.3/19.7
      Water/glycerol29.7/24.036.1/33.431.7/036.2/31.433.6/27.7
      Root mean square deviations
      Bonds (Å)0.0080.0080.0090.0170.008
      Angles (degrees)1.21.11.21.61.1
      Ramachandran
      Favored (%)98.3497.1897.8896.9497.41
      Allowed (%)1.662.591.653.062.12
      Outliers (%)0.000.230.470.000.47
      PDB code4YR04YR24YR34YP34YQW
      a Data shown in parentheses are from the highest resolution shell.
      b R-merge: R linear = SUM(ABS(I − 〈I〉))/SUM(I).
      Figure thumbnail gr5
      FIGURE 5Electron density maps for the active sites of mutant ternary complexes. The Fourier (2FoFc) sum electron density is at the 1σ threshold. A, R61M·G·dCTP; B, Q38A/R61A·G·dCTP; C, R61M·8-oxoG·dCTP; D, Q38A/R61A·8-oxoG·dCTP; E, R61M·8-oxoG·dATP. The metal ions (Ca2+) are shown in red in each panel.
      Figure thumbnail gr6
      FIGURE 6Active site views of dCTP insertion opposite unmodified G by wild-type and mutant hpol η. A, C, E, and G, views from the major groove side; B, D, F, and H, views of from the top of base pairings. A and B, wild-type hpol η·G·dCMPNPP (PDB 4O3N); C and D, R61M·G·dCTP; E and F, Q38A/R61A·G·dCTP; G and H, superimposed structures of wild-type hpol η, R61M, and Q38A/R61A with the incoming dCTP opposite template G. The metal ions (Mg2+ in A and B; Ca2+ in C–H) are represented in red in each panel.
      Arg-61 of wild-type hpol η engages in favorable Coulombic interaction with the α-phosphate of the incoming dCTP. The electrostatic force was lost after mutating it to the hydrophobic Met, although the size of the side chain was similar to Arg. Interestingly, in the ternary Q38A/R61A·G·dCTP complex, one water molecule was located above the base ring of dCTP and likely to form a lone pair-π or H-π interaction with the nucleobase (Fig. 6) (
      • Egli M.
      • Sarkhel S.
      Lone pair-aromatic interactions: to stabilize or not to stabilize.
      ).
      In the minor groove, the Nϵ atom of Gln-38 connected with N3 of the template G through a hydrogen bond and a water molecule-bridged Oϵ of Gln-38 and N2 of G, thus stabilizing the template G to form a base pair with the incoming dCTP. When Gln-38 was mutated to Ala, these interactions were disrupted, and the bridging water molecule was not present in the Q38A/R61A·G·dCTP structure (Fig. 6).

      X-ray Crystallography of Mutant hpol η Inserting dCTP Opposite 8-oxoG

      To investigate the roles of Arg-61 and Gln-38 in bypassing 8-oxoG, mutant hpol η was crystallized with dCTP opposite 8-oxoG in the presence of Ca2+. Fourier (2FoFc) sum electron density maps are shown in Fig. 5. Similar to the structures depicting dCTP insertion opposite unmodified G, wild-type hpol η and the mutants provided a scaffold for dCTP pairing with 8-oxoG in the Watson-Crick geometry, with 8-oxoG in the anti conformation (Fig. 7). The distances between the primer terminal 3′-OH and the Pα of dCTP were 3.2 or 3.4 Å in the complex with wild-type hpol η (
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ), 3.3 or 3.9 Å with the R61M mutant, and 3.9 Å with Q38A/R61A, respectively.
      Figure thumbnail gr7
      FIGURE 7Active site views of dCTP insertion opposite 8-oxoG by wild-type and mutant hpol η. A, C, E, and G are views of active sites from the major groove side; base pairings from the top are shown in B, D, F, and H. A and B, wild-type hpol η·8-oxoG·dCMPNPP (PDB 4O3P); C and D, R61M·8-oxoG·dCTP; E and F, Q38A/R61A·8-oxoG·dCTP; G and H, superimposed structures of wild-type hpol η, R61M, and Q38A/R61A with the incoming dCTP opposite 8-oxoG. The metal ions (Mg2+ in A and B; Ca2+ in C–H) are shown in red.
      Similar to dCTP insertion opposite G by wild-type hpol η, the side chain of Arg-61 is involved in an electrostatic interaction with the α-phosphate of dCTP in the wild-type hpol η·8-oxoG·dCTP complex, but this interaction was destroyed when replacing Arg-61 with Met. Interestingly, when Arg-61 was mutated to Ala, water molecules were lodged on top of dCTP. One of the water molecules was on top of the nucleobase of the dCTP, whereas the position of the other matched that of Nη of Arg-61 from the wild-type hpol η·8-oxoG·dCTP complex, allowing it to donate a hydrogen bond to the α-phosphate of the incoming dCTP (Fig. 7).
      In the minor groove of the wild-type ternary complex, Nϵ of Gln-38 was engaged in a hydrogen bond with N3 of 8-oxoG. After superimposition, a water molecule in the Q38A/R61A·8-oxoG·dCTP complex was observed near the Nϵ of Gln-38 in the wild-type hpol η·8-oxoG·dCTP complex. Further investigation revealed a hydrogen bonding interaction between this water and the N3 of 8-oxoG. In addition, in the wild-type hpol η·8-oxoG·dCTP complex, a water molecule bridged both the Oϵ of Gln-38 and N2 of 8-oxoG by hydrogen bonds. This bridging water hydrogen-bonds with the Nϵ-mimicking water as well as N2 of 8-oxoG in the Q38A/R61A·8-oxoG·dCTP complex (Fig. 7).

      X-ray Crystallography of Mutant hpol η Inserting dATP Opposite 8-oxoG

      To explore the role of Arg-61 in the fidelity of hpol η, we crystallized the hpol η R61M mutant with dATP opposite 8-oxoG. Similar to the wild-type hpol η·8-oxoG·dATP complex, 8-oxoG adopted the syn conformation and formed a Hoogsteen pair with the incoming dATP when Arg-61 was mutated to Met. The distance from the 3′-OH of the primer end to Pα of dATP was 3.7 Å, compared with 3.1 or 3.3 Å in the complex with wild-type hpol η (
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ) (Fig. 8).
      Figure thumbnail gr8
      FIGURE 8Active site views of dATP misinsertion opposite 8-oxoG by wild-type and mutant hpol η. A, C, and E, active site views from the major groove side. B, D, and F show base pairings from the top. A and B, wild-type hpol η·8-oxoG·dAMPNPP (PDB code 4O3O); C and D, R61M·8-oxoG·dATP; E and F, superimposed structures of wild-type hpol η and the R61M mutant with incoming dATP opposite template 8-oxoG. Metal ions (Mg2+ in A and B; Ca2+ in C and D) are shown in red.
      The methionine side chain in the point mutant R61M exhibited two conformations in the ternary structure. One of the conformations partially superimposed with the side chain of Arg-61 in the wild-type hpol η·8-oxoG·dATP complex. However, due to the hydrophobic nature of the Met side chain, the electrostatic interaction seen in the wild-type complex with the incoming dATP was lost. The Nϵ of Gln-38 from either wild-type or R61M mutant hpol η donated a hydrogen bond to the O8 atom of 8-oxoG in the minor groove (Fig. 8).

      Multimeric Forms of hpol η

      Wild-type hpol η and the mutants R61M and Q38A/R61A were purified by gel filtration chromatography (Superdex-75 10/300 GL) prior to crystallization. Two distinct peaks, eluting with tR of ∼8.1 and 11.3 ml, were observed in the chromatogram in each case, suggesting the presence of proteins with different molecular masses (Fig. 9A). The second peak was eluted with the molecular mass of monomeric hpol η, as established using the standards BSA (67 kDa) and ovalbumin (43 kDa). The proteins from the two peaks had the same mobility in SDS-PAGE (data not shown), indicating that the protein in the first peak was a multimeric form of hpol η. The R61M mutant had the largest ratio of peak 1 (multimer) over peak 2 (monomer) (Fig. 9A), and kinetic assays with minimal protein dilution following the chromatography (final dilution ratio 1:4 (v/v) for the reaction) were used to measure their catalytic activities (burst phase, pre-steady state). The protein from peak 2 (monomer) showed a 1.5-fold higher burst rate compared with that from peak 1 (multimer) for dCTP insertion opposite template G. The burst amplitudes were 65 and 202 nm (compared with the input concentrations of 566 and 772 nm for the peak 1 and 2 proteins), respectively. In addition, both peaks of the R61M mutant were crystallized with DNA and incoming dCTP with no dramatic structural differences (data not shown). To characterize the multimeric composition, the mutant R61M, as well as two markers, thyroglobulin (670 kDa) and BSA (67 kDa), were used to calibrate a Superdex-200 10/300 GL gel filtration column. The mutant enzyme was eluted in a peak corresponding to monomeric hpol η, a broad peak with molecular mass in the range of 400–600 kDa, and the void peak, indicating the presence of an even higher fold ensemble (Fig. 9B).
      Figure thumbnail gr9
      FIGURE 9Gel filtration chromatography of wild-type and mutant hpol η. A, wild-type and mutant hpol η were chromatographed on a Superdex-75 10/300 GL gel filtration column. B, hpol η R61M mutant plus two standard protein markers, thyroglobulin (670 kDa) and BSA (67 kDa), were loaded on a Superdex-200 10/300 GL gel filtration column, respectively. UV absorbance was monitored at 280 nm.

      Discussion

      The fidelity of some DNA polymerases is determined largely by a single key residue. For example, Arg-332 in the archbacterial Sulfolobus solfataricus Y-family DNA polymerase Dpo4 directly interacts with the O8 atom of 8-oxoG and determines its fidelity in the bypass of 8-oxoG (
      • Eoff R.L.
      • Irimia A.
      • Angel K.C.
      • Egli M.
      • Guengerich F.P.
      Hydrogen bonding of 7,8-dihydro-8-oxodeoxyguanosine with a charged residue in the little finger domain determines miscoding events in Sulfolobus solfataricus DNA polymerase Dpo4.
      ). Structures of hpol η show that two highly conserved residues, Arg-61 and Gln-38, are important in the catalytic reaction (
      • Zhao Y.
      • Biertümpfel C.
      • Gregory M.T.
      • Hua Y.J.
      • Hanaoka F.
      • Yang W.
      Structural basis of human DNA polymerase η-mediated chemoresistance to cisplatin.
      ,
      • Gregory M.T.
      • Park G.Y.
      • Johnstone T.C.
      • Lee Y.S.
      • Yang W.
      • Lippard S.J.
      Structural and mechanistic studies of polymerase η bypass of phenanthriplatin DNA damage.
      ,
      • Zhao Y.
      • Gregory M.T.
      • Biertümpfel C.
      • Hua Y.J.
      • Hanaoka F.
      • Yang W.
      Mechanism of somatic hypermutation at the WA motif by human DNA polymerase η.
      ,
      • Silverstein T.D.
      • Jain R.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Structural basis for error-free replication of oxidatively damaged DNA by yeast DNA polymerase η.
      ,
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ,
      • Nakamura T.
      • Zhao Y.
      • Yamagata Y.
      • Hua Y.J.
      • Yang W.
      Watching DNA polymerase η make a phosphodiester bond.
      ,
      • Biertümpfel C.
      • Zhao Y.
      • Kondo Y.
      • Ramón-Maiques S.
      • Gregory M.
      • Lee J.Y.
      • Masutani C.
      • Lehmann A.R.
      • Hanaoka F.
      • Yang W.
      Structure and mechanism of human DNA polymerase η.
      ,
      • Silverstein T.D.
      • Johnson R.E.
      • Jain R.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Structural basis for the suppression of skin cancers by DNA polymerase η.
      ,
      • Patra A.
      • Zhang Q.
      • Lei L.
      • Su Y.
      • Egli M.
      • Guengerich F.P.
      Structural and kinetic analysis of nucleoside triphosphate incorporation opposite an abasic site by human translesion DNA polymerase η.
      ,
      • Ummat A.
      • Silverstein T.D.
      • Jain R.
      • Buku A.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Human DNA polymerase η is pre-aligned for dNTP binding and catalysis.
      ,
      • Schorr S.
      • Schneider S.
      • Lammens K.
      • Hopfner K.P.
      • Carell T.
      Mechanism of replication blocking and bypass of Y-family polymerase η by bulky acetylaminofluorene DNA adducts.
      ). In order to delineate their roles for the activity and fidelity of hpol η, we mutated the two residues to amino acids with different side chain properties. The unmodified G and a common DNA lesion, 8-oxoG, were used in DNA templates in our study.
      The misincorporation frequencies of wild-type and hpol η mutants for dATP insertion opposite unmodified G were very small, all <4%, as judged from the steady-state kinetic assay results. However, the misincorporation frequency was increased 36-fold for mutant Q38L, 13-fold for R61K, 8-fold for Q38A, and 7-fold for Q38A/R61A compared with that for wild-type hpol η, whereas mutants R61A and R61M had misincorporation frequencies similar to that of the wild-type protein in incorporating dATP opposite G (Fig. 2A and Table 1). On the other hand, because 8-oxoG can readily form a syn conformation due to a steric effect and pair with A, the dATP insertion frequency was increased 280-fold for wild-type hpol η compared with that opposite unmodified G (
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ). Interestingly, mutant R61K had the highest misincorporation frequency (0.60) for dATP insertion opposite 8-oxoG, whereas R61M had the lowest value (0.079) compared with wild-type hpol η (0.28). The other mutants had slightly reduced misincorporation frequency (Fig. 2B and Table 2). In addition, previous reports also showed that the template base/lesion as well as the flanking sequence also affected the misincorporation frequency for wild-type or mutant hpol η (
      • Zhao Y.
      • Gregory M.T.
      • Biertümpfel C.
      • Hua Y.J.
      • Hanaoka F.
      • Yang W.
      Mechanism of somatic hypermutation at the WA motif by human DNA polymerase η.
      ,
      • Suarez S.C.
      • Beardslee R.A.
      • Toffton S.M.
      • McCulloch S.D.
      Biochemical analysis of active site mutations of human polymerase η.
      ). Altogether, we conclude that active site residues, the template base/lesion, and the flanking DNA sequence synergistically determine the misincorporation frequency of hpol η.
      It was unexpected that the R61A mutant would be more active than R61M; however, x-ray crystal structures of the corresponding complexes provide a potential explanation. Polarized water molecules mimic the side chain of Arg-61 and partially compensate for its functions in the R61A mutant. In order to understand how these water molecules participate in the nucleotidyl transfer reaction, the Q38A/R61A·8-oxoG·dCTP complex was compared with the other reported wild-type hpol η structures. A third metal ion (Mg2+) has been reported to be involved in the catalytic process, whereas the side chain of Arg-61 of wild-type hpol η adopted the conformation facing toward the major groove (Fig. 10A) (
      • Nakamura T.
      • Zhao Y.
      • Yamagata Y.
      • Hua Y.J.
      • Yang W.
      Watching DNA polymerase η make a phosphodiester bond.
      ). After superimposition, the water interacting with the α-phosphate in the Q38A/R61A·8-oxoG·dCTP complex partially overlapped with a water molecule coordinating with the third Mg2+ (Fig. 10, B and C). Therefore, the water in the mutant hpol η may not only play a role in mimicking the missing side chain of Arg-61 in the ground state but may also interact with the third metal ion during the catalytic process.
      Figure thumbnail gr10
      FIGURE 10Comparison of wild-type hpol η during the catalytic process with Q38A/R61A·8-oxoG·dCTP. A, the ternary structure of wild-type hpol η during catalytic process (PDB code 4ECT (
      • Nakamura T.
      • Zhao Y.
      • Yamagata Y.
      • Hua Y.J.
      • Yang W.
      Watching DNA polymerase η make a phosphodiester bond.
      )); B and C, active site views of superimposed structures of wild-type hpol η complex and Q38A/R61A·8-oxoG·dCTP from the major groove and on top of the base pair.
      Because H2O and monovalent Na+ may look very similar in electron density maps (
      • Wouters J.
      Cation-π (Na+-Trp) interactions in the crystal structure of tetragonal lysozyme.
      ), close investigation of the coordination, occupancy, and B factor was conducted (for those water molecules). In particular, the water molecule on top of the base ring of the incoming dCTP was of interest. This water is 2.9 Å from the centroid of the aromatic ring in the Q38A/R61A·G·dCTP complex, and the corresponding distance in the Q38A/R61A·8-oxoG·dCTP complex is 3.1 Å, indicating the possibility of the formation of a lone pair-π or H-π interaction between H2O and the base ring (
      • Egli M.
      • Sarkhel S.
      Lone pair-aromatic interactions: to stabilize or not to stabilize.
      ). To distinguish H2O from a monovalent metal ion, the former was replaced by a Na+ ion with half-occupancy in the Q38A/R61A·8-oxoG·dCTP model structure. After refinement using the program Phenix, the occupancy of Na+ was 0.38, and the B factor dropped from 35.6 to 21 A2 compared with a fully occupied water (data not presented). Thus, we cannot completely exclude the possibility that the electron density peak represents a partially occupied Na+ instead of H2O on top of the base ring. Soaking crystals with K+ or Rb+ and collecting anomalous data may help to further resolve the issue, although K+ and Rb+ have larger ionic radii than Na+ (
      • Tereshko V.
      • Wilds C.J.
      • Minasov G.
      • Prakash T.P.
      • Maier M.A.
      • Howard A.
      • Wawrzak Z.
      • Manoharan M.
      • Egli M.
      Detection of alkali metal ions in DNA crystals using state-of-the-art x-ray diffraction experiments.
      ).
      On the other hand, the electrostatic interaction with the incoming dNTP was lost when Arg-61 was mutated to the hydrophobic Met. In addition, the side chain of R61M was too big to allow room for Arg-61-mimicking water molecules, as seen in the ternary structures with the mutation R61A. As a result, hpol η with the single substitution R61A was more active than with R61M. Similar results were observed with the Q38A and Q38L mutants (TABLE 1, TABLE 2 and FIGURE 6, FIGURE 7).
      A second unexpected result was observed from the pre-steady-state kinetic data; the burst amplitudes for wild type hpol η inserting dCTP opposite unmodified G or 8-oxG were ∼100 or 70% of the input, respectively, whereas the amplitude was <21% for either the R61M or Q38A/R61A mutant. The result indicated the presence of either an inactive form(s) of mutant protein or an equilibrium of reactive and nonreactive ternary complexes (
      • Guengerich F.P.
      Interactions of carcinogen-bound DNA with individual DNA polymerases.
      ). Interestingly, gel filtration chromatography showed the presence of both multimeric and monomeric forms of wild type or mutants of hpol η. Both forms of the proteins were active and formed single crystals (data not shown; however, the extent of the multimeric state is not known at the high concentration used for crystal formation). These results suggest that hpol η may assemble to multimers in vivo, and the two mutants showed an increased tendency to multimeric assembly compared with wild-type hpol η (Fig. 9), although extensive cellular studies would be needed for further conclusions about physiological relevance. The reason for this change in behavior is unknown, considering that the mutations are in the interior of the protein. Interestingly, multimeric forms of the other polymerases have been reported previously. For example, S. solfataricus Dpo4 can form a dimer on DNA in vitro (
      • Lin H.K.
      • Chase S.F.
      • Laue T.M.
      • Jen-Jacobson L.
      • Trakselis M.A.
      Differential temperature-dependent multimeric assemblies of replication and repair polymerases on DNA increase processivity.
      ). Small angle x-ray scattering and ultracentrifugation results reveal that mammalian DNA polymerase β binds with DNA in ratios of both 2:1 and 1:1 in solution (
      • Tang K.H.
      • Niebuhr M.
      • Aulabaugh A.
      • Tsai M.D.
      Solution structures of 2:1 and 1:1 DNA polymerase-DNA complexes probed by ultracentrifugation and small-angle x-ray scattering.
      ,
      • Tang K.H.
      • Tsai M.D.
      Structure and function of 2:1 DNA polymerase. DNA complexes.
      ). Recently, an A-family DNA polymerase (polymerase θ) has been reported to be able to form a dimer, which may be important for polymerase θ to participate in microhomology-mediated end joining (
      • Kent T.
      • Chandramouly G.
      • McDevitt S.M.
      • Ozdemir A.Y.
      • Pomerantz R.T.
      Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase θ.
      ). Considering those examples, it is possible that the multimeric form(s) of hpol η plays a specific role in certain biological processes, although further investigation will be required.
      hpol η has been shown to have active site prealignment, dNTP-metal ion binding, and third metal ion binding steps during its catalytic cycle (
      • Nakamura T.
      • Zhao Y.
      • Yamagata Y.
      • Hua Y.J.
      • Yang W.
      Watching DNA polymerase η make a phosphodiester bond.
      ,
      • Ummat A.
      • Silverstein T.D.
      • Jain R.
      • Buku A.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      • Aggarwal A.K.
      Human DNA polymerase η is pre-aligned for dNTP binding and catalysis.
      ). In our study, the biochemical and structural results provide direct evidence that the conserved active site residues Arg-61 and Gln-38 synergistically contribute to enzyme efficiency and fidelity, further delineating the catalytic mechanism of the nucleotidyl transfer reaction catalyzed by hpol η.

      Acknowledgments

      We thank P. S. Pallan for advice on some of the crystallography work and K. Trisler for assistance in preparation of the manuscript. Vanderbilt University is a member institution of the Life Sciences Collaborative Access Team at Sector 21 of the Advanced Photon Source (Argonne, IL). Use of the Advanced Photon Source at Argonne National Laboratory was supported by the United States Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract DE-AC02-06CH11357.

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