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J. Biol. Chem., Vol. 281, Issue 4, 2358-2372, January 27, 2006
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From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
Received for publication, October 5, 2005 , and in revised form, November 16, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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95% incorporation of dCTP > dATP opposite 8-oxoG. Pre-steady-state kinetics showed faster rates of dCTP incorporation opposite 8-oxoG than G. The measured Kd,dCTP was 15-fold lower for an oligonucleotide containing 8-oxoG than with G. Extension beyond an 8-oxoG:C pair was similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. The Ea for dCTP insertion opposite 8-oxoG was lower than for opposite G. Crystal structures of Dpo4 complexes with oligonucleotides were solved with C, A, and G nucleoside triphosphates placed opposite 8-oxoG. With ddCTP, dCTP, and dATP the phosphodiester bonds were formed even in the presence of Ca2+. The 8-oxoG:C pair showed classic Watson-Crick geometry; the 8-oxoG:A pair was in the syn:anti configuration, with the A hybridized in a Hoogsteen pair with 8-oxoG. With dGTP placed opposite 8-oxoG, pairing was not to the 8-oxoG but to the 5' C (and in classic Watson-Crick geometry), consistent with the low frequency of this frameshift event observed in the catalytic assays. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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8-OxoG2 is a major lesion arising from oxidative stress in lower organisms and eukaryotes, although the actual reported levels vary considerably because of artifacts in measurement (7, 8). This lesion is of interest not only in consideration of cancer (9, 10) but has also been associated with aging (11), infertility (12), and hepatitis (13). Although 8-oxoG can be oxidized further to spiro products that are even more mutagenic in some assay systems (14, 15), the relevance of those products is unclear in that they remain to be identified in vivo, with the exception of base excision repair-deficient Escherichia coli exposed to high concentrations of chromate (16).
The molecular basis of miscoding by 8-oxoG has been the subject of investigation since the discovery of this lesion in DNA. Originally the lesion was reported as 8-hydroxyguanine; NMR studies (17) defined the structure as the tautomer 8-oxoG, with chemistry of the N-7 atom changed from that of a nucleophile to an amide nitrogen. In all biological systems examined, a proclivity for binding to or insertion of A is observed. The Tm for the 8-oxoG:A pair is 
6 °C lower than a normal T:A pair, but the Tm for the 8-oxoG:C pair in the same context is 35 °C lower than for a G:C base pair (18). Thus one might expect pairing of an A with 8-oxoG to be favored over that of a C (with 8-oxoG) from thermodynamic considerations. NMR and x-ray diffraction studies of 8-oxoG pairing in oligonucleotides have been reported, and the similarity of an 8-oxoG:A pair to a T:A pair has been noted (18–20). Another feature of the binding of 8-oxoG to A in double-stranded oligonucleotides is the anti to syn shift of the 8-oxoG glycosidic bond, yielding a flip of the base to change the position of the atoms and facilitate pairing to A.
Although the insertion of A opposite 8-oxoG is the most common outcome (and subsequently yields a G to T transition), the biological response varies considerably (21). In early work in the field, Shibutani et al. (22) noted considerable variation among polymerases in their tendencies to insert dATP versus dCTP. Although the parameters used in the literature vary, the extent of misincorporation ranges from about 5 to 95% among different DNA polymerases (22–30). On the basis of steady-state kinetic parameters, E. coli polymerases I and II (exo-), bacteriophage pol T7, rat pol 
, and bovine pol 
insert dATP opposite 8-oxoG in 30–50% of the events (23–25, 28, 29, 31), and DNA polymerase pol 
(22), human immunodeficiency virus type 1 reverse transcriptase (24), and Bacillus stearothermophilus pol BF (27) are particularly prone to insert dATP > dCTP. Of the DNA polymerases reported, only RB69 (26) and Saccharomyces cerevisiae pol 
(30, 32) insert dCTP in 95% of the incorporation events instead of dATP. Furthermore, the patterns of incorporation of 8-oxo-dGTP opposite template C versus A do not match the patterns of incorporation of dCTP versus dATP opposite 8-oxoG, indicating the asymmetry of the system (25).
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(32). Four x-ray structures of DNA polymerases bound to oligonucleotides containing 8-oxoG have been reported to date (26–29), and some details of these vary, e.g. the localized inversion of the dCTP phosphate in pol (28).
Most of the work with 8-oxoG done to date has been with replicative DNA polymerases and relatively little has been done with the more recently discovered translesion polymerases, except for the yeast pol studies already mentioned (30, 32). A recent Carlson and Washington (30) article with S. cerevisiae pol is of note in that pre-steady-state kinetics indicated that the efficiency (kpol/Kd) for dCTP opposite 8-oxoG was still 50% that of the opposite G, which is unusual compared with our own work with replicative polymerases (23, 24, 31). Archebacter DNA polymerase Dpo4 is a model for translesion synthesis (for a recent discussion of mechanisms, see Ref. 35). We analyzed the interaction of Dpo4 with 8-oxoG using MS, steady-state and pre-steady-state kinetics, and x-ray crystallography. Although the translesion DNA polymerases are often assumed to be rather error prone, we find that Dpo4 shows high fidelity in inserting dCTP opposite 8-oxoG and that this pair is very efficiently extended. Consistent with the activity data, the crystal structures of ternary complexes of Dpo4 with dCTP and ddCTP opposite 8-oxoG reveal standard Watson-Crick geometry of the pairs. Conversely, dATP forms a Hoogsteen pair with 8-oxoG, with the latter adopting syn geometry. All three complexes belong to the "Type I" crystal form described for the ternary complexes with native DNA (36). Thus, 8-oxoG constitutes the single template base present at the active site. However, in both complexes with dCTP and ddCTP the primer was extended by a single C and, in the complex with dATP, the structural data indicate the presence of a second nucleoside triphosphate immediately adjacent to the active site. The complexes with dGTP and ddGTP belong to the "Type II" form (36), with two template bases present at the active site, whereby 8-oxoG is skipped and the nucleoside triphosphates engage in a Watson-Crick base pair with the C 5'-adjacent to the modified base. This arrangement is similar to that found in the ternary complex of dGTP with a template-primer construct containing 1,N2-ethenodeoxyguanosine (37) and helps to rationalize the low frequency frameshift with G opposite 8-oxoG in the in vitro primer extension studies.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Polymerization Assays and Gel Electrophoresis—A 32P-labeled primer, annealed to either an unmodified or adducted template, was extended in the presence of the individual dNTPs (Scheme 1). In the preliminary "run-on" assays, each reaction was initiated by adding 2 µl of dNTP-Mg2+ solution (final concentrations of 250 µM of each dNTP and 5 mM MgCl2) to a preincubated E·DNA complex (final concentrations of 50 mM Tris-HCl (pH 7.8), 100 nM DNA duplex, 100 nM Dpo4, 5 mM DTT, 50 µg of bovine serum albumin ml-1, 50 mM NaCl, and 5% glycerol (v/v)) at 37 °C, yielding a total reaction volume of 8 µl. After varying lengths of time, reactions were quenched with 50 µl of 20 mM EDTA (pH 9.0) in 95% formamide (v/v).
Aliquots (3 µl) were separated by electrophoresis using denaturing gels containing 8.0 M urea and 16% acrylamide (w/v) (from a 19:1 acrylamide:bisacrylamide solution (w/w), AccuGel, National Diagnostics, Atlanta, GA) with 80 mM Tris borate buffer (pH 7.8) containing 1 mM EDTA. The gel was exposed to a phosphorimager screen (Imaging Screen K, Bio-Rad) overnight. The bands (representing extension of the primer) were visualized with a phosphorimaging system (Bio-Rad, Molecular Imager® FX) using the manufacturer's Quantity One Software, version 4.3.0.
Steady-state Kinetics—Unless indicated otherwise, all Dpo4 reactions were performed at 37 °C in 50 mM Tris-HCl buffer (pH 7.8) containing 5% glycerol (v/v), 5 mM DTT, 50 mM NaCl, and 50µg of bovine serum albumin ml-1. For both unmodified and modified templates, the molar ratio of the primer-template to enzyme was 10:1 to 40:1, and the reactions were done at eight dNTP concentrations (reaction time of 20 s to 3 min).
LC-MS Analysis of Oligonucleotide Products from Dpo4 Reactions—Dpo4 reactions were performed at 37 °C for 4 h in 50 mM Tris-HCl buffer (pH 7.8) containing 5% glycerol (v/v), 5 mM DTT, 50 mM NaCl, 5 mM MgCl2, and 100 µg of bovine serum albumin ml-1. The reactions were done with 10 µM oligonucleotide substrate, 5 µM Dpo4, and four dNTPs at 1 mM each, in a final reaction volume of 100 µl. The reaction was terminated by extraction of excess dNTPs using a spin column (Bio-Spin 6 chromatography column, Bio-Rad). To the above filtrate (120 µl), concentrated Tris-HCl, DTT, and EDTA were added to restore the initial concentrations, and uracil DNA glycosylase solution was added (20 units) (37). The reaction was incubated at 37 °C for 6 h to hydrolyze the uracil residue on the extended primer. The final reaction mixture was then heated at 95 °C for 1 h in the presence of 0.25 M piperidine, followed by removal of solvent by lyophilization. The dried residues were dissolved in 100 µl of H2O for the following MS analysis.
MS was performed on a DecaXP ion trap instrument (ThermoFinnigan, San Jose, CA). Separation of oligonucleotides was carried out with a Jupiter microbore column (1.0 x 150 mm, 5 µm, Phenomenex, Torrance, CA). Buffer A contained 10 mM NH4CH3CO2 (pH 6.8) and 2% CH3CN (v/v); buffer B contained 10 mM NH4CH3CO2 (pH 6.8) and 95% CH3CN (v/v). The following gradient program was used with a flow rate of 1. 0 ml min-1: 0–2 min, hold at 100% A; 2–20 min, linear program to 100% B; 20–30 min, hold at 100% B; 30–31 min, linear program to 100% A; 31–40 min, hold at 100% A (for next injection). The desired oligonucleotide products were eluted at 8 min. A pre-column "Tee" set-up was applied, with only 10% of the total flow infused to the ion source. The fast flow rate helped reduce the equilibration time of the column, and thus the retention time of the oligonucleotides was consistent between runs. Samples were infused using an autosampler, with 8 µl withdrawn from a 50-µl reaction. Electrospray conditions were: source voltage, 3.4 kV; source current, 8.5 µA; sheath gas flow rate setting, 28.2; auxiliary sweep gas flow rate setting, 4.3; capillary voltage, -49 V; capillary temperature, 230 °C; and tube lens voltage, -67 V. MS/MS conditions were: normalized collision energy, 35%; activation Q, 0.250; time, 30 ms; 1 scan. Product ion spectra were acquired over the m/z 250–2000 range. The abundant ions from LC-MS spectra were selected for CID analysis, and the cut-off was set above 15% of the most abundant ion. When more than one ion came from a single species, the peak responding to the doubly charged parent ion was chosen for fragmentation analysis. The calculations of the CID fragmentations of a certain oligonucleotide sequence were done using a program linked to the Mass Spectrometry Group of Medicinal Chemistry at the University of Utah.
Pre-steady-state Kinetics—Pre-steady-state kinetics were performed using a KinTek model RQF-3 chemical quench flow apparatus (KinTek Corp., Austin, TX) with 50 mM Tris-HCl (pH 7.8) buffer in the drive syringes. The reactions were initiated by mixing the pre-equilibrated polymerase-DNA complex (containing 100 mM Tris-HCl, pH 7.8, 200 nM 32P-labeled DNA duplex, 5 mM DTT, 100 µg of bovine serum albumin ml-1, 50, 200, or 300 nM Dpo4, 50 mM NaCl, and 5% glycerol (v/v)) in sample syringe A (12.5 µl) with either dNTP-Mg2+ (40–400 µM dNTP, depending on the oligonucleotide sequence) or 10 mM MgCl2 from syringe B (10.9 µl) at 37 °C. The reactions were quenched with 0.6 M EDTA (pH 9.0) in syringe C after reaction times that varied from 0.1 to 30 s. Reactions were combined with 500 µl of formamide dye solution (20 mM EDTA, 95% formamide (v/v), and 0.05% bromphenol blue (w/v)), and the components were separated by electrophoresis (3 µl of the sample) on a denaturing gel, with analysis as described for gel electrophoresis of polymerization reactions. The resulting plot (of product concentration versus time) was fit to the burst equation, y = A(1 - e-kpt) + ksst, where A represents burst amplitude, kp is the pre-steady-state rate of nucleotide incorporation, t is time, and kss is the steady-state rate of nucleotide incorporation, and analyzed using GraphPad Prism version 3.0a (San Diego, CA). In subsequent analyses with multiple dNTP concentrations, the results were fit to the hyperbolic expression kp = (kpol·[dNTP])/([dNTP] + Kd, dNTP), using GraphPad Prism.
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X-ray Diffraction Data Collection and Processing—Diffraction data sets for Dpo4 ternary complex crystals were collected at 110 K using a synchrotron radiation wavelength of 1.0 Å on beamlines at the Advanced Photon Source (APS), Argonne, IL (SER-CAT BM-22, Dpo4-dGTP = Dpo4-dG; IMCA-CAT ID-17, Dpo4-dCTP = Dpo4-dC and Dpo4-ddCTP = Dpo4-ddC; DND-CAT ID-5, Dpo4-ddGTP = Dpo4-ddG), or at the Advanced Light Source (ALS), Berkeley, CA (8.2.1, Dpo4-dATP = Dpo4-dA) (Table 1). The high resolution limit of diffraction for these crystals lies within a range of 2.27 to 2.6 Å. Data to 3.13-Å resolution were also collected for the Dpo4-ddA complex but this structure was not refined. Indexing and scaling with the Dpo4-dG and Dpo4-ddG complexes were performed using the program X-GEN (38); for the Dpo4-dA, Dpo4-dC, and Dpo4-ddC complexes the program XDS (39) was used. Autoindexing and systematic absences indicated the space group P21212 for all structures. The data were further processed using CCP4 package programs, including the truncate procedure performed with TRUNCATE (40). The resulting data sets for the Dpo4-dG and Dpo4-ddG complexes are of very good quality as indicated by R-merge values of 7.4 and 6.2%, respectively. The Dpo4-dA, Dpo4-dC, and Dpo4-ddC data sets show slightly inferior quality because of high mosaicity, the R-merge values are 9.4, 12.0, and 12.5%, respectively (see Table 1 for statistics of data processing and data quality). It should also be noted that the crystals formed by the Dpo4-dA, -dC, and -ddC ternary complexes in general had a higher tendency to be twinned.
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The Dpo4-dA, Dpo4-dC, and Dpo4-ddC structures were solved by molecular replacement with the program Phaser (41), using the refined 2.27-Å resolution structure of the Dpo4-ddG complex, devoid of all side chains and solvent molecules, as the search model. The positioned models gave values for Rotation Function Z of 27.3 (for Dpo4-dA), 27.2 (for Dpo4-dC), and 24.8 (for Dpo4-ddC). The values for the Translation Function Z were 17.9, 16.0, and 14.3, respectively, at 3.5-Å resolution. The locations of the models containing the side chains were optimized by several rounds of rigid body refinement. Data of increasing resolution were included in the refinement until the diffraction limit was reached.
The TURBO-FRODO program (version OpenGL.1) was used for manual model rebuilding into 
A maps computed using modified A coefficients (42). The initial difference Fourier maps showed clear negative density (higher than 5.0 r.m.s. deviation) for Ca2+ ions as well as for the (d)dNTP used in the crystallizations. Unambiguous density was also observed for the DNA duplex except for the first three template nucleotides (5'-TCA) that were partly or fully disordered in all structures. Therefore, in the absence of density, the 5'-terminal nucleotides of the template were completely omitted from the models in some of the structures (see Table 1 for model composition in each case). Clear density was seen in the initial difference Fourier electron density maps for a 14th nucleotide added by the polymerase to the 13-mer primer in the Dpo4-dC and Dpo4-ddC structures.
Refinements were performed using the program CNS (43). Five percent of the reflections were excluded from the refinement to calculate the cross-validation residual R-free. Water oxygen atoms were added into positive regions (higher than 3.0 standard deviation) of (Fo - Fc) Fourier difference electron density during the manual model rebuilding steps. Current statistics of the refined models for all structures are presented in Table 1.
The standard procedures in CNS (43) and PROCHECK (44) were used to inspect the quality and stereochemistry of the five models. The crystallographic figures were prepared using Pymol. Atomic coordinates and measured structure factor amplitudes for the Dpo4-dG, -dA, -dC, -ddG, and -ddC complexes have been deposited in the Protein Data Bank with accession codes 2c22, 2c2d, 2c2e, 2c28, and 2c2r, respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Full-length Extension of Oligonucleotide Primers by Dpo4 in the Presence of All Four dNTPs—Initial experiments with Dpo4 were done with a primer-template complex based on earlier studies (37), with a uracil residue placed in the primer strand in place of T to facilitate subsequent MS analysis (see below) (Fig. 1). In these qualitative experiments done with a single high concentration of the dNTP mixture, extension of the primer to full-length product was similar with templates containing either 8-oxoG or G in the same position (Fig. 1).
Steady-state Kinetic Analysis of dNTP Insertion and Extension by Dpo4—Preliminary analysis indicated that Dpo4 inserted dNTPs opposite 8-oxoG in the order C > A > G > T (results not shown). The system was analyzed in more detail using steady-state kinetics (Fig. 2, Table 2). When insertion was opposite an unmodified G, insertion of dATP or dGTP was >103 less efficient than dCTP (Table 2). For insertion opposite 8-oxoG, the order was dCTP > dATP > dGTP, with an order of magnitude or more difference in catalytic efficiency (kcat/Km) in each pair of comparisons. The insertion of dCTP opposite G and 8-oxoG showed similar catalytic efficiency, even with differences in the trends for kcat and Km (Table 2).
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4-fold less efficient (Table 2, Fig. 2, bottom panel), in contrast to most reports with DNA polymerases (22–26, 33, 34) with the exception of yeast pol (32). MS Analysis of Dpo4 Oligonucleotide Products—In recent work with Dpo4 (37) and other DNA polymerases, simple electrophoretic gel analyses of polymerization have been found to be deficient in the analysis of patterns of nucleotide incorporation, in that some unusual events (e.g. multiple dNTP additions) are observed in incubations with single dNTPs that are not common in studies with all four dNTPs present. In the approach used here, a primer is fully extended and MS is used to analyze the composition of the product (37). A uracil in the primer, several bases 5' of the first site of incorporation and paired with A, appears not to modify the polymerization process. This uracil allows the extended primer to be cut with uracil DNA glycosylase to make it smaller and facilitate MS/CID analysis in an ion-trap instrument.
The product of extension of primer 5 (Scheme 1) placed opposite template 2 (containing 8-oxoG) was analyzed as described. The products eluted from HPLC together at tR 8.5 min (Fig. 3A), and analysis of the peak yielded ions at m/z 1087.3 and 724.6, which were -2 and -3 ions, respectively (Fig. 3B). Reconstruction of the m/z 1087.3 HPLC profile yielded the chromatogram shown in Fig. 3C, and CID analysis of the m/z 1087.3 ion produced the fragmentation pattern shown in Fig. 3D. Comparison of the major ions with those possible for several oligonucleotides (supplementary data Table 1S) indicated the sequence 5'-pTCCGTGA-3', corresponding to the insertion of C opposite the 8-oxoG in the template.
Further inspection of the initial mass spectrum shown in Fig. 3B indicated the presence of an ion at m/z 1099.0. This m/z value corresponds to an Mr of 2200.0 and a -2 charge, consistent with a product containing an A inserted opposite the 8-oxoG. CID analysis of this peak in the manner described above provided further evidence for this assignment (supplementary data Figs. 1S, A and B, and Table S2). The expected -3 ion was not observed in this case. Reconstruction analysis (as in Fig. 3C) and comparisons indicate that the extent of dATP incorporation is 5% that of dCTP, a result consistent with that predicted by comparison of the catalytic efficiencies in the steady-state kinetic analysis with individual dNTPs (Table 2).
The insertion analysis was also done with a longer oligonucleotide to establish if the proclivity to insert dCTP opposite 8-oxoG is general (the longer oligonucleotide system was also used to facilitate incorporation studies done at higher temperatures, see below). Analysis of the product indicated only incorporation of C and no A, G, or T (supplementary data Fig. 2S and Table 3S) (with the limit of detection realistically being 2–3%).
Pre-steady-state Kinetic Analysis of Insertion of dNTPs Opposite 8-OxoG—The steady-state analyses (see below) suggested that insertion of dCTP opposite 8-oxoG was as efficient as opposite G, but steady-state rates can be misleading in that they are often dominated by product off-rates (50). Pre-steady-state analysis of dCTP incorporation was done (Fig. 4). The preliminary results indicated that the initial rate of polymerization was at least as fast opposite 8-oxoG as opposite G, although the apparent steady-state rate was somewhat lower, as in the work presented in Table 2. With both G and 8-oxoG, the extent of the burst phase was quantitative with the Dpo4 concentration (estimated from UV analysis of the protein (37)). This result is only consistent with the rate-limiting step occurring after formation of the product (51, 52).
Further analysis was done using single turnover conditions, in which the concentration of Dpo4 was in excess of the oligonucleotide pair and the dNTP concentration was varied (52). The results (Fig. 5, A and B) were fit to hyperbolic plots (Fig. 5, C and D). For incorporation of dCTP opposite G, the values kpol = 1.1 s-1 and Kd,dCTP = 420 µM were estimated. For dCTP incorporation opposite 8-oxoG, the values kpol = 1.6 s-1 and Kd,dCTP = 27 µM were estimated (Fig. 5, B and D). Thus, incorporation of dCTP appears to be inherently more efficient opposite 8-oxoG than G.
Determination of Ea for dCTP Insertion Opposite G and 8-OxoG by Dpo4—The experiment shown in Fig. 4 was repeated at various temperatures in the presence of a high concentration of dCTP (Fig. 6). The assays were done at temperatures ranging from 20 to 50 °C using a longer oligonucleotide pair with a higher calculated Tm (68 °C) to facilitate the studies done at higher temperatures. This oligonucleotide pair (6:7, Scheme 1) had been analyzed for insertion preferences using MS (supplementary data Fig. 2S). The results were fit to the Arrhenius equation kp = Ae-Ea/RT, where A is a reaction-specific constant, Ea is the activation energy, R is the gas constant (1.99 cal deg-1 mol-1), and T is the absolute temperature (Fig. 7). The estimates of Ea for insertion opposite G and 8-oxoG were 8.4 and 4.2 kcal mol-1, respectively.
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(32). The system was examined using pre-steady-state kinetic analysis (Fig. 7). The kp measured at a single high concentration of dGTP was 4-fold higher for insertion beyond the 8-oxoG:C pair than the 8-oxoG:A pair (Fig. 7). The kp value for the 8-oxoG:C pair was even higher than for the corresponding G:C pair. The kss value for the 8-oxoG:C extension was less than for the other two oligonucleotides examined. X-ray Crystal Structures of Dpo4 Complexes—To gain insight into the pairing modes of 8-oxoG at the active site of Dpo4 and potentially elucidate the structural bases of the biochemical observations, we determined crystal structures of five ternary Dpo4-DNA-(d)dNTP complexes at resolutions between 2.27 and 2.6 Å. All crystals were obtained in the presence of Ca2+, and potential traces of Mg2+ were masked with EDTA. Thus, the polymerase was expected to be inactive under the crystallization conditions used, as Ca2+ is a known inhibitor of DNA polymerases (53–56). To grow crystals of ternary complexes we used the 18-mer template 2, modified with 8-oxoG (Scheme 1) (37), and the native DNA primer 1 (13-mer, Scheme 1). Both 2'-deoxyribonucleoside (dNTPs) and 2',3'-dideoxyribonucleoside triphosphates (ddNTPs) were co-crystallized with Dpo4 and the template-primer duplex. In previous crystallization experiments with Dpo4 complexes (37) we found that crystal quality often varied significantly, depending on whether dNTP or ddNTP was used for preparing the complex. The ddNTPs are normally inhibitors of Dpo4 activity; thus, single extension of the primer by a ddNTP is much less efficient relative to the corresponding dNTP prevented even in the presence of Mg2+ and despite availability of a 3'-OH at the primer terminus (data not shown).
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The Dpo4-dG and Dpo4-ddG complexes are of the Type II form (36), with two template bases lodged at the active site of the polymerase (8-oxoG and the 5' adjacent C, Fig. 8, A and D). Both structures display close similarity to the structure of the ternary complex with a DNA of identical sequence but with 1,N2-ethenoG in place of 8-oxoG (37), consistent with the straightforward phasing of the Dpo4-dG and -ddG structures via molecular replacement, using the 1,N2-ethenoG complex as the search model. The 8-oxoG is stacked inside the duplex but does not pair with the base from the incoming nucleoside triphosphate. Instead, the latter is inserted opposite the 5' adjacent template C, leaving a gap to the 3'-terminal primer base that is itself paired with the template G that follows 8-oxoG. The base moieties of (d)dGTP and the 3'-terminal cytidine in the primer are subtly tilted around 8-oxoG, thus reducing the distance between the terminal 3'-hydroxyl group of the primer and the -phosphate group (Fig. 8, A and D). Conversely, the Dpo4-dA, -dC, and -ddC complexes belong to the Type I form (36), in which 8-oxoG of the template and nucleoside triphosphate constitute the single base pair at the active site (Fig. 8, B, C, and E).
The active site conformations classified as Types I and II represent only one difference between the Dpo4-dG and -ddG structures on the one hand and the Dpo4-dA, -dC, and -ddC structures on the other. Analysis of the latter structures revealed the presence of an additional nucleotide at or adjacent to the polymerase active site. In the Dpo4-dA structure, electron density is consistent with a second dATP molecule nested against the minor groove of the primer-template duplex, such that its triphosphate moiety is directed into the active site (Fig. 9A). The nucleobase of the second dATP practically dissects the minor groove, but only a single hydrogen bond to a base from the template strand is formed (N-6 to O-2 of T-9, 3.41 Å). The 3'-OH of this dATP is hydrogen bonded to the side chain of Ser103 and to the amide nitrogen from the same residue (Fig. 10B). The short distance (2.51 Å) between the terminal 3'-hydroxyl oxygen of the primer strand and the -phosphorus of the second dATP indicates the possibility that a small fraction of the primer strand has actually been extended in the crystal structure. By comparison, the distance between the 3'-OH and the -phosphorus of the dATP molecule located opposite 8-oxoG at the active site is much longer (6.51 Å; Fig. 9A).
In both the Dpo4-dC and -ddC structures, the electron density reveals 14-mer primers (13-mer 1, Scheme 1, extended by a single C). The orientations of C14 in the two complexes are very similar to the nucleoside portion of dATP in the Dpo4-dA complex (Fig. 9). The contact between N-4 of C14 and O-2 of template T9 in Dpo4-dC is slightly long (3.95 Å). Instead hydrogen bonds now exist between the exocyclic amino group of C14 and O-2 of T11 from the primer and O-4' of the adjacent residue T12 (3.55 and 2.86 Å, respectively; Fig. 9B). The 3'-hydroxyl oxygen of C14 is hydrogen bonded to the backbone carbonyl oxygen of Ala102 (3.42 Å, Fig. 10C). However, because the backbone curls around between C13 and C14, the 3'-OH of C14 is far removed from the -phosphorus of the dCTP paired with 8-oxoG (10.1 Å, Fig. 10C).
The presence of a second dATP in the Dpo4-dA structure and extended primers in the Dpo4-dC and -ddC structures are accompanied by subtle rearrangements of the metal ions normally coordinated at and adjacent to the active site (Figs. 11 and 12). In some cases, additional ions are observed. Thus, in the Dpo4-dA complex, five Ca2+ ions stabilize the 8 total negative charges of the two dATP molecules and adjacent phosphates of the primer strand (Fig. 10B); in the Dpo4-ddC complex, four Ca2+ ions are located at the active site as compared with the three ions normally seen in Dpo4 binary and ternary complexes (37). Comparisons between the individual complex structures as well as between the structures of DNA duplexes in the Dpo4 complexes alone reveal various deviations (Figs. 11 and 12). In general, the r.m.s. deviations between superimposed C atom positions for Type II complexes (Dpo4-dG, -ddG, and 1JXL (36)) are 0.4 Å. For those between C atoms of Type I complexes (Dpo4-dA, -dC, -ddC, and 1JX4 (36)) they are about 0.8 Å. From the illustrations in Figs. 11 and 12 it is also apparent that the orientation of the DNA primer-template duplex in the Dpo4-dG complex differs only minimally from the one in the complex with native DNA (Fig. 12A). However, the superimposed structures of Type I complexes show considerable deviations (Fig. 11) and the changes in DNA orientation seen in the Dpo4-dA complex relative to the reference structure (1JX4, Fig. 11B) somewhat exceed those found for the Dpo4-dC (Fig. 11C) and -ddC complexes (Fig. 11D). It is clear that these deviations are in part because of the presence of a second nucleoside triphosphate (Dpo4-dA) or the need to accommodate an extended primer (Dpo4-(d)dC).
The observation in the structures of Dpo4 activity with the 8-oxoG-modified template and either dCTP or ddCTP under the crystallization conditions used (i.e. in the absence of Mg2+) is unexpected. We conducted single nucleotide extension experiments with the 13-mer primer 1 paired with 18-mer template 2 in the presence of Ca2+ and demonstrated that Dpo4 is indeed active with dCTP, ddCTP, and dATP, albeit much more slowly than with Mg2+ but at a rate significant enough for the time frame used for crystallization (data not shown). It is noteworthy in view of the more efficient incorporation opposite 8-oxoG of dCTP relative to dATP (Table 2) that it is the Dpo4-dC and -ddC complexes that exhibit primers extended by a single residue in the crystal structures. Moreover, the in vitro primer extension studies with individual dNTPs or ddNTPs and Ca2+ show that dCTP and ddCTP are inserted most efficiently opposite 8-oxoG (a more detailed discussion of these results and their correlation with the structures of Dpo4 complexes will be published elsewhere).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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4-fold more efficient (kcat/Km) than for an 8-oxoG:A pair. Analysis of the crystal structures shows that, in contrast to the normal Watson-Crick base pairing and geometry, the interaction of A with a template 8-oxoG has the 8-oxoG in the abnormal syn conformation and the A in the normal anti configuration (Fig. 10). In a crystal containing dGTP, a very unfavorable substrate, the G was paired not with the adduct (8-oxoG) but with the 5' base of the adduct in the template (C), a phenomenon seen with Dpo4 and some other DNA adducts (37, 47).
The results of the LC-MS/MS analysis (Fig. 3 and supplementary data) of the products of the Dpo4-catalyzed polymerization of the oligonucleotide pair 5:2 (Scheme 1, Fig. 1) are semi-quantitatively consistent with the results of the steady-state (Fig. 2, Table 2) and pre-steady-state (Figs. 5 and 6) kinetic assays, in that incorporation of C is observed in 95% of the events and insertion and extension beyond A are observed the remainder of the time. The incorporation of dCTP >> dATP was also observed in another sequence context using LC-MS/MS analysis (supplementary data Fig. 2S) and is not considered to be a sequence-specific phenomenon.
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90% of the incorporation events in the oligonucleotides examined. Of the other studies available with various polymerases, including bovine pols and , E. coli pols I (Klenow fragment) and II (exo-), yeast pol , bacteriophage pol T7, and pols BF and RB69 (22–27, 29, 30, 34, 57), the ratio of dATP:dCTP incorporation varies (21) and only Dpo4 (this work) and yeast (and probably human) pol (32) have been found to preferentially extend 8-oxoG:C pairs relative to 8-oxoG:A pairs. It is of interest to contrast this behavior of Dpo4 in placing dCTP opposite template 8-oxoG with the report of the same enzyme to exclusively insert 8-oxo dGTP opposite template A, instead of C (58). These latter results, reminiscent of the reported asymmetry of the 8-oxoG pairs demonstrated earlier with other polymerases (25), clearly show that the patterns cannot be explained only in the context of strength of base pairing. The only two DNA polymerases that had been reported to show a high tendency to insert dCTP opposite 8-oxoG, instead of dATP, are RB69 (26) and S. cerevisiae pol (30, 32). These enzymes insert dATP in only 5% of the incorporation events, as judged by the available data. Dpo4 yields a similar value of 2% (Table 2, Fig. 3, and supplemental data). In the case of RB69, a crystal structure is available with dCTP bound, but not with dATP (26). No structure is available for the yeast pol complex; pre-steady-state kinetic measurements indicate the same Kd for dATP as for dCTP, with the difference being in the kpol (30). In our own work, the Kd for dCTP binding opposite 8-oxoG was considerably lower than opposite G, an unexpected result (Fig. 5). Even with a model DNA polymerase such as Dpo4, the mechanisms underlying discrimination of nucleotides and appropriate pairing are still the source of investigation and discussion (35). Possible mechanisms include optimization base stacking and metal alignment and coordination. The new structural data sheds light on the above observations regarding 8-oxoG. In the Dpo4-dC complex (the situation in Dpo4-ddC is similar), Arg332 is hydrogen bonded to O-8 of 8-oxoG (Fig. 10C). In Dpo4-dA where 8-oxoG adopts a syn conformation, formation of this hydrogen bond is prevented as O-8 is now rotated away from the border of the major groove into the minor groove and N-3 is too far away to allow interaction with Arg332 (Fig. 10B). The existence of this hydrogen bond has important consequences for the relative efficiencies of dCTP and dATP insertion opposite a template 8-oxoG because it changes the energetics of the syn/anti equilibrium in 8-oxoG. The anti conformation of 8-oxoG is accompanied by sterically unfavorable contacts between the 8-oxygen and the deoxyribose moiety. The syn orientation of the nucleobase becomes more favorable in 8-oxoG compared with G because it relieves the steric strain, hence the preferred incorporation of dATP in the Hoogsteen mode opposite 8-oxoG by the majority of polymerases including most Y-class polymerases. Likewise, incorporation of 8-oxo dGTP is strongly preferred opposite template-A by all polymerases including Dpo4. Obviously, the interaction between Arg332 and O-8 will shift the syn/anti equilibrium for 8-oxoG in the template toward the anti conformation. Instead of the Hoogsteen face, 8-oxoG will normally present its Watson-Crick face to the nucleoside triphosphate at the active site of Dpo4 and thus pair preferably with dCTP. Conversely, 8-oxo-dGTP is expected to adopt a syn conformation in Dpo4 and pair with template A in the Hoogsteen mode. The apparent contradiction constituted by deviating pairing modes of 8-oxoG in the template and as a nucleotide substrate of Dpo4 can therefore be fully resolved thanks to the structural data.
The interaction between Arg332 and 8-oxoG in the DNA template is unique, first because none of the four natural bases features an acceptor functionality at the edge of the major groove and, second, because the little finger domains (residues 244–341 in Sulfolobus solfataricus Dpo4) exhibit a high degree of sequence diversity. Judging from a structure-based sequence alignment (36), E. coli UmuC (pol V), E. coli DinB (pol IV), human pol (Rad30A), pol 
(Rad30B), and pol 
(DinB1), and S. cerevisiae pol (Rad30) do not have an Arg in position 332. In both human pols and , Arg332 is replaced by His, but it is unlikely that His can engage in the same interaction as Arg at the active site of Dpo4.
Our crystal structures of Dpo4 complexes with DNA templates containing 8-oxoG may also provide a rationalization of the preferential extension of 8-oxoG:C pairs relative to 8-oxoG:A pairs. In the Type II structure found for the Dpo4-dG complex, Arg332 also forms a hydrogen bond to O-8 of 8-oxoG (Fig. 10A), although the template base is shifted down relative to its position in the Dpo4-dA and -dC complexes (Fig. 10, B and C, respectively). Thus, Arg332 is capable of slightly changing its orientation and interacts with an 8-oxoG that is being replicated and also with one that has moved to the -1 position following strand relocation. In both orientations, the side chain will afford stabilizing interactions with backbone phosphates (Fig. 10). As is the case for the corresponding pair in a replicating mode, the syn 8-oxoG:A pair, now shifted down by a single step, cannot interact with Arg332 via O-8. The conservation of this interaction with 8-oxoG during and after replication most likely stabilizes local conformation and orientation of the template-primer duplex and may thus contribute favorably to the extension of the 8-oxoG:C pair compared with that of the 8-oxoG:A pair.
The catalytic efficiency (as reflected in the steady-state parameter kcat/Km) for insertion of dCTP opposite 8-oxoG is similar to that opposite G (Table 2). The steady-state Km for the 8-oxoG reaction was 6-fold lower, and the kcat was about 
that for insertion of dCTP opposite G (Table 2, Fig. 2, top panel). A similar pattern was observed for the steady-state kinetics of extension beyond 8-oxoG:C versus a G:C pair (Table 2). The basis for these results is not clear, in that the exact meaning of steady-state kinetic parameters in DNA polymerase reactions is not very obvious (50). These results are consistent with the differences in the kss component in the pre-steady-state kinetic assays, as might be expected (Figs. 4 and 5). One result that is rather striking is the much lower Kd,dCTP value measured for incorporation opposite 8-oxoG (27 µM) versus G (420 µM) (Fig. 5, C and D). In principle, this value, determined using single-turnover conditions (Fig. 5, A and B), should reflect the dissociation constant for the substrate dCTP in the enzyme intermediate poised for phosphodiester bond formation (52).
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The crystal structures provide some insight into the mechanisms used by this enzyme, particularly in regards to the favorable transition state barrier for insertion of dCTP opposite 8-oxoG relative to G. Formation of the fortuitous hydrogen bond between Arg332 and O-8 of 8-oxoG more or less freezes the template nucleoside in the anti conformation. Therefore, this interaction stabilizes the orientation of the template base at the active site. A more rigid template base is consistent with facilitated pairing to the substrate and a lower activation energy as long as it supports a geometry that is conductive to phosphodiester bond formation. Whether the Arg322-8-oxoG interaction explains the tighter binding between 8-oxoG and dCTP compared with the G:dCTP pair is more difficult to answer at this point, although the hypothesis can be tested. The presence of the carbonyl group at position 8 of G not only changes the N-7 atom from a hydrogen bond acceptor to a donor, but has additional stereoelectronic effects that influence nucleoside conformation and Watson-Crick hydrogen bonding strengths. Modulation of these effects by strong hydrogen bonds between O-8 and Arg may contribute to the higher stability of the 8-oxoG:dCTP pair at the Dpo4 active site.
The crystal structures of the ternary Dpo4-dA and Dpo4-(d)dC complexes reported here are unusual in that they unexpectedly reveal additional nucleotides near the active site. In the case of Dpo4-dA, a second dATP molecule is lodged in the minor groove of the template-primer duplex in the immediate vicinity of the substrate dATP opposite 8-oxoG (Figs. 9A and 10B). In the Dpo4-dC and -ddC complexes the 13-mer primers were extended by a single C under the crystallization conditions used (Fig. 9, B and C). However, in both structures the primer strand turns sharply between C13 and C14 and thus allows accommodation of dCTP (or ddCTP) opposite 8-oxoG. Therefore, these complexes represent mixed substrate-product complexes and they demonstrate that Dpo4 may even be capable of carrying out two insertions opposite the same template base (8-oxoG in our case). Such behavior is consistent with a product isolated from in vitro primer extension reactions opposite a template with a single 1,N2-ethenoG adduct that can be explained by Dpo4 consecutively inserting two Ts opposite the same template A (37). The crystal structures of the Dpo4-dC and -ddC complexes indicate that the primer strand may bulge out into the minor groove prior to addition of the next nucleotide and that the channel formed by Dpo4 around the primer-template duplex provides enough space to tolerate the bulge. The disruption of the 8-oxoG:C pair at the active site and the looped-out conformation of (d)dC that allows binding of another (d)dCTP opposite the lesion is most likely the result of the particular metal ion (Ca2+) used in the crystallization. However, the observed interaction between Arg332 and O-8 of 8-oxoG in the anti conformation and the conclusions regarding its roles in the efficient insertion of dCTP opposite 8-oxoG and the asymmetry of the efficiency of incorporation (mostly insertion of 8-oxo dGTP opposite template dA versus mostly insertion of dCTP opposite template 8-oxo dG) is independent of the nature of the divalent metal ion. We will report details of the differences between Mg2+ and Ca2+ as cofactors of Dpo4 in a forthcoming manuscript.3
) in the template (oligomer 2 of 
), or 8-oxoG:A pair (
) (G or 8-oxoG in template strand). The following parameters were estimated: G:C, kp = 0.69 s-1 and kss = 0.050 s-1; 8-oxoG:C, kp = 1.9 s-1 and kss = 0.018 s-1; 8-oxoG:A, kp = 0.45 s-1 and kss = 0.030 s-1. The concentration of Dpo4, primer-template complex, and dCTP were 50 nM, 150 nM, and 500 µM, respectively.
-phosphate groups of (d)dNTPs are highlighted in cyan, gray, and white, respectively.
