Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES

Abstract

Previous work has shown that Y-family DNA polymerases tolerate large DNA adducts, but a substantial decrease in catalytic efficiency and fidelity occurs during bypass of N2,N2-dimethyl (Me2)-substituted guanine (N2,N2-Me2G), in contrast to a single methyl substitution. Therefore, it is unclear why the addition of two methyl groups is so disruptive. The presence of N2,N2-Me2G lowered the catalytic efficiency of the model enzyme Sulfolobus solfataricus Dpo4 16,000-fold. Dpo4 inserted dNTPs almost at random during bypass of N2,N2-Me2G, and much of the enzyme was kinetically trapped by an inactive ternary complex when N2,N2-Me2G was present, as judged by a reduced burst amplitude (5% of total enzyme) and kinetic modeling. One crystal structure of Dpo4 with a primer having a 3′-terminal dideoxycytosine (Cdd) opposite template N2,N2-Me2G in a post-insertion position showed Cdd folded back into the minor groove, as a catalytically incompetent complex. A second crystal had two unique orientations for the primer terminal Cdd as follows: (i) flipped into the minor groove and (ii) a long pairing with N2,N2-Me2G in which one hydrogen bond exists between the O-2 atom of Cdd and the N-1 atom of N2,N2-Me2G, with a second water-mediated hydrogen bond between the N-3 atom of Cdd and the O-6 atom of N2,N2-Me2G. A crystal structure of Dpo4 with dTTP opposite template N2,N2-Me2G revealed a wobble orientation. Collectively, these results explain, in a detailed manner, the basis for the reduced efficiency and fidelity of Dpo4-catalyzed bypass of N2,N2-Me2G compared with mono-substituted N2-alkyl G adducts. Previous work has shown that Y-family DNA polymerases tolerate large DNA adducts, but a substantial decrease in catalytic efficiency and fidelity occurs during bypass of N2,N2-dimethyl (Me2)-substituted guanine (N2,N2-Me2G), in contrast to a single methyl substitution. Therefore, it is unclear why the addition of two methyl groups is so disruptive. The presence of N2,N2-Me2G lowered the catalytic efficiency of the model enzyme Sulfolobus solfataricus Dpo4 16,000-fold. Dpo4 inserted dNTPs almost at random during bypass of N2,N2-Me2G, and much of the enzyme was kinetically trapped by an inactive ternary complex when N2,N2-Me2G was present, as judged by a reduced burst amplitude (5% of total enzyme) and kinetic modeling. One crystal structure of Dpo4 with a primer having a 3′-terminal dideoxycytosine (Cdd) opposite template N2,N2-Me2G in a post-insertion position showed Cdd folded back into the minor groove, as a catalytically incompetent complex. A second crystal had two unique orientations for the primer terminal Cdd as follows: (i) flipped into the minor groove and (ii) a long pairing with N2,N2-Me2G in which one hydrogen bond exists between the O-2 atom of Cdd and the N-1 atom of N2,N2-Me2G, with a second water-mediated hydrogen bond between the N-3 atom of Cdd and the O-6 atom of N2,N2-Me2G. A crystal structure of Dpo4 with dTTP opposite template N2,N2-Me2G revealed a wobble orientation. Collectively, these results explain, in a detailed manner, the basis for the reduced efficiency and fidelity of Dpo4-catalyzed bypass of N2,N2-Me2G compared with mono-substituted N2-alkyl G adducts. Cellular DNA is continuously attacked by physical agents and by various endogenous and exogenous chemicals to produce DNA damage products, including abasic sites (1Schaaper R.M. Glickman B.W. Loeb L.A. Cancer Res. 1982; 42: 3480-3485PubMed Google Scholar, 2Nakamura J. Walker V.E. Upton P.B. Chiang S.Y. Kow Y.W. Swenberg J.A. Cancer Res. 1998; 58: 222-225PubMed Google Scholar), deamination products (3Wink D.A. Kasprzak K.S. Maragos C.M. Elespuru R.K. Misra M. Dunams T.M. Cebula T.A. Koch W.H. Andrews A.W. Allen J.S. Keefer L.K. Science. 1991; 254: 1001-1003Crossref PubMed Scopus (1129) Google Scholar, 4Nguyen T. Brunson D. Crespi C.L. Penman B.W. Wishnok J.S. Tannenbaum S.R. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 3030-3034Crossref PubMed Scopus (946) Google Scholar), oxidized adducts (e.g. 8-oxoG 3The abbreviations used are: 8-oxoG7,8-dihydro-8-oxodeoxyguanosineEtethylG2′-deoxyguanosineLCliquid chromatographyMemethylMSmass spectrometryMS/MStandem mass spectrometryN2-NaphGN2-(2-naphthyl)methylGpol(DNA) polymerasePDBProtein Data Bank. 3The abbreviations used are: 8-oxoG7,8-dihydro-8-oxodeoxyguanosineEtethylG2′-deoxyguanosineLCliquid chromatographyMemethylMSmass spectrometryMS/MStandem mass spectrometryN2-NaphGN2-(2-naphthyl)methylGpol(DNA) polymerasePDBProtein Data Bank. (5Kuchino Y. Mori F. Kasai H. Inoue H. Iwai S. Miura K. Ohtsuka E. Nishimura S. Nature. 1987; 327: 77-79Crossref PubMed Scopus (733) Google Scholar, 6Aruoma O.I. Halliwell B. Dizdaroglu M. J. Biol. Chem. 1989; 264: 13024-13028Abstract Full Text PDF PubMed Google Scholar, 7Cheng K.C. Cahill D.S. Kasai H. Nishimura S. Loeb L.A. J. Biol. Chem. 1992; 267: 166-172Abstract Full Text PDF PubMed Google Scholar)), alkyl lesions (particularly O6-alkyl G (8Searle C.E. Chemical Carcinogens. 2nd Ed. American Chemical Society, Washington, DC1984Google Scholar, 9Friedberg E.C. Walker G.C. Siede W. Wood R.D. Schultz R.A. Ellenberger T. DNA Repair and Mutagenesis. 2nd Ed. American Society for Microbiology, Washington, DC2006Google Scholar)), UV-induced pyrimidine dimers, and 6-4 photoproducts (10Mouret S. Baudouin C. Charveron M. Favier A. Cadet J. Douki T. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 13765-13770Crossref PubMed Scopus (511) Google Scholar, 11Douki T. Cadet J. Biochemistry. 2001; 40: 2495-2501Crossref PubMed Scopus (264) Google Scholar), bis-electrophile-induced exocyclic DNA products (12Mao H. Schnetz-Boutaud N.C. Weisenseel J.P. Marnett L.J. Stone M.P. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 6615-6620Crossref PubMed Scopus (124) Google Scholar, 13Guengerich F.P. Langouët S. Mican A.N. Akasaka S. Müller M. Persmark M. IARC Sci. Publ. 1999; 150: 137-145Google Scholar, 14de los Santos C. Zaliznyak T. Johnson F. J. Biol. Chem. 2001; 276: 9077-9082Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 15Stone M.P. Cho Y.J. Huang H. Kim H.Y. Kozekov I.D. Kozekova A. Wang H. Minko I.G. Lloyd R.S. Harris T.M. Rizzo C.J. Acc. Chem. Res. 2008; 41: 793-804Crossref PubMed Scopus (143) Google Scholar), and inter- and intra-strand DNA cross-links (9Friedberg E.C. Walker G.C. Siede W. Wood R.D. Schultz R.A. Ellenberger T. DNA Repair and Mutagenesis. 2nd Ed. American Society for Microbiology, Washington, DC2006Google Scholar, 15Stone M.P. Cho Y.J. Huang H. Kim H.Y. Kozekov I.D. Kozekova A. Wang H. Minko I.G. Lloyd R.S. Harris T.M. Rizzo C.J. Acc. Chem. Res. 2008; 41: 793-804Crossref PubMed Scopus (143) Google Scholar). Even with all of the cellular DNA repair systems available, some DNA adducts are present in cells and lead to misincorporation, mutation, and blockage when they interact with DNA polymerases, potentially causing cell death, aging, and cancer (9Friedberg E.C. Walker G.C. Siede W. Wood R.D. Schultz R.A. Ellenberger T. DNA Repair and Mutagenesis. 2nd Ed. American Society for Microbiology, Washington, DC2006Google Scholar). 7,8-dihydro-8-oxodeoxyguanosine ethyl 2′-deoxyguanosine liquid chromatography methyl mass spectrometry tandem mass spectrometry N2-(2-naphthyl)methylG (DNA) polymerase Protein Data Bank. 7,8-dihydro-8-oxodeoxyguanosine ethyl 2′-deoxyguanosine liquid chromatography methyl mass spectrometry tandem mass spectrometry N2-(2-naphthyl)methylG (DNA) polymerase Protein Data Bank. The details of how DNA polymerases interact with these modifications in the DNA are important in understanding the biochemistry relevant to these adverse events. DNA polymerases are complex enzymes; they must be able to bind not only DNA but also all four of the canonical dNTPs. The dilemma is how polymerases “sense” which DNA base is in the active site and how the process selects the appropriate base prior to the phosphodiester bond formation step, which “seals” the choice unless exonuclease or repair processes intervene later. The details of this sensing, and the obligate conformational changes necessary to amplify the energy involved in recognition (16Petruska J. Goodman M.F. Boosalis M.S. Sowers L.C. Cheong C. Tinoco Jr., I. Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 6252-6256Crossref PubMed Scopus (289) Google Scholar), are still controversial (17Showalter A.K. Tsai M.D. Biochemistry. 2002; 41: 10571-10576Crossref PubMed Scopus (126) Google Scholar, 18Joyce C.M. Benkovic S.J. Biochemistry. 2004; 43: 14317-14324Crossref PubMed Scopus (281) Google Scholar, 19Guengerich F.P. Chem. Rev. 2006; 106: 420-452Crossref PubMed Scopus (95) Google Scholar, 20Johnson K.A. J. Biol. Chem. 2008; 283: 26297-26301Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The situation can be much more mechanistically complex when a modified base is present in the DNA substrate (19Guengerich F.P. Chem. Rev. 2006; 106: 420-452Crossref PubMed Scopus (95) Google Scholar). The N-2 atom of guanine is susceptible to attack by a wide variety of chemicals, including formaldehyde (21Yasui M. Matsui S. Ihara M. Laxmi Y.R. Shibutani S. Matsuda T. Nucleic Acids Res. 2001; 29: 1994-2001Crossref PubMed Google Scholar), acetaldehyde (22Terashima I. Matsuda T. Fang T.W. Suzuki N. Kobayashi J. Kohda K. Shibutani S. Biochemistry. 2001; 40: 4106-4114Crossref PubMed Scopus (47) Google Scholar, 23Liu X. Lao Y. Yang I.Y. Hecht S.S. Moriya M. Biochemistry. 2006; 45: 12898-12905Crossref PubMed Scopus (34) Google Scholar, 24Lao Y. Hecht S.S. Chem. Res. Toxicol. 2005; 18: 711-721Crossref PubMed Scopus (30) Google Scholar, 25Zhang S. Villalta P.W. Wang M. Hecht S.S. Chem. Res. Toxicol. 2006; 19: 1386-1392Crossref PubMed Scopus (88) Google Scholar), styrene oxide (26Forgacs E. Latham G. Beard W.A. Prasad R. Bebenek K. Kunkel T.A. Wilson S.H. Lloyd R.S. J. Biol. Chem. 1997; 272: 8525-8530Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), α-hydroxy-N-nitrosopyrrolidine (27Wang M. Lao Y. Cheng G. Shi Y. Villalta P.W. Hecht S.S. Chem. Res. Toxicol. 2007; 20: 625-633Crossref PubMed Scopus (15) Google Scholar), oxidation products of heterocyclic aromatic amines (28Turesky R.J. Rossi S.C. Welti D.H. Lay Jr., J.O. Kadlubar F.F. Chem. Res. Toxicol. 1992; 5: 479-490Crossref PubMed Scopus (161) Google Scholar), and various polycyclic aromatic hydrocarbons (29Meehan T. Straub K. Nature. 1979; 277: 410-412Crossref PubMed Scopus (268) Google Scholar, 30Cheng S.C. Hilton B.D. Roman J.M. Dipple A. Chem. Res. Toxicol. 1989; 2: 334-340Crossref PubMed Scopus (374) Google Scholar), leading to the formation of various N2-alkyl G adducts. Pyridyloxobutyl-derived N2-alkyl G adducts have been detected in rats after chronic treatment with N′-nitrosonornicotine (31Lao Y. Yu N. Kassie F. Villalta P.W. Hecht S.S. Chem. Res. Toxicol. 2007; 20: 246-256Crossref PubMed Scopus (55) Google Scholar). N2-EtG was detected in granulocyte and lymphocyte DNA and urine of alcoholic patients (32Fang J.L. Vaca C.E. Carcinogenesis. 1997; 18: 627-632Crossref PubMed Scopus (245) Google Scholar, 33Matsuda T. Terashima I. Matsumoto Y. Yabushita H. Matsui S. Shibutani S. Biochemistry. 1999; 38: 929-935Crossref PubMed Scopus (81) Google Scholar), and misincorporation opposite N2-EtG by Escherichia coli DNA polymerase I/Klenow fragment (exonuclease−) has been reported (22Terashima I. Matsuda T. Fang T.W. Suzuki N. Kobayashi J. Kohda K. Shibutani S. Biochemistry. 2001; 40: 4106-4114Crossref PubMed Scopus (47) Google Scholar). Crotonaldehyde- and acetaldehyde-derived 1,N2-propanodeoxyguanosine adducts have also been characterized in human tissue DNA (25Zhang S. Villalta P.W. Wang M. Hecht S.S. Chem. Res. Toxicol. 2006; 19: 1386-1392Crossref PubMed Scopus (88) Google Scholar). Even 2′-O-substitutions that differ by a single methylene moiety can exhibit very different RNA affinities in terms of modified DNAs (34Pattanayek R. Sethaphong L. Pan C. Prhavc M. Prakash T.P. Manoharan M. Egli M. J. Am. Chem. Soc. 2004; 126: 15006-15007Crossref PubMed Scopus (30) Google Scholar). In addition to exogenous agents, S- adenosyl-l-methionine-dependent methyltransferases can both mono- and di-methylate the N-2 atom of guanine in RNA (35Martin J.L. McMillan F.M. Curr. Opin. Struct. Biol. 2002; 12: 783-793Crossref PubMed Scopus (452) Google Scholar). N2-MeG and N2,N2-Me2G have been detected in tRNA, rRNA, and small nuclear RNA molecules from prokaryotic, archaeal, and eukaryotic systems (36Limbach P.A. Crain P.F. McCloskey J.A. Nucleic Acids Res. 1994; 22: 2183-2196Crossref PubMed Scopus (476) Google Scholar, 37Edqvist J. Stråby K.B. Grosjean H. Biochimie. 1995; 77: 54-61Crossref PubMed Scopus (36) Google Scholar). The N2,N2-Me2G modification occurs at position 26 in at least 103 eukaryotic tRNAs, with N2-MeG occurring in at least 17 tRNAs (25Zhang S. Villalta P.W. Wang M. Hecht S.S. Chem. Res. Toxicol. 2006; 19: 1386-1392Crossref PubMed Scopus (88) Google Scholar, 37Edqvist J. Stråby K.B. Grosjean H. Biochimie. 1995; 77: 54-61Crossref PubMed Scopus (36) Google Scholar, 38Steinberg S. Misch A. Sprinzl M. Nucleic Acids Res. 1993; 21: 3011-3015Crossref PubMed Scopus (233) Google Scholar). It has been postulated that the presence of N2,N2-Me2G at positions 10 and 26 of tRNA helps prevent +1 frameshifting during translation events, but whether this is because N2,N2-Me2G at position 26 stabilizes the tertiary structure of the region near the D-stem and the anti-codon stem or because the modifications influence tRNA positioning in the ribosome is unclear (39Armengaud J. Urbonavicius J. Fernandez B. Chaussinand G. Bujnicki J.M. Grosjean H. J. Biol. Chem. 2004; 279: 37142-37152Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). N2-Methylation of guanine is important in the context of genomic replication because some retroviruses can use host RNA to prime viral genomic synthesis (40Marquet R. Isel C. Ehresmann C. Ehresmann B. Biochimie. 1995; 77: 113-124Crossref PubMed Scopus (198) Google Scholar), and N2-MeG has been shown to inhibit avian myeloblastosis virus reverse transcriptase activity on E. coli 16 S rRNA (37Edqvist J. Stråby K.B. Grosjean H. Biochimie. 1995; 77: 54-61Crossref PubMed Scopus (36) Google Scholar). Structure-function relationships with various N2-alkyl G lesions have been studied in oligonucleotides in this laboratory with a number of DNA polymerases. Even a Me or Et group is quite inhibitory to the processive DNA polymerases HIV-1 reverse transcriptase and bacteriophage DNA polymerase T7 (41Choi J.Y. Guengerich F.P. J. Biol. Chem. 2004; 279: 19217-19229Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The polymerization activity of Klenow fragment was only slightly attenuated, but misincorporation was observed (21Yasui M. Matsui S. Ihara M. Laxmi Y.R. Shibutani S. Matsuda T. Nucleic Acids Res. 2001; 29: 1994-2001Crossref PubMed Google Scholar, 22Terashima I. Matsuda T. Fang T.W. Suzuki N. Kobayashi J. Kohda K. Shibutani S. Biochemistry. 2001; 40: 4106-4114Crossref PubMed Scopus (47) Google Scholar). With human Y-family DNA polymerases, the rates of dNTP incorporation with the four normal dNTPs are slower, and processivity is lower than with the processive A- and B-family DNA polymerases. However, the presence of alkyl (and aralkyl) moieties at the N-2 atom of G is much less inhibitory to Y-family DNA polymerases (42Choi J.Y. Guengerich F.P. J. Mol. Biol. 2005; 352: 72-90Crossref PubMed Scopus (80) Google Scholar, 43Choi J.Y. Guengerich F.P. J. Biol. Chem. 2006; 281: 12315-12324Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 44Choi J.Y. Angel K.C. Guengerich F.P. J. Biol. Chem. 2006; 281: 21062-21072Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 45Choi J.Y. Guengerich F.P. J. Biol. Chem. 2008; 283: 23645-23655Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 46Zhang H. Eoff R.L. Kozekov I.D. Rizzo C.J. Egli M. Guengerich F.P. J. Biol. Chem. 2009; 284: 3563-3576Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and with some DNA polymerases even a bulky (6-benzo[a]pyrenyl)methyl moiety is tolerated reasonably well (43Choi J.Y. Guengerich F.P. J. Biol. Chem. 2006; 281: 12315-12324Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 44Choi J.Y. Angel K.C. Guengerich F.P. J. Biol. Chem. 2006; 281: 21062-21072Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 45Choi J.Y. Guengerich F.P. J. Biol. Chem. 2008; 283: 23645-23655Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Dpo4, a prototypic Y-family DNA polymerase from the crenarchaeon Sulfolobus solfataricus, was even less sensitive to the effect of increasing bulk than the human Y-family enzymes (46Zhang H. Eoff R.L. Kozekov I.D. Rizzo C.J. Egli M. Guengerich F.P. J. Biol. Chem. 2009; 284: 3563-3576Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The presence of an Et group has only a limited effect in slowing the Y-family DNA polymerases (42Choi J.Y. Guengerich F.P. J. Mol. Biol. 2005; 352: 72-90Crossref PubMed Scopus (80) Google Scholar, 43Choi J.Y. Guengerich F.P. J. Biol. Chem. 2006; 281: 12315-12324Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 44Choi J.Y. Angel K.C. Guengerich F.P. J. Biol. Chem. 2006; 281: 21062-21072Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 45Choi J.Y. Guengerich F.P. J. Biol. Chem. 2008; 283: 23645-23655Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 46Zhang H. Eoff R.L. Kozekov I.D. Rizzo C.J. Egli M. Guengerich F.P. J. Biol. Chem. 2009; 284: 3563-3576Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 47Pence M.G. Blans P. Zink C.N. Hollis T. Fishbein J.C. Perrino F.W. J. Biol. Chem. 2009; 284: 1732-1740Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), and small conformational changes occurring in pol ι have been shown to accommodate the lesion (47Pence M.G. Blans P. Zink C.N. Hollis T. Fishbein J.C. Perrino F.W. J. Biol. Chem. 2009; 284: 1732-1740Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). However, distributing the same amount of overall bulk into two 1-carbon units, i.e. N2,N2-Me2G (Fig. 1), has a dramatic effect in slowing incorporation of dCTP by human Y-family pol η and κ (42Choi J.Y. Guengerich F.P. J. Mol. Biol. 2005; 352: 72-90Crossref PubMed Scopus (80) Google Scholar, 44Choi J.Y. Angel K.C. Guengerich F.P. J. Biol. Chem. 2006; 281: 21062-21072Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) but not (human) pol ι or Rev1 (43Choi J.Y. Guengerich F.P. J. Biol. Chem. 2006; 281: 12315-12324Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 45Choi J.Y. Guengerich F.P. J. Biol. Chem. 2008; 283: 23645-23655Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), the latter two of which use non-“Watson-Crick” pairing in alignments for phosphodiester bond formation (48Nair D.T. Johnson R.E. Prakash L. Prakash S. Aggarwal A.K. Science. 2005; 309: 2219-2222Crossref PubMed Scopus (201) Google Scholar, 49Nair D.T. Johnson R.E. Prakash S. Prakash L. Aggarwal A.K. Nature. 2004; 430: 377-380Crossref PubMed Scopus (259) Google Scholar, 50Nair D.T. Johnson R.E. Prakash L. Prakash S. Aggarwal A.K. Structure. 2008; 16: 239-245Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In the case of Dpo4 and the bulky lesion N2-NaphG, we were able to characterize the crystal structures of two forms and provide evidence for the existence of multiple conformations of the polymerase·oligonucleotide·dNTP complex (46Zhang H. Eoff R.L. Kozekov I.D. Rizzo C.J. Egli M. Guengerich F.P. J. Biol. Chem. 2009; 284: 3563-3576Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The dramatic attenuation of dCTP incorporation opposite N2,N2-Me2G compared with N2-MeG with human pol η and κ (44Choi J.Y. Angel K.C. Guengerich F.P. J. Biol. Chem. 2006; 281: 21062-21072Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) is striking and was also observed in preliminary experiments with Dpo4. The small size and strong blocking nature of this adduct rendered N2,N2-Me2G useful in structural and kinetic investigations regarding why this simple adduct is so inhibitory. The results allow for some general conclusions that have relevance in the context of DNA polymerases bound to damaged DNA and their equilibria between catalytically active and nonproductive species. Unlabeled dNTPs were purchased from Amersham Biosciences; (Sp)-dCTPαS was obtained from Biolog Life Science Institute (Bremen, Germany), and [γ-32P]ATP (specific activity 3 × 103 Ci mmol−1) was from PerkinElmer Life Sciences. T4 polynucleotide kinase was purchased from New England Biolabs (Beverly, MA). Dpo4 was expressed and purified as described elsewhere (51Zang H. Goodenough A.K. Choi J.Y. Irimia A. Loukachevitch L.V. Kozekov I.D. Angel K.C. Rizzo C.J. Egli M. Guengerich F.P. J. Biol. Chem. 2005; 280: 29750-29764Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Unmodified 13-mer, 14-mer, 24-mer, 25-mer, and 36-mer (Table 1) were purchased from Midland Certified Reagent Co. (Midland, TX). The 18-mer and 36-mer templates containing N2-MeG or N2,N2-Me2G were synthesized and characterized by capillary gel electrophoresis and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (43Choi J.Y. Guengerich F.P. J. Biol. Chem. 2006; 281: 12315-12324Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Extinction coefficients for the oligonucleotides, estimated by the Borer method (52Borer P.N. Fasman G.D. Handbook of Biochemistry and Molecular Biology. 3rd Ed. CRC Press, Inc., Cleveland, OH1975: 589-590Google Scholar), were as follows: 13-mer, ϵ260 = 112 mm−1 cm−1; 14-mer, ϵ260 = 122 mm−1 cm−1; 18-mer, ϵ260 = 157 mm−1 cm−1; 24-mer, ϵ260 = 224 mm−1 cm−1; 25-mer, ϵ260 = 232 mm−1 cm−1; and 36-mer, ϵ260 = 310 mm−1 cm−1 (43Choi J.Y. Guengerich F.P. J. Biol. Chem. 2006; 281: 12315-12324Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar).TABLE 1Oligodeoxynucleotides used in this study24-mer5′-GCCTCGAGCCAGCCGCAGACGCAG25C-mer5′-GCCTCGAGCCAGCCGCAGACGCAGC25T-mer5′-GCCTCGAGCCAGCCGCAGACGCAGT36-mer3′-CGGAGCTCGGTCGGCGTCTGCGTCG*CTCCTGCGGCT13Cdd-mer5′-GGGGGAAGGATTCdd14Cdd-mer5′-GGGGGAAGGATTCCdd18G*C-mer3′-CCCCCTTCCTAAG(N2,N2-Me2G)CACT18G*T-mer3′-CCCCCTTCCTAAG(N2,N2-Me2G)TACT Open table in a new tab The 5′-end of the 24-mer primer was labeled with [γ-32P]ATP using T4 polynucleotide kinase at 37 °C for 30 min. After removal of excess [γ-32P]ATP using a Bio-Spin 6 column (Bio-Rad), the labeled primer and unmodified (G) or modified (N2-MeG or N2,N2-Me2G) template (molar ratio 1:1) were heated at 95 °C for 5 min and then slowly cooled to room temperature to form the 24-mer/36-mer duplexes, which were used for all steady-state and pre-steady-state kinetic experiments. Standard DNA polymerization reactions with Dpo4 were carried out in 50 mm Tris-HCl buffer (pH 7.5 at 25 °C) containing 50 mm NaCl, 5 mm dithiothreitol, 100 μg of bovine serum albumin ml−1, and 5% (v/v) glycerol at 37 °C (42Choi J.Y. Guengerich F.P. J. Mol. Biol. 2005; 352: 72-90Crossref PubMed Scopus (80) Google Scholar, 43Choi J.Y. Guengerich F.P. J. Biol. Chem. 2006; 281: 12315-12324Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 44Choi J.Y. Angel K.C. Guengerich F.P. J. Biol. Chem. 2006; 281: 21062-21072Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 45Choi J.Y. Guengerich F.P. J. Biol. Chem. 2008; 283: 23645-23655Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). All reactions were initiated by mixing dNTP/MgCl2 (final MgCl2 concentration of 5 mm) solution to preincubated Dpo4/DNA mixtures. After reaction, 5-μl aliquots were quenched with EDTA/formamide solution (50 μl of 20 mm EDTA in 95% formamide (v/v) with 0.5% bromphenol blue (w/v) and 0.05% xylene cyanol (w/v)). Products were resolved using 20% polyacrylamide (w/v) denaturing gel electrophoresis (containing 8 m urea) and visualized and quantitated by phosphorimaging analysis using a Bio-Rad Molecular Imager FX instrument and Quantity One software. The primer was extended by adding four dNTPs (100 μm each) and 5 mm MgCl2 to the incubated mixture of 100 nm DNA and Dpo4 (0, 0.5, 2, or 10 nm) under standard assay conditions. After 20 min, reactions were quenched and processed as described above. Steady-state single-base incorporation experiments were performed by adding a single dNTP at varying concentrations (12 points) and MgCl2 to the Dpo4·DNA complexes, incubated in the reaction buffer. The molar ratio of Dpo4 to DNA was <0.1, and primer conversion to product was kept <20% by adjusting the polymerase concentration and incorporation time (53Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Crossref PubMed Scopus (403) Google Scholar). Reactions were quenched and products were analyzed and quantitated; graphs of the incorporation rates versus dNTP concentration were fit to a hyperbolic equation to yield kcat and Km values using nonlinear regression in GraphPad Prism version 3.0 (GraphPad, San Diego). Rapid quench experiments were performed in a model RQF-3 KinTek Quench Flow apparatus (KinTek, Austin, TX) with 50 mm Tris-HCl (pH 7.4) aqueous solution in the drive syringes. Reactions with excess DNA (including phosphorothioate analysis) were initiated by rapid mixing of 70 nm Dpo4, 120 nm DNA mixtures (12.5 μl) with 1 mm dNTP (or (Sp)-dCTPαS), 5 mm MgCl2 (10.9 μl). Reactions with excess Dpo4 were initiated by mixing 200 nm Dpo4, 100 nm DNA mixtures (12.5 μl) with dCTP (varying concentrations)·5 mm MgCl2 complex (10.9 μl). After the reactions were quenched by the addition of 0.6 m EDTA from the central syringe line after varying times, the products were analyzed and quantitated. The reactions with excess DNA or with excess Dpo4 were fit to Equations 1 or 2, respectively (where t is time), to obtain a burst amplitude A, burst rate kp, and steady-state velocity kss, y=A(1−e−kpt)+ksst(eq. 1) y=A(1−e−kpt)(eq. 2) Plots of kp versus dCTP concentration were fit to hyperbolic Equation 3, kp=kpol[dCTP]/([dCTP]+Kd,dCTP)(eq. 3) to estimate kpol and Kd,dCTP, where kpol is the maximal rate of nucleotide incorporation, and Kd,dCTP is an equilibrium dissociation constant for dCTP in the active form of the polymerase (54Patel S.S. Wong I. Johnson K.A. Biochemistry. 1991; 30: 511-525Crossref PubMed Scopus (472) Google Scholar, 55Johnson K.A. Methods Enzymol. 1995; 249: 38-61Crossref PubMed Scopus (194) Google Scholar). All nonlinear regression analysis used GraphPad Prism version 3.0. The primer was extended by incubating Dpo4 (5 μm), unlabeled DNA (10 μm), a mixture of all four dNTPs (1 mm each), and MgCl2 (5 mm) in Tris-HCl buffer (pH 7.5, final volume 100 μl) at 37 °C for 4 h. Each reaction was terminated by extraction of the remaining dNTPs using a Bio-Spin 6 chromatography column, and concentrated Tris-HCl, dithiothreitol, and EDTA were added to restore the concentrations to 50, 5, and 1 mm, respectively. E. coli uracil DNA glycosylase (20 units, Sigma) was then added; the solution was incubated at 37 °C for 6 h to hydrolyze the uracil residues on the extended primer and then heated at 95 °C for 1 h in the presence of 0.25 m piperidine, followed by removal of the solvent by in vacuo centrifugation. The dried sample was resuspended in 100 μl of H2O for mass spectrometry analysis. LC-MS/MS analysis was performed using a Waters Acquity UPLC system (Waters, Milford, MA) connected to a Finnigan LTQ mass spectrometer (Thermo Fisher Scientific, Waltham, MA) operating in the electrospray ionization negative ion mode. An Acquity UPLC BEH octadecylsilane (C18) column (1.7 μm, 1.0 × 100 mm) was used with the following LC conditions (all at 50 °C) with Buffer A (10 mm NH4CH3CO2 plus 2% CH3CN (v/v)) and Buffer B (10 mm NH4CH3CO2 plus 95% CH3CN (v/v)). The conditions used were similar to those reported previously (46Zhang H. Eoff R.L. Kozekov I.D. Rizzo C.J. Egli M. Guengerich F.P. J. Biol. Chem. 2009; 284: 3563-3576Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 57Eoff R.L. Angel K.C. Egli M. Guengerich F.P. J. Biol. Chem. 2007; 282: 13573-13584Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The calculations of the collision-induced dissociation fragmentations of oligonucleotide sequences were done using a program linked to the Mass Spectrometry Group (Medicinal Chemistry) of the University of Utah. Attempts to crystallize Dpo4 in the presence of N2,N2-Me2G-substituted primer-template DNA and an incoming dCTP were unsuccessful. However, we did obtain diffraction quality crystals for three complexes. For correct pairing opposite N2,N2-Me2G (Dpo4 + N2,N2-Me2G·14Cdd + dGTP, post-insertion complex, DMG-1), the DNA duplex contained the 18G*C-mer template and the 14Cdd-mer primer, with Cdd being a dideoxy residue (Table 1). The Dpo4/DNA mixture (1:1.2 molar ratio, in 20 mm Tris-HCl buffer (pH 7.5, 25 °C) containing 60 mm NaCl, 4% glycerol (v/v), and 5 mm β-mercaptoethanol) was placed on ice for 1 h prior to incubation with 5 mm MgCl2 and 1 mm dGTP. The final Dpo4 concentration was 8–9 mg ml−1 for all three setups. The crystals were grown using a sitting drop, vapor-diffusion method with the reservoir solution containing 10–15% polyethylene glycol 3350 (w/v), 30 mm NaCl, 100 mm MgCl2, and 3% glycerol (v/v). Droplets consisted of a 1:0.5 or 1:1.5 (v/v) mixture of the Dpo4·DNA·Mg2+·dGTP complex and the reservoir solutions and were equilibrated against the reservoir solutions. A complex of Dpo4 bound to N2,N2-Me2G-substituted primer-template DNA (Dpo4-N2,N2-Me2G·14Cdd, binary com