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• W2000553427 abstract "We examined the effect of a single O6-methylguanine (O6-MeG) template residue on catalysis by a model Y family polymerase, Dpo4 from Sulfolobus solfataricus. Mass spectral analysis of Dpo4-catalyzed extension products revealed that the enzyme accurately bypasses O6-MeG, with C being the major product (∼70%) and T or A being the minor species (∼20% or ∼10%, respectively), consistent with steady-state kinetic parameters. Transient-state kinetic experiments revealed that kpol, the maximum forward rate constant describing polymerization, for dCTP incorporation opposite O6-MeG was ∼6-fold slower than observed for unmodified G, and no measurable product was observed for dTTP incorporation in the pre-steady state. The lack of any structural information regarding how O6-MeG paired in a polymerase active site led us to perform x-ray crystallographic studies, which show that “wobble” pairing occurs between C and O6-MeG. A structure containing T opposite O6-MeG was solved, but much of the ribose and pyrimidine base density was disordered, in accordance with a much higher Km,dTTP that drives the difference in efficiency between C and T incorporation. The more stabilized C:O6-MeG pairing reinforces the importance of hydrogen bonding with respect to nucleotide selection within a geometrically tolerant polymerase active site. We examined the effect of a single O6-methylguanine (O6-MeG) template residue on catalysis by a model Y family polymerase, Dpo4 from Sulfolobus solfataricus. Mass spectral analysis of Dpo4-catalyzed extension products revealed that the enzyme accurately bypasses O6-MeG, with C being the major product (∼70%) and T or A being the minor species (∼20% or ∼10%, respectively), consistent with steady-state kinetic parameters. Transient-state kinetic experiments revealed that kpol, the maximum forward rate constant describing polymerization, for dCTP incorporation opposite O6-MeG was ∼6-fold slower than observed for unmodified G, and no measurable product was observed for dTTP incorporation in the pre-steady state. The lack of any structural information regarding how O6-MeG paired in a polymerase active site led us to perform x-ray crystallographic studies, which show that “wobble” pairing occurs between C and O6-MeG. A structure containing T opposite O6-MeG was solved, but much of the ribose and pyrimidine base density was disordered, in accordance with a much higher Km,dTTP that drives the difference in efficiency between C and T incorporation. The more stabilized C:O6-MeG pairing reinforces the importance of hydrogen bonding with respect to nucleotide selection within a geometrically tolerant polymerase active site. Of the myriad forms that covalent modification of DNA can take, alkylation of the purine/pyrimidine bases is one of the most extensively studied (1Loveless A. Nature. 1969; 223: 206-207Crossref PubMed Scopus (900) Google Scholar, 2Lawley P.D. Searle C.E. Chemical Carcinogens. 2nd Ed. American Chemical Society, Washington, D. C.1984: 325-484Google Scholar). The term “alkylating agent” encompasses a variety of known carcinogenic chemicals ranging from the spontaneously reactive nitrogen and sulfur mustards (e.g. mechlorethamine) and N-alkyl-N-nitrosoureas to metabolically activated compounds such as cyclophosphamide and N-nitrosamines (3Wheeler G.P. Cancer Res. 1962; 22: 651-688PubMed Google Scholar). Guanine is generally considered the most easily oxidized of the bases, and the N7 position is the most nucleophilic atom of guanine. One prevalent form of guanine oxidation occurs at the O6 position, with a simple and widely studied process being addition of a methyl group to form O6-MeG 2The abbreviations used are: MeG, methylguanine; CID, collision-induced dissociation; dCTPαS, 2′-deoxycytidine 5′-O-(1-thiotriphosphate); DTT, di-thiothreitol; LC, liquid chromatography; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight; MS, mass spectrometry; pol, (DNA) polymerase; pol T7-, bacteriophage pol T7 exonuclease-deficient; RT, reverse transcriptase; HIV, human immunodeficiency virus. (4Margison G.P. Santibanez Koref M.F. Povey A.C. Mutagenesis. 2002; 17: 483-487Crossref PubMed Scopus (171) Google Scholar). Methylation of the O6 atom results in alternate pairing schemes that include a O6-MeG:C “wobble” pairing and a pseudo-“Watson-Crick” O6-MeG:T pair (Fig. 1), and the relevance of O6-MeG to mutagenesis is well established (5Delaney J.C. Essigmann J.M. Biochemistry. 2001; 40: 14968-14975Crossref PubMed Scopus (47) Google Scholar, 6Dodson L.A. Foote R.S. Mitra S. Masker W.E. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7440-7444Crossref PubMed Scopus (49) Google Scholar, 7Dosanjh M.K. Singer B. Essigmann J.M. Biochemistry. 1991; 30: 7027-7033Crossref PubMed Scopus (63) Google Scholar). Cells can repair O6-MeG by recognition and/or removal of the lesion through either the mismatch repair pathway or through the actions of (O6-alkylguanine DNA alkyltransferase) (8Pegg A.E. Roberfroid M. von Bahr C. Foote R.S. Mitra S. Bresil H. Likhachev A. Montesano R. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 5162-5165Crossref PubMed Scopus (136) Google Scholar, 9Pegg A.E. Dolan M.E. Scicchitano D. Morimoto K. Environ. Health Perspect. 1985; 62: 109-114Crossref PubMed Scopus (56) Google Scholar, 10Yoshioka K. Yoshioka Y. Hsieh P. Mol. Cell. 2006; 22: 501-510Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). O6-MeG is even observed in the DNA of the general population, although the level measured between studies has varied (4Margison G.P. Santibanez Koref M.F. Povey A.C. Mutagenesis. 2002; 17: 483-487Crossref PubMed Scopus (171) Google Scholar, 11Georgiadis P. Samoli E. Kaila S. Katsouyanni K. Kyrtopoulos S.A. Cancer Epidemiol. Biomarkers Prev. 2000; 9: 299-305PubMed Google Scholar). Substantially increased levels of O6-MeG are found in patients treated with chemotherapeutic regimes that include methylating agents (4Margison G.P. Santibanez Koref M.F. Povey A.C. Mutagenesis. 2002; 17: 483-487Crossref PubMed Scopus (171) Google Scholar, 12Kyrtopoulos S.A. Souliotis V.L. Valavanis C. Boussiotis V.A. Pangalis G.A. Environ. Health Perspect. 1993; 99: 143-147PubMed Google Scholar, 13Middleton M.R. Lee S.M. Arance A. Wood M. Thatcher N. Margison G.P. Int. J. Cancer. 2000; 88: 469-473Crossref PubMed Scopus (63) Google Scholar). Of the enzymes associated with what has commonly been referred to as “translesion synthesis,” the Y-family DNA polymerases are thought to represent the major constituent present during post-replication repair of covalently modified DNA (14Langston L.D. O'Donnell M. Mol. Cell. 2006; 23: 155-160Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 15Ohmori H. Friedberg E.C. Fuchs R.P. Goodman M.F. Hanaoka F. Hinkle D. Kunkel T.A. Lawrence C.W. Livneh Z. Nohmi T. Prakash L. Prakash S. Todo T. Walker G.C. Wang Z. Woodgate R. Mol. Cell. 2001; 8: 7-8Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar, 16Plosky B.S. Woodgate R. Curr. Opin. Genet. Dev. 2004; 14: 113-119Crossref PubMed Scopus (98) Google Scholar). Four human Y-family polymerases are known (η, ι, κ, and Rev1), and representatives also occur in other eukaryotic, archaeal, and prokaryotic systems (17Prakash S. Johnson R.E. Prakash L. Annu. Rev. Biochem. 2005; 74: 317-353Crossref PubMed Scopus (830) Google Scholar). Current models for translesion synthesis across damaged DNA during replication propose a dynamic exchange between two general groups of polymerases, namely the high fidelity replicative polymerases that perform the vast majority of incorporation events and the Y-family enzymes (14Langston L.D. O'Donnell M. Mol. Cell. 2006; 23: 155-160Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 16Plosky B.S. Woodgate R. Curr. Opin. Genet. Dev. 2004; 14: 113-119Crossref PubMed Scopus (98) Google Scholar). In mammalian systems the coordination of the four Y-family polymerases, at sites of damage or otherwise, is less than clear at this point. For all of these reasons, the one or more mechanisms by which specialized polymerases bypass damaged DNA is an area of intense focus. Several crystal structures of the Dpo4 DNA polymerase from Sulfolobus solfataricus in complex with covalently modified DNA have served as a major source of structural information regarding how Y-family polymerases bypass damaged DNA templates (18Ling H. Boudsocq F. Plosky B.S. Woodgate R. Yang W. Nature. 2003; 424: 1083-1087Crossref PubMed Scopus (202) Google Scholar, 19Ling H. Boudsocq F. Woodgate R. Yang W. Cell. 2001; 107: 91-102Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, 20Ling H. Sayer J.M. Plosky B.S. Yagi H. Boudsocq F. Woodgate R. Jerina D.M. Yang W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2265-2269Crossref PubMed Scopus (163) Google Scholar, 21Ling H. Boudsocq F. Woodgate R. Yang W. Mol. Cell. 2004; 13: 751-762Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 22Zang 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, 23Zang H. Irimia A. Choi J.Y. Angel K.C. Loukachevitch L.V. Egli M. Guengerich F.P. J. Biol. Chem. 2006; 281: 2358-2372Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Rigorous kinetic analysis of Dpo4 catalysis performed with unmodified DNA indicates that the enzyme bears all of the hallmarks of a “translesion” polymerase, namely low efficiency (“low” kpol and “high” KD,dCTP), low processivity (∼16 incorporation events prior to dissociation), and low “fidelity” (one mistake every few thousand insertions) (24Fiala K.A. Suo Z. Biochemistry. 2004; 43: 2116-2125Crossref PubMed Scopus (111) Google Scholar, 25Fiala K.A. Suo Z. Biochemistry. 2004; 43: 2106-2115Crossref PubMed Scopus (110) Google Scholar). However, within the context of the cell these attributes are not at all surprising, because copying undamaged DNA does not appear to be the major function of these enzymes. An investigation of Dpo4-catalyzed bypass of a ubiquitous product of oxidative damage, 7,8-dihydro-8-oxodeoxyguanosine, revealed that Dpo4 efficiency is increased ∼2-fold during lesion bypass (23Zang H. Irimia A. Choi J.Y. Angel K.C. Loukachevitch L.V. Egli M. Guengerich F.P. J. Biol. Chem. 2006; 281: 2358-2372Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The increased catalytic efficiency is in direct contrast to results obtained with T7- and other high fidelity polymerases, where catalysis is, in general, greatly inhibited for both C and A incorporation events (26Furge L.L. Guengerich F.P. Biochemistry. 1997; 36: 6475-6487Crossref PubMed Scopus (97) Google Scholar, 27Furge L.L. Guengerich F.P. Biochemistry. 1998; 37: 3567-3574Crossref PubMed Scopus (33) Google Scholar). In the present study, transient-state kinetic approaches were combined with mass spectral analysis of incorporation/extension products and x-ray crystallography. The results clearly illustrate that Dpo4 favors C incorporation followed by correct extension of at least 4 bp, with T and some A incorporations occurring as minor products. The rate-constant defining Dpo4-catalyzed incorporation of dCTP, kpol, is ∼6-fold slower for incorporation opposite O6-MeG relative to G. The basis for the decreased rate was revealed by the crystal structure to be formation of a wobble base pairing between O6-MeG and C. From these results some of the mechanistic distinctions between polymerase subfamilies and the subsequent influence of those distinctions upon whether C or T is paired opposite O6-MeG become apparent. Materials—Dpo4 was expressed in Escherichia coli and purified to electrophoretic homogeneity as described previously (25Fiala K.A. Suo Z. Biochemistry. 2004; 43: 2106-2115Crossref PubMed Scopus (110) Google Scholar). All unlabeled dNTPs were obtained from Amersham Biosciences (Piscataway, NJ), Sp-dCTPαS was purchased from Biolog Life Science Institute (Bremen, Germany), and [γ-32P]ATP was purchased from PerkinElmer Life Sciences. All oligonucleotides used in this work were synthesized by Midland Certified Reagent Co. (Midland, TX) and purified using high-performance liquid chromatography by the manufacturer, with analysis by matrix-assisted laser desorption time-of-flight MS. The 13-base primer sequence used in the kinetic and mass spectral analyses was 5′-GGGGGAAGGATTC-3′. The 14-base primer sequences used in the indicated kinetic assays and the crystal structures was 5′-GGGGGAAGGATTCC-3′ for the O6-MeG:C structure and 5′-GGGGGAAGGATTCT-3′ for the O6-MeG:T structure. The template DNA sequence used in all of the kinetic and mass spectral assays and in the O6-MeG:C and O6-MeG:dATP structures was 5′-TCATXGAATCCTTCCCCC-3′, where X = Gor O6-MeG, as indicated. A second template sequence, used for the O6-MeG:T structure, was 5′-TCACXGAATCCTTCCCCC-3′, where X = O6-MeG. Polymerization Assays and Gel Electrophoresis—A 32P-labeled primer, annealed to either an unmodified or adducted template oligonucleotide, was extended in the presence of the indicated dNTP(s). Each reaction was initiated by adding 2 μl of dNTP·Mg2+ (250 μm dNTP and 5 mm MgCl2) solution to a preincubated Dpo4·DNA complex (25-100 nm). The reaction was carried out at 37 °C in Tris-HCl (pH 7.8 at 22 °C) buffer containing 50 mm NaCl, 1.0 mm DTT, and 50 μg μl-1 bovine serum albumin. At the indicated time, 5-μl aliquots were quenched with 50 μl of 500 mm EDTA, pH 9.0. The samples were then mixed with 100 μl of a 95% formamide/20 mm EDTA solution and were separated on a 20% polyacrylamide (w/v)/7 m urea gel. Products were visualized and quantified using a phosphorimaging screen and Quantity One™ software, respectively (Bio-Rad, Hercules, CA). Formation of an 18-base extension product from a 13-base primer was quantified by fitting the data to Equation 1, f18mer(t)=A1-∑r=1n((kobs)t)r-1(r-1)!e-(kobs)t+k2t(Eq. 1) where A is the amount of product formed during the first binding event between Dpo4 and DNA, kobs is the an observed rate constant defining nucleotide incorporation, n is the number of incorporation events required to observe product formation, k2 is the steady-state rate of nucleotide incorporation, and t is time. All statistical values given indicate the standard error. Steady-state Kinetics—Dpo4-catalyzed single nucleotide incorporation was measured over a range of dNTP concentrations. All reactions were carried out at 37 °C in 50 mm Tris-HCl (pH 7.8 at 25 °C) buffer containing 50 mm NaCl, 1.0 mm DTT, 50 μg μl-1 bovine serum albumin, and 5% glycerol (v/v). Dpo4 (10 nm) was preincubated with DNA (100 nm), and the reaction was initiated by adding dNTP·Mg2+. Aliquots were quenched with 500 mm EDTA (pH 9.0) after varying incubation times. The initial portion of the velocity curve was fit to a linear equation in the program GraphPad Prism (GraphPad, San Diego, CA). The resulting velocity was plotted as a function of dNTP concentration and then fit to a hyperbola, correcting for enzyme concentration to obtain an estimate of kcat and Km,dNTP. Pre-steady-state Kinetics—All pre-steady-state experiments were performed using a KinTek RQF-3 model chemical quench-flow apparatus (KinTek Corp., Austin, TX) with 50 mm Tris-HCl (pH 7.8 at 25 °C) buffer in the drive syringes. Initially, all RQF experiments were carried out at 37 °C in a buffer containing 50 mm Tris-HCl, pH 7.8 (at 25 °C), 50 mm NaCl, 5 mm DTT, 100 μg μl-1 bovine serum albumin, and 5% (v/v) glycerol. Subsequent experiments indicated that increasing the concentration of glycerol in the reaction mixture resulted in considerably more product in the first binding event for Dpo4-catalyzed incorporation of dCTP opposite O6-MeG (supplemental Fig. S4). Therefore, the pre-steady-state reactions were repeated using reaction buffer containing 35% glycerol (v/v). Polymerase catalysis was stopped via addition of 500 mm EDTA (pH 9.0). Where indicated, competitor primer/template DNA (1 μm 13/18-mer) was included in the right syringe as a trap for protein, thereby creating single-turnover conditions even under enzyme limiting conditions. Substrate and product DNA was separated by electrophoresis on a 20% polyacrylamide (w/v)/7 m urea gel. The products were then visualized using phosphorimaging and quantitated using Quantity One™ software (Bio-Rad). Results obtained under single-turnover conditions were fit to Equation 2, y=A(1-e-kobst)(Eq. 2) where A is the product formed in first binding event, kobs is the rate constant defining polymerization under the conditions used for the experiment being analyzed, and t is time. Results obtained under conditions that allowed a second round of Dpo4·DNA binding and polymerase action were fit to Equation 3, y=A(1-e-kobst)+ksst(Eq. 3) where kss represents a steady-state velocity of nucleotide incorporation. Liquid Chromatography Mass Spectrometry Analysis of Oligonucleotide Products from Dpo4 Reactions—Dpo4 (5 μm) was preincubated with primer/template DNA (10 μm), and the reaction was initiated by addition of dNTP (1 mm each) and Mg2+ (5 mm) for a final volume of 100 μl. Dpo4 catalysis was allowed to proceed at 37 °C for 4 h in 50 mm Tris-HCl (pH 7.8 at 25 °C) buffer containing 50 mm NaCl, 1 mm DTT, 50 μg μl-1 bovine serum albumin, and 5% glycerol (v/v). The reaction was terminated by extraction of the remaining dNTPs by using a size-exclusion chromatography column (Bio-Spin 6 chromatography column, Bio-Rad). Concentrated stocks of Tris-HCl, DTT, and EDTA were added to restore the concentrations to 50 mm, 5 mm, and 1 mm, respectively. Next, E. coli uracil DNA glycosylase (20 units, Sigma-Aldrich) was added and the solution was incubated at 37 °C for 6 h to hydrolyze the uracil residue on the extended primer (22Zang 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). The reaction mixture was then heated at 95 °C for 1 h in the presence of 0.25 m piperidine, followed by removal of the solvent by centrifugation under vacuum. The dried sample was re-suspended in 100 μl of H2O for MS analysis. LC-MS/MS analysis was performed on a Waters Aquity ultraperformance liquid chromatography system (Waters, Milford, MA) connected to a Finnigan LTQ mass spectrometer (ThermoElectron Corp., San Jose, CA), operating in the electrospray ionization negative ion mode. An Aquity ultraperformance liquid chromatography BEH octadecylsilane (C18) column (1.7 μm, 1.0 mm × 100 mm) was used with the following LC conditions: buffer A contained 10 mm NH4CH3CO2 plus 2% CH3CN (v/v), and buffer B contained 10 mm NH4CH3CO2 plus 95% CH3CN (v/v). The following gradient program was used with a flow rate of 150 μl min-1: 0-3 min, linear gradient from 100% A to 97%A/3% B (v/v); 3-4.5 min, linear gradient to 80% A/20% B (v/v); 4-5.5 min, linear gradient to 100% B; 5-5.5 min, hold at 100% B; 5.5-6.5 min, linear gradient to 100% A; 6.5-9.5 min, hold at 100% A. The temperature of the column was maintained at 50 °C. Samples were infused with an autosampler system. Electrospray ionization conditions were as follows: source voltage, 4 kV; source current, 100 μA; auxillary gas flow rate setting, 20; sweep gas flow rate setting, 5; sheath gas flow setting, 34; capillary voltage, -49 V; capillary temperature, 350 °C; and tube lens voltage, -90 V. MS/MS conditions were as follows: normalized collision energy, 35%; activation Q, 0.250; and activation time, 30 ms. Product ion spectra were acquired over the range m/z 345-2000. The doubly (negatively) charged species were generally used for CID analysis. The calculations of the CID fragmentations of oligonucleotide sequences were done using a program linked to the Mass Spectrometry Group of Medicinal Chemistry at the University of Utah (www.medlib.med.utah.edu/massspec). The nomenclature used in supplemental Tables S1-S3 has been described previously (28Christian N.P. Reilly J.P. Mokler V.R. Wincott F.E. Ellington A.D. J. Am. Soc. Mass Spectrom. 2001; 12: 744-753Crossref PubMed Scopus (25) Google Scholar). Crystallization of Dpo4·DNA Complexes—Dpo4 was concentrated to ∼300-550 μm (∼12-22 mg ml-1) using a spin concentrator with a 104 Mr cutoff filter (Amicon) in 50 mm Tris-HCl (pH 7.4 at 25 °C) buffer containing 200 mm NaCl, 5 mm β-mercaptoethanol, and 10% glycerol (v/v). Dpo4 was then mixed with DNA (1:1.2 molar ratio), incubated at 37 °C for 10 min, centrifuged at 104 rpm for 5 min (Eppendorf, centrifuge 5415C) to remove insoluble material, and then placed on ice for 1 h prior to incubation with 1 mm d(N)TP and 5 mm CaCl2. Crystals were grown using the sitting drop, vapor-diffusion method by mixing 1 μl of complex with 1 μl of solution containing 5-10% polyethylene glycol 3350 (w/v), and 100 mm Ca(OAc)2, and equilibrated against a well solution containing 25 mm Tris-HCl (pH 7.4 at 25 °C) buffer, 5-10% polyethylene glycol 3350 (w/v), 100 mm Ca(OAc)2, and 2.5% glycerol (v/v). Crystals were soaked in mother liquor containing an additional 25% polyethylene glycol 3350 (w/v) and 15% ethylene glycol (v/v), and then swiped through paratone-N (Hampton Research, Aliso Viejo, CA) and flash frozen in a stream of liquid nitrogen. X-ray Diffraction Data Collection and Processing—Diffraction data sets for Dpo4 ternary O6-MeG:C and O6-MeG:dATP complexes were collected at 100 K using a radiation wavelength of 1.54 Å on a Bruker Microstar (Bruker AXS, Madison, WI) system housed in the Center for Structural Biology at Vander-bilt. Data sets for the O6-MeG:T were collected at 110 K using synchrotron radiation wavelength of 0.98 Å on the X25 beam-line at the National Synchrotron Light Source, Brookhaven, NY. Indexing and scaling were performed using HKL2000 (29Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38570) Google Scholar). All three structures indexed to the same space group and had very similar unit cell parameters. Structure Determination and Refinement—The refined Dpo4-dG model (23Zang H. Irimia A. Choi J.Y. Angel K.C. Loukachevitch L.V. Egli M. Guengerich F.P. J. Biol. Chem. 2006; 281: 2358-2372Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) was used as a starting model for the O6-MeG:C structure, the refined O6-MeG:C model was used and the starting model for the O6-MeG:T structure, and the Dpo4-2 model (22Zang 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) was used as a starting model for the O6-MeG:dATP structure. In each instance, several rounds of rigid body refinement of the diffraction data, with gradually increasing resolution, optimized the initial positions of the models. The model was refined further using the CNS Solve package (version 1.1) (30Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar), including simulated annealing, gradient minimization, and refinement of individual isotropic temperature factors. Individual occupancy refinement was necessary to establish the final model for the active site thymidine residue in the O6-MeG:T structure. Manual model building was performed using TURBO. 3C. Cambillau and A. Roussel (1997) Turbo Frodo, Version OpenGL. 1, Université Aix-Marseille II, Marseille, France. Extension of Oligonucleotide Primers by Dpo4 in the Presence of All Four dNTPs—A time course performed under enzyme-limiting conditions provides a general measure of how Dpo4 catalysis is affected by O6-MeG (Fig. 2). An observed rate constant defining five incorporation events can be measured by following the appearance of the fully extended 18-mer primer and fitting the data to Equation 1, where n = 5. The amount of product formed in the first binding event (i.e. the “burst” amplitude) is diminished by roughly 25% relative to what is observed with the DNA control indicating that fewer Dpo4 molecules are able to fully extend the primer in the first attempt (Fig. 2C). It is important to note that, in the absence of any other evidence (see below), the exact identity of the fully extended product is unknown. Dpo4 Catalysis in the Presence of a Single dNTP—In the next set of experiments, Dpo4 catalysis was allowed to proceed in the presence of a single nucleotide. Dpo4 can incorporate each of the four dNTPs across from O6-MeG (supplemental Fig. S1). Steady-state kinetic assays were then employed as a first quantitative measure of the preferential mechanism for Dpo4 insertion opposite O6-MeG. The relative catalytic efficiency of nucleotide incorporation by Dpo4 was determined by varying the concentration of dNTP in the reaction solution (Table 1). Dpo4-catalyzed incorporation of dCTP opposite O6-MeG is inhibited ∼103-fold relative to unmodified DNA, but the enzyme is ∼3-, ∼6-, and ∼14-fold more efficient at correct incorporation of C opposite O6-MeG compared with incorrect incorporation of T, A, and G, respectively.TABLE 1Steady-state kinetic parameters for one-base incorporation by Dpo4Oligomer pairPrimer-template pairdNTPkcatKm,dNTPΔEfficiency relative to dCTP:Gs−1μm13-merdCTP0.58 ± 0.013.0 ± 0.218-mer-1-G-13-merdCTP0.071 ± 0.005340 ± 70950-fold less18-mer-1-O6MeG-13-merdTTP0.11 ± 0.012100 ± 5003600-fold less18-mer-1-G-13-merdTTP0.088 ± 0.0031200 ± 1202600-fold less18-mer-1-O6MeG-13-merdATP0.012 ± 0.001225 ± 223600-fold less18-mer-1-G-13-merdATP0.0022 ± 0.000171 ± 166000-fold less18-mer-1-O6MeG-13-merdGTP0.012 ± 0.001760 ± 13012000-fold less18-mer-1-G-13-merdGTP0.008 ± 0.001590 ± 7013500-fold less18-mer-1-O6MeG- Open table in a new tab LC-MS/MS Analysis of Full-length Extension Products—Unambiguous identification of full-length extension products resulting from Dpo4 catalysis was carried out as described previously (23Zang H. Irimia A. Choi J.Y. Angel K.C. Loukachevitch L.V. Egli M. Guengerich F.P. J. Biol. Chem. 2006; 281: 2358-2372Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), with slight modifications that are described under “Experimental Procedures.” MS results for Dpo4-catalyzed incorporation opposite and extension past O6-MeG modified template DNA are summarized in Fig. 3. Two major ions were observed at m/z 1078.6 and 719.1, corresponding with the -2 and -3 ions, respectively (Fig. 3B). The total ion trace for the m/z 1079 ion MS/MS is shown in Fig. 3C, and CID analysis of the m/z 1079 ion resulted in the fragmentation pattern shown in Fig. 3D. The major ions in the fragmentation pattern are consistent with the sequence, 5′-pTCCATGA-3′ (supplemental Table S1), which corresponds to the insertion of C opposite O6-MeG and accurate full-length extension of the primer. A second pair of ions was detected at m/z 1086.1 and 723.8, both of which are consistent with the -2 and -3 charge states of a parent ion representing T insertion opposite O6-MeG followed by accurate full-length extension. CID provided a fragmentation pattern consistent with this sequence assignment (supplemental Fig. S2 and Table S2). The third ion pair, 1090.7 and 726.8, was identified as A insertion products, again followed by accurate full-length extension (supplemental Fig. S3 and Table S3). Comparison of the selected ion counts (for the ions corresponding to all three products) indicates that correct incorporation of C opposite O6-MeG comprises roughly 70% of the full-length extension products observed in the reaction mixture. Misincorporation of T accounted for ∼20% of the products, and A accounted for the remaining ∼10%, consistent with the steady-state parameters. Transient-state Kinetic Analysis for Dpo4 Bypass of O6-MeG—Pre-steady-state experiments were performed under enzyme-limiting conditions in the presence of dCTP alone. The presence of O6-MeG in the template strand reduces the amount of product generated in the first catalytic turnover by roughly 3-fold under the experimental conditions used here (Fig. 4). The kobs value is 4.4-fold slower for incorporation opposite O6-MeG compared with undamaged DNA at this particular concentration of nucleoside triphosphate (1 mm dCTP). Previous studies suggest that phosphoryl transfer (i.e. the “chemistry” step) is not the rate-limiting step that defines correct dNTP incorporation by Dpo4 (24Fiala K.A. Suo Z. Biochemistry. 2004; 43: 2116-2125Crossref PubMed Scopus (111) Google Scholar). One approach to determining the overall contribution of “chemistry” to the polymerase catalytic cycle involves substituting sulfur for one of the oxygen atoms in the α-phosphate group. In principle, substitution of oxygen with a less electronegative sulfur atom makes bond breakage (and subsequent phosphoryl transfer) more difficult. If phosphoryl transfer is the rate-limiting step in the multistep polymerase catalytic cycle then the sulfur substitution experiment will exhibit a decreased rate of nucleotide incorporation, although interpretation of such changes are a matter of some debate (32Joyce C.M. Benkovic S.J. Biochemistry. 2004; 43: 14317-14324Crossref PubMed Scopus (281) Google Scholar). The measured reduction in kobs upon substitution of sulfur for oxygen (“thio” effect) for unmodified G was ∼1.9 (Fig. 4A), which is similar to previous phosphorothioate substitution experiments with Dpo4 that resulted in a thio effect of ∼1.4 (24Fiala K.A. Suo Z. Biochemistry. 2004; 43: 2116-2125Crossref PubMed Scopus (111) Goo" @default.
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• W2000553427 title "Sulfolobus solfataricus DNA Polymerase Dpo4 Is Partially Inhibited by “Wobble” Pairing between O6-Methylguanine and Cytosine, but Accurate Bypass Is Preferred" @default.
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