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• W2237287274 abstract "Ribonucleotides and 2′-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2′-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2′-OH group. Y-family human DNA polymerase η (hpol η) is of interest because of its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol η maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2′-deoxyguanosine and cyclobutane pyrimidine dimer but with rates that are 103-fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that the hpol η scaffolds the incoming rNTP to pair with the template base (dG) or 7,8-dihydro-8-oxo-2′-deoxyguanosine with a significant propeller twist. As a result, the 2′-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and Pα of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18. This is the first crystal structure of a DNA polymerase with an incoming rNTP opposite a DNA lesion. Ribonucleotides and 2′-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2′-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2′-OH group. Y-family human DNA polymerase η (hpol η) is of interest because of its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol η maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2′-deoxyguanosine and cyclobutane pyrimidine dimer but with rates that are 103-fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that the hpol η scaffolds the incoming rNTP to pair with the template base (dG) or 7,8-dihydro-8-oxo-2′-deoxyguanosine with a significant propeller twist. As a result, the 2′-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and Pα of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18. This is the first crystal structure of a DNA polymerase with an incoming rNTP opposite a DNA lesion. RNA and DNA are fundamental to life in all forms. The two nucleic acid polymers are composed of ribonucleotides and 2′-deoxyribonucleotides as the basic units, respectively. However, ribonucleotides have been found in DNA; they constitute a large proportion of the “DNA lesions” in the genome. In mice, they have been shown to be collectively the most frequently occurring DNA lesions, even more than abasic sites and 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG). 2The abbreviations used are: 8-oxodG, 7,8-dihydro-8-oxo-2′-deoxyguanosine; h, human; pol, DNA polymerase; CPD, cyclobutane pyrimidine dimer; PDB, Protein Data Bank; dAMPNPP, 2′-deoxyadenosine-5′-[(α,β)-imido]triphosphate; dCMPNPP, 2′-deoxycytidine-5′-[(α,β)-imido]triphosphate. The presence of ribonucleotides in DNA increases the possibility of spontaneous hydrolysis, causing DNA to break more frequently. These ribonucleotides are mainly removed from DNA by the RNase H2 pathway (1Reijns M.A. Rabe B. Rigby R.E. Mill P. Astell K.R. Lettice L.A. Boyle S. Leitch A. Keighren M. Kilanowski F. Devenney P.S. Sexton D. Grimes G. Holt I.J. Hill R.E. et al.Enzymatic removal of ribonucleotides from DNA is essential for mammalian genome integrity and development.Cell. 2012; 149: 1008-1022Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar2Rydberg B. Game J. Excision of misincorporated ribonucleotides in DNA by RNase H (type 2) and FEN-1 in cell-free extracts.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16654-16659Crossref PubMed Scopus (155) Google Scholar, 3Vaisman A. Woodgate R. Redundancy in ribonucleotide excision repair: competition, compensation, and cooperation.DNA Repair. 2015; 29: 74-82Crossref PubMed Scopus (20) Google Scholar, 4Sparks J.L. Chon H. Cerritelli S.M. Kunkel T.A. Johansson E. Crouch R.J. Burgers P.M. RNase H2-initiated ribonucleotide excision repair.Mol. Cell. 2012; 47: 980-986Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar5Li Y.F. Breaker R.R. Kinetics of RNA degradation by specific base catalysis of transesterification involving the 2′-hydroxyl group.J. Am. Chem. Soc. 1999; 121: 5364-5372Crossref Scopus (415) Google Scholar). DNA polymerases introduce ribonucleotides into DNA by misinserting them (6Nick McElhinny S.A. Kumar D. Clark A.B. Watt D.L. Watts B.E. Lundström E.B. Johansson E. Chabes A. Kunkel T.A. Genome instability due to ribonucleotide incorporation into DNA.Nat. Chem. Biol. 2010; 6: 774-781Crossref PubMed Scopus (292) Google Scholar). Concentrations of cellular rNTPs are 1–6 orders of magnitude higher than those of dNTPs, depending on the cell type and the stage of the cell cycle (6Nick McElhinny S.A. Kumar D. Clark A.B. Watt D.L. Watts B.E. Lundström E.B. Johansson E. Chabes A. Kunkel T.A. Genome instability due to ribonucleotide incorporation into DNA.Nat. Chem. Biol. 2010; 6: 774-781Crossref PubMed Scopus (292) Google Scholar7Traut T.W. Physiological concentrations of purines and pyrimidines.Mol. Cell. Biochem. 1994; 140: 1-22Crossref PubMed Scopus (1290) Google Scholar, 8Nick McElhinny S.A. Watts B.E. Kumar D. Watt D.L. Lundström E.B. Burgers P.M. Johansson E. Chabes A. Kunkel T.A. Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4949-4954Crossref PubMed Scopus (304) Google Scholar9Chabes A. Stillman B. Constitutively high dNTP concentration inhibits cell cycle progression and the DNA damage checkpoint in yeast Saccharomyces cerevisiae.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1183-1188Crossref PubMed Scopus (104) Google Scholar). To discriminate dNTP from rNTP, a steric gate residue (typically a tyrosine or phenylalanine) is generally conserved in DNA polymerases and thought to create a clash with the extra hydroxyl group of the incoming rNTP (10Joyce C.M. Choosing the right sugar: how polymerases select a nucleotide substrate.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 1619-1622Crossref PubMed Scopus (190) Google Scholar11DeLucia A.M. Chaudhuri S. Potapova O. Grindley N.D. Joyce C.M. The properties of steric gate mutants reveal different constraints within the active sites of Y-family and A-family DNA polymerases.J. Biol. Chem. 2006; 281: 27286-27291Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 12Vaisman A. Kuban W. McDonald J.P. Karata K. Yang W. Goodman M.F. Woodgate R. Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity.Nucleic Acids Res. 2012; 40: 6144-6157Crossref PubMed Scopus (34) Google Scholar, 13DeLucia A.M. Grindley N.D. Joyce C.M. An error-prone family Y DNA polymerase (DinB homolog from Sulfolobus solfataricus) uses a “steric gate” residue for discrimination against ribonucleotides.Nucleic Acids Res. 2003; 31: 4129-4137Crossref PubMed Scopus (67) Google Scholar, 14Niimi N. Sassa A. Katafuchi A. Grúz P. Fujimoto H. Bonala R.R. Johnson F. Ohta T. Nohmi T. The steric gate amino acid tyrosine 112 is required for efficient mismatched-primer extension by human DNA polymerase κ.Biochemistry. 2009; 48: 4239-4246Crossref PubMed Scopus (29) Google Scholar, 15Yang G. Franklin M. Li J. Lin T.C. Konigsberg W. A conserved Tyr residue is required for sugar selectivity in a pol α DNA polymerase.Biochemistry. 2002; 41: 10256-10261Crossref PubMed Scopus (75) Google Scholar, 16Kirouac K.N. Suo Z. Ling H. Structural mechanism of ribonucleotide discrimination by a Y-family DNA polymerase.J. Mol. Biol. 2011; 407: 382-390Crossref PubMed Scopus (15) Google Scholar17Donigan K.A. McLenigan M.P. Yang W. Goodman M.F. Woodgate R. The steric gate of DNA polymerase ι regulates ribonucleotide incorporation and deoxyribonucleotide fidelity.J. Biol. Chem. 2014; 289: 9136-9145Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Although limited in extent, ribonucleotide incorporation has still been observed by a variety of DNA polymerases, from replicative ones with relatively high fidelity and small active sites (e.g. pol ϵ and pol δ) to error-prone X-family pol λ and pol β and Y-family pol ι (6Nick McElhinny S.A. Kumar D. Clark A.B. Watt D.L. Watts B.E. Lundström E.B. Johansson E. Chabes A. Kunkel T.A. Genome instability due to ribonucleotide incorporation into DNA.Nat. Chem. Biol. 2010; 6: 774-781Crossref PubMed Scopus (292) Google Scholar, 8Nick McElhinny S.A. Watts B.E. Kumar D. Watt D.L. Lundström E.B. Burgers P.M. Johansson E. Chabes A. Kunkel T.A. Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4949-4954Crossref PubMed Scopus (304) Google Scholar, 17Donigan K.A. McLenigan M.P. Yang W. Goodman M.F. Woodgate R. The steric gate of DNA polymerase ι regulates ribonucleotide incorporation and deoxyribonucleotide fidelity.J. Biol. Chem. 2014; 289: 9136-9145Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar18Gosavi R.A. Moon A.F. Kunkel T.A. Pedersen L.C. Bebenek K. The catalytic cycle for ribonucleotide incorporation by human DNA pol λ.Nucleic Acids Res. 2012; 40: 7518-7527Crossref PubMed Scopus (39) Google Scholar, 19Clausen A.R. Murray M.S. Passer A.R. Pedersen L.C. Kunkel T.A. Structure-function analysis of ribonucleotide bypass by B family DNA replicases.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 16802-16807Crossref PubMed Scopus (38) Google Scholar, 20Clausen A.R. Zhang S. Burgers P.M. Lee M.Y. Kunkel T.A. Ribonucleotide incorporation, proofreading and bypass by human DNA polymerase δ.DNA Repair. 2013; 12: 121-127Crossref PubMed Scopus (94) Google Scholar21Cavanaugh N.A. Beard W.A. Wilson S.H. DNA polymerase β ribonucleotide discrimination: insertion, misinsertion, extension, and coding.J. Biol. Chem. 2010; 285: 24457-24465Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Compared with other DNA polymerases, those in the Y-family are known for high misinsertion rates and tolerance of DNA adducts in the template strand by relying on their spacious active sites (22Sale J.E. Lehmann A.R. Woodgate R. Y-family DNA polymerases and their role in tolerance of cellular DNA damage.Nat. Rev. Mol. Cell Biol. 2012; 13: 141-152Crossref PubMed Scopus (470) Google Scholar, 23Yang W. An overview of Y-family DNA polymerases and a case study of human DNA polymerase η.Biochemistry. 2014; 53: 2793-2803Crossref PubMed Scopus (123) Google Scholar24Kunkel T.A. DNA replication fidelity.J. Biol. Chem. 2004; 279: 16895-16898Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). Y-family human DNA polymerase η (hpol η) is of particular interest, as it is the only known DNA polymerase directly related to a human genetic disorder, mainly due to its unique role in translesion synthesis past the UV-induced DNA lesion cyclobutane pyrimidine dimer (CPD). Patients defective in hpol η, the result of which is a form of xeroderma pigmentosum (XP-V), are typically highly sensitive to UV light, with increased incidence of skin and other types of cancer and (for some of the individuals) neurodegeneration (25Inui H. Oh K.S. Nadem C. Ueda T. Khan S.G. Metin A. Gozukara E. Emmert S. Slor H. Busch D.B. Baker C.C. DiGiovanna J.J. Tamura D. Seitz C.S. Gratchev A. et al.Xeroderma pigmentosum-variant patients from America, Europe, and Asia.J. Invest. Dermatol. 2008; 128: 2055-2068Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar26Masutani C. Kusumoto R. Yamada A. Dohmae N. Yokoi M. Yuasa M. Araki M. Iwai S. Takio K. Hanaoka F. The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase η.Nature. 1999; 399: 700-704Crossref PubMed Scopus (1146) Google Scholar, 27Johnson R.E. Kondratick C.M. Prakash S. Prakash L. hRAD30 mutations in the variant form of xeroderma pigmentosum.Science. 1999; 285: 263-265Crossref PubMed Scopus (672) Google Scholar28Biertümpfel 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.Nature. 2010; 465: 1044-1048Crossref PubMed Scopus (253) Google Scholar). hpol η is also involved in translesion synthesis of other DNA lesions, e.g. 8-oxodG (29Patra 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.J. Biol. Chem. 2014; 289: 16867-16882Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 30Haracska L. Yu S.L. Johnson R.E. Prakash L. Prakash S. Efficient and accurate replication in the presence of 7,8-dihydro-8-oxoguanine by DNA polymerase η.Nat. Genet. 2000; 25: 458-461Crossref PubMed Scopus (303) Google Scholar), abasic sites (31Patra 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.J. Biol. Chem. 2015; 290: 8028-8038Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 32Haracska L. Washington M.T. Prakash S. Prakash L. Inefficient bypass of an abasic site by DNA polymerase η.J. Biol. Chem. 2001; 276: 6861-6866Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), and cisplatin or other therapeutic drug-induced DNA damage (33Zhao Y. Biertümpfel C. Gregory M.T. Hua Y.J. Hanaoka F. Yang W. Structural basis of human DNA polymerase η-mediated chemoresistance to cisplatin.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 7269-7274Crossref PubMed Scopus (126) Google Scholar34Gregory 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.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 9133-9138Crossref PubMed Scopus (47) Google Scholar, 35Vaisman A. Masutani C. Hanaoka F. Chaney S.G. Efficient translesion replication past oxaliplatin and cisplatin GpG adducts by human DNA polymerase η.Biochemistry. 2000; 39: 4575-4580Crossref PubMed Scopus (196) Google Scholar, 36Bassett E. King N.M. Bryant M.F. Hector S. Pendyala L. Chaney S.G. Cordeiro-Stone M. The role of DNA polymerase η in translesion synthesis past platinum-DNA adducts in human fibroblasts.Cancer Res. 2004; 64: 6469-6475Crossref PubMed Scopus (87) Google Scholar, 37Albertella M.R. Green C.M. Lehmann A.R. O'Connor M.J. A role for polymerase η in the cellular tolerance to cisplatin-induced damage.Cancer Res. 2005; 65: 9799-9806Crossref PubMed Scopus (184) Google Scholar38Chen Y.W. Cleaver J.E. Hanaoka F. Chang C.F. Chou K.M. A novel role of DNA polymerase η in modulating cellular sensitivity to chemotherapeutic agents.Mol. Cancer Res. 2006; 4: 257-265Crossref PubMed Scopus (108) Google Scholar). In this study, we investigated the ability of hpol η to incorporate ribonucleotides into DNA and crystallized hpol η with incoming rNTPs opposite both undamaged DNA and an 8-oxodG lesion. Our results demonstrate that hpol η can incorporate ribonucleotides into DNA with relatively high selectivity but low efficiency, even when the template strand contains the DNA lesion 8-oxodG or CPD. The x-ray crystal structures show that the incoming rNTP at the hpol η active site adopts a slightly different orientation relative to dNTP to avoid a clash with the steric gate residue, without significantly disrupting the pairing with the template dG or 8-oxodG. The latter appears to be the first crystal structure of an incoming rNTP opposite a DNA lesion within a DNA polymerase. Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA) or TriLink BioTechnologies (San Diego) and purified by HPLC by the manufacturers. rNTPs and dNTPs were purchased from New England Biolabs (Ipswich, MA). These experiments were conducted with the catalytic core of hpol η or the Y92A mutant (1–432 amino acids), and the former has been shown to have similar catalytic activity as the full-length protein in vitro (33Zhao Y. Biertümpfel C. Gregory M.T. Hua Y.J. Hanaoka F. Yang W. Structural basis of human DNA polymerase η-mediated chemoresistance to cisplatin.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 7269-7274Crossref PubMed Scopus (126) Google Scholar). The hpol η and the Y92A mutant (1–432 amino acids) were expressed and purified as reported previously (39Su Y. Patra A. Harp J.M. Egli M. Guengerich F.P. 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.J. Biol. Chem. 2015; 290: 15921-15933Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Polyethylene glycol monomethyl ether 2000 (from Hampton Research, Aliso Viejo, CA) was used for crystallization. The fluorescently labeled primer 5′-6-carboxyfluorescein-CGG GCT CGT AAG CGT CAT-3′ was annealed with each of the following template oligonucleotides at a 1:1 molar ratio by heating to 95 °C and slowly cooling: 1) 5′-TCA TGA TGA CGC TTA CGA GCC CG-3′; 2) 5′-TCA TTA TGA CGC TTA CGA GCC CG-3′; 3) 5′-TCA T(8-oxodG)A TGA CGC TTA CGA GCC CG-3′; 4) 5′-TCA (CPD)A TGA CGC TTA CGA GCC CG-3′ (CPD indicates cis-syn thymine dimer). These annealed fluorescent substrates were used in extension, single nucleotide incorporation, and steady-state kinetic assays. The following oligonucleotides were annealed at equal molar ratios for crystallization: 5′-AGC GTC AT-3′ and 5′-CAT GAT GAC GCT-3′; 5′-AGC GTC AT-3′ and 5′-CAT (8-oxodG)AT GAC GCT-3′. The extension assays were conducted with 5 μm DNA substrate (oligonucleotide), 1.2 μm hpol η, and 4 mm dNTP or rNTP mixtures (1 mm for each dNTP or rNTP) in 40 mm Tris-HCl buffer (pH 7.5) containing 10 mm dithiothreitol (DTT), 0.1 mg/ml bovine serum albumin (BSA), 5% glycerol (v/v), 5 mm MgCl2, and 100 mm KCl at 37 °C for 5, 30, 55, and 240 min. Single nucleotide incorporation assays were conducted using the same reaction buffer but with 5 μm annealed DNA substrate, 500 nm hpol η, and 1 mm each of dNTP or rNTP at 37 °C for 5, 30, and 55 min. For steady-state kinetic assays, 5 μm DNA substrate was incubated with 1.5 to 500 nm hpol η or the Y92A mutant as well as varying concentrations of each dNTP or rNTP at 37 °C for 5 min, in the same reaction buffer as extension and single base incorporation assay. Reactions were stopped by the addition of a quench buffer (90% formamide (v/v) and 10 mm EDTA). Products were separated on 18% (w/v) denaturing polyacrylamide gels and visualized using a Typhoon system (GE Healthcare). The data for steady-state kinetic assays were fit to a (hyperbolic) Michaelis-Menten equation using Prism software (GraphPad, La Jolla, CA). Each DNA substrate and hpol η were mixed at a molar ratio of 1.1 to 1, before adding CaCl2 (final concentration 3.33 mm) and excess amount of rNTP. To obtain crystals by hanging drop vapor diffusion, 1 μl of each protein/DNA mixture was mixed with 1 μl of reservoir solution, which contained 100 mm sodium MES (pH 6.0), 5 mm CaCl2, and 16–22% (w/v) polyethylene glycol monomethyl ether 2000, followed by equilibration with 500 μl of the reservoir solution at 18 °C. The crystals were washed through cryoprotectant (composed of 3 volumes of reservoir solution and 1 volume of glycerol) before flash freezing in liquid nitrogen. Diffraction data were collected on the 21-ID-F or 21-ID-G beamline at the Advanced Photon Source (Life Sciences Collaborative Access Team, Argonne National Laboratory, Argonne, IL). Data were integrated and scaled using HKL2000 (40Otwinowski Z. Minor W. Processing of x-ray diffraction data collected in oscillation mode.Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38527) Google Scholar), processed with Phaser MR (41McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. Phaser crystallographic software.J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (14444) Google Scholar) for molecular replacement phasing (PDB code 4O3N as the search model for hpol η·dG·rCTP, and PDB code 4O3P for hpol η·dG·rCTP and hpol η·dG·rATP) (29Patra 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.J. Biol. Chem. 2014; 289: 16867-16882Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), and refined with PHENIX (42Adams 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. et al.PHENIX: a comprehensive Python-based system for macromolecular structure solution.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 213-221Crossref PubMed Scopus (16439) Google Scholar). Model building was performed with ARP/ω ARP classic (43Langer G.G. Hazledine S. Wiegels T. Carolan C. Lamzin V.S. Visual automated macromolecular model building.Acta Crystallogr. D Biol. Crystallogr. 2013; 69: 635-641Crossref PubMed Scopus (53) Google Scholar, 44Murshudov 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.Acta Crystallogr. D Biol. Crystallogr. 2011; 67: 355-367Crossref PubMed Scopus (5952) Google Scholar) and COOT (45Emsley P. Lohkamp B. Scott W.G. Cowtan K. Features and development of Coot.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 486-501Crossref PubMed Scopus (17085) Google Scholar). All illustrations were generated with the program UCSF Chimera (46Pettersen 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.J. Comput. Chem. 2004; 25: 1605-1612Crossref PubMed Scopus (27950) Google Scholar). To assess the ribonucleotide incorporation ability of hpol η, we compared the extension of the primers in the presence of all four dNTPs or rNTPs with different incubation times. The primer was extended at 5 min and reached full-length after 30 min in the presence of rNTPs, compared with 5 min for full-length extension with dNTPs (Fig. 1A). To estimate the fidelity of ribonucleotide incorporation, hpol η was incubated with annealed undamaged DNA substrate as well as individual dNTP or rNTP. Under our reaction conditions, multiple nucleotides were added to the primer in the presence of a single dNTP in an error-prone manner. However, only one or two nucleotide extensions was observed with a single rNTP. Among the four ribonucleotides, rCTP (able to Watson-Crick pair with template dG) was the most efficient one for insertion (Fig. 1B). To further measure the efficiency and fidelity of rNTP incorporation by hpol η, steady-state kinetic experiments were conducted. The catalytic efficiency (kcat/Km) for rCTP insertion opposite template dG was 770-fold less than that for dCTP and 2–5-fold less relative to those for the mismatched dNTPs. However, rates of incorporation of the other ribonucleotides (other than rCTP) were very low and could not be measured experimentally. In addition, to investigate the effect of the template base, another set of DNA substrates with dT in the template instead of dG was included in the steady-state kinetic study. The change for catalytic efficiency between rATP and dATP insertion opposite template dT was 3400-fold, slightly higher than that between rCTP and dCTP opposite dG (770-fold, Table 1).TABLE 1Steady-state kinetics for insertion of nucleoside triphosphates opposite dG and dT by hpol ηTemplatedNTP/rNTPKmkcatkcat/KmfaMisinsertion frequency is as follows: f = (kcat/Km)incorrect/(kcat/Km)correct.1/fbFold change is 1/f.μmmin−1μm−1 min−1dGdCTP2.4 ± 0.3119 ± 350 ± 611dATP84 ± 911 ± 10.13 ± 0.020.0026380dGTP58 ± 712 ± 10.21 ± 0.030.0042240dTTP170 ± 1658 ± 20.34 ± 0.030.0068150rCTP188 ± 1412 ± 10.064 ± 0.0070.0013770dTdCTP127 ± 819 ± 10.15 ± 0.010.0048210dATP2.5 ± 0.277 ± 231 ± 311dGTP9.1 ± 0.627 ± 13.0 ± 0.20.09710dTTP132 ± 1527 ± 10.20 ± 0.020.0065150rATP278 ± 372.5 ± 0.10.0090 ± 0.00120.000293400a Misinsertion frequency is as follows: f = (kcat/Km)incorrect/(kcat/Km)correct.b Fold change is 1/f. Open table in a new tab We conducted primer extension assays by hpol η with 8-oxodG in the template strand in the presence of dNTPs or rNTPs. The pattern for primer extension past 8-oxodG was very similar to that against undamaged DNA (Figs. 1A and 2A). The primer was partially extended to full length by 30 min with the mixture of all four rNTPs, whereas hpol η elongated the primer to full length within 5 min with dNTPs. In single nucleotide insertion assays, only one rC was added to the primer within 5 min, although in the presence of the other individual rNTPs significantly elongated primers were only observed after 30 min of incubation (Fig. 2, A and B). In steady-state kinetic assays, hpol η incorporated dCTP opposite template 8-oxodG 2300-fold more efficiently than rCTP and 43,000-fold more than rATP. The difference between the catalytic efficiencies for rCTP and rATP incorporation was about 19, compared with 5 for dCTP and dATP, indicating that during nucleotide incorporation hpol η has similar base selectivity regardless of the sugar (Table 2).TABLE 2Steady-state kinetics for insertion of nucleoside triphosphates opposite 8-oxodG by hpol ηTemplatedNTP/rNTPKmkcatkcat/Kmf1/fμmmin−1μm−1 min−18-oxodGdCTP1.7 ± 0.381 ± 348 ± 911dATP8.4 ± 1.077 ± 39.2 ± 1.10.195.3dGTP18 ± 229 ± 11.6 ± 0.20.03330dTTP140 ± 1144 ± 10.31 ± 0.030.0065150rCTP292 ± 426.1 ± 0.30.021 ± 0.0030.000442300rATP445 ± 400.51 ± 0.020.0011 ± 0.00010.00002343000 Open table in a new tab Given that hpol η is capable of bypassing CPD by inserting the correct nucleotide dA (26Masutani C. Kusumoto R. Yamada A. Dohmae N. Yokoi M. Yuasa M. Araki M. Iwai S. Takio K. Hanaoka F. The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase η.Nature. 1999; 399: 700-704Crossref PubMed Scopus (1146) Google Scholar, 28Biertümpfel 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.Nature. 2010; 465: 1044-1048Crossref PubMed Scopus (253) Google Scholar), we investigated the incorporation of ribonucleotides against this lesion. hpol η extended the primer past CPD in a manner similar to that observed opposite undamaged DNA or 8-oxodG. With only rATP, more than 50% of the primers were elongated by two nucleotides after 5 min, much faster than with the other single rNTPs (Fig. 3, A and B). Quantitatively, rATP insertion was 1400-fold less efficient than dATP opposite CPD (Table 3).TABLE 3Steady-state kinetics for insertion of nucleoside triphosphates opposite CPD by hpol ηTemplatedNTP/rNTPKmkcatkcat/Kmf1/fμmmin−1μm−1 min−1CPDdCTP31 ± 327 ± 10.87 ± 0.090.02638dATP1.7 ± 0.257 ± 134 ± 411dGTP34 ± 340 ± 11.2 ± 0.10.03529dTTP23 ± 232 ± 11.4 ± 0.10.04124rATP295 ± 357.0 ± 0.30.024 ± 0.0030.000711400 Open table in a new tab To understand the mechanism of ribonucleotide insertion by hpol η, we co-crystallized hpol η with an incoming rCTP positioned opposite template dG in the presence of Ca2+ (Table 4). The final Fourier (2Fo − Fc) sum electron density map (with a threshold of 1σ) is shown in Fig. 4A. The incoming rCTP formed a Watson-Crick base pair with the template dG, but a significant propeller twist was observed. The dihedral angle between the two base planes was 27°. In addition, the base pair was slightly shifted toward the major groove, compared with the dG:dCTP pair at the hpol η active site. Phe-18 was identified as the steric gate residue, and the distance between either 2′-OH or 3′-OH of rCTP and their closest atoms of the Phe-18 side chain was 3.2 Å (Fig. 5). In comparison with the hpol η·dG:dCTP structure (PDB code 4O3N) (29Patra 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.J. Biol. Chem. 2014; 289: 16867-16882Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), the position of the phenyl ring of Phe-18 was almost identical. In addition, the distance between the 3′-OH of the primer end (two conformations) to Pα of the incoming rCTP was 4.5 or 4.3 Å, about 1 Å further than that in the hpol η·dG:dCTP structure (3.5 or 3.3 Å), providing an explanation for the 103-fold lower catalytic efficiency of the polymerization reaction (Fig. 5).TABLE 4Crystal data, data collection parameters, and structure refinement statisticsComplexhpol η·dG:rCTPhpol η·(8-oxodG):rCTPhpol η·(8-oxodG):rATPData collectionWavelength (Å)0.978560.978720.97856Space groupP61P61P61Resolution (Å)50.00-1.66 (1.69-1.66)aData shown in parentheses are from the highest resolution shell.50.00-1.78 (1.81-1.78)50.00-1.75 (1.78-1.75)Unit cell a = b, c (Å)99.2" @default.
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• W2237287274 title "Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η" @default.
• W2237287274 cites W1085957732 @default.
• W2237287274 cites W1522788044 @default.
• W2237287274 cites W1531823089 @default.
• W2237287274 cites W1539796472 @default.
• W2237287274 cites W1964261732 @default.
• W2237287274 cites W1970184264 @default.
• W2237287274 cites W1973839692 @default.
• W2237287274 cites W1975872149 @default.
• W2237287274 cites W1979278914 @default.
• W2237287274 cites W1980745500 @default.
• W2237287274 cites W1993705842 @default.
• W2237287274 cites W1999240709 @default.
• W2237287274 cites W2022265644 @default.
• W2237287274 cites W2025055438 @default.
• W2237287274 cites W2030961393 @default.
• W2237287274 cites W2032445148 @default.
• W2237287274 cites W2037845691 @default.
• W2237287274 cites W2038390954 @default.
• W2237287274 cites W2042344660 @default.
• W2237287274 cites W2045074917 @default.
• W2237287274 cites W2046018151 @default.
• W2237287274 cites W2054377678 @default.
• W2237287274 cites W2056802283 @default.
• W2237287274 cites W2059767272 @default.
• W2237287274 cites W2063705661 @default.
• W2237287274 cites W2064639767 @default.
• W2237287274 cites W2065286203 @default.
• W2237287274 cites W2074050403 @default.
• W2237287274 cites W2077036683 @default.
• W2237287274 cites W2078507463 @default.
• W2237287274 cites W2086322923 @default.
• W2237287274 cites W2091262927 @default.
• W2237287274 cites W2094249206 @default.
• W2237287274 cites W2104547359 @default.
• W2237287274 cites W2123061667 @default.
• W2237287274 cites W2123334025 @default.
• W2237287274 cites W2124026197 @default.
• W2237287274 cites W2125224457 @default.
• W2237287274 cites W2126847533 @default.
• W2237287274 cites W2132008457 @default.
• W2237287274 cites W2132629607 @default.
• W2237287274 cites W2139039510 @default.
• W2237287274 cites W2142892199 @default.
• W2237287274 cites W2152112318 @default.
• W2237287274 cites W2159211495 @default.
• W2237287274 cites W2163341755 @default.
• W2237287274 cites W2164402320 @default.
• W2237287274 cites W2168068318 @default.
• W2237287274 cites W2180229411 @default.
• W2237287274 cites W2327611812 @default.
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