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1.
The Y family DNA polymerase yeast pol eta inserts pyrene deoxyribose monophosphate (dPMP) in preference to A opposite an abasic site, the 3'-T of a thymine dimer, and a normal T with almost equal efficiency. In contrast, pol A family polymerases such as Klenow fragment and T7 DNA polymerase only insert dPMP efficiently opposite an abasic site and the 3'-T of a thymine dimer but not opposite undamaged DNA. Pyrene nucleotide is also an efficient chain-terminating inhibitor of DNA synthesis by pol eta but not by Klenow fragment or T7 DNA polymerase. To better understand the origin of the efficiency and sequence specificity of dPMP insertion by pol eta, the kinetics of dPMP insertion opposite various templates have been determined. In one sequence context, the efficiency of dPMP insertion increases 4.6-fold opposite G < A < T < C, suggesting that the templating nucleotide modulates dPMP insertion efficiency by having to destack prior to dPTP binding. The efficiency of insertion of dPMP opposite T in the same sequence context increases 7-fold for primers terminating in G < A < C < T and is similar to that observed for nontemplated blunt-end extension, suggesting that stacking interactions between the pyrene and the primer terminus are also important. On heterogeneous templates, the average selectivity for dPMP insertion relative to the complementary dNMP decreases in the order of dAMP > dGMP > dTMP > dCMP, from a high of 5.8 when dAMP is to be inserted following a T to a low of 0.5 when dCMP is to be inserted following a C. The relative preference for dPMP insertion at a given site can be largely explained by the energetic cost of destacking the templating base and stacking of pyrene nucleotide relative to that of stacking and base pairing the complementary nucleotide. Thus, pyrene nucleotide represents a novel class of nucleotide-based chain-terminating DNA synthesis inhibitors whose base portion consists of a hydrophobic, non-hydrogen bonding, base-pair mimic. 相似文献
2.
Polymerase eta is a member of the Y family of DNA polymerases which is able to bypass thymine dimers efficiently and in a relatively error-free manner. To elucidate the mechanism of dimer bypass, the efficiency of dAMP and pyrene nucleotide insertion opposite the thymine dimer and its N3-methyl derivatives was determined. Pol eta inserts pyrene nucleotide with greater efficiency than dAMP opposite the 3'-T of an undimerized or dimerized T and is an effective inhibitor of DNA synthesis by pol eta. Substitution of the N3H of the 3'-T of an undimerized T or a dimerized T with a methyl group has little effect on the insertion efficiency of pyrene nucleotide but greatly inhibits the insertion of dAMP. Together, these results suggest that the error-free insertion of dAMP opposite the 3'-T of the cis-syn thymine dimer happens by way of a loosely held dimer in the active site which can be displaced from the active site by pyrene nucleotide. In contrast, pol eta cannot insert pyrene nucleotide opposite the 5'-T of the dimer, whereas it can insert dAMP with efficiency comparable to that opposite the 3'-T. The inability to insert pyrene nucleotide opposite the 5'-T of the dimer is consistent with the idea that while the polymerase binds loosely to a templating nucleotide, it binds tightly to the nucleotide to its 3'-side. Overall, the results show a marked difference from similar studies on pol I family polymerases, and suggest mechanisms by which this Y family polymerase can process damaged DNA efficiently. 相似文献
3.
DNA polymerase catalysis in the absence of Watson-Crick hydrogen bonds: analysis by single-turnover kinetics 总被引:1,自引:0,他引:1
Potapova O Chan C DeLucia AM Helquist SA Kool ET Grindley ND Joyce CM 《Biochemistry》2006,45(3):890-898
We report the first pre-steady-state kinetic studies of DNA replication in the absence of hydrogen bonds. We have used nonpolar nucleotide analogues that mimic the shape of a Watson-Crick base pair to investigate the kinetic consequences of a lack of hydrogen bonds in the polymerase reaction catalyzed by the Klenow fragment of DNA polymerase I from Escherichia coli. With a thymine isostere lacking hydrogen-bonding ability in the nascent pair, the efficiency (k(pol)/Kd) of the polymerase reaction is decreased by 30-fold, affecting the ground state (Kd) and transition state (k(pol)) approximately equally. When both thymine and adenine analogues in the nascent pair lack hydrogen-bonding ability, the efficiency of the polymerase reaction is decreased by about 1000-fold, with most of the decrease attributable to the transition state. Reactions using nonpolar analogues at the primer-terminal base pair demonstrated the requirement for a hydrogen bond between the polymerase and the minor groove of the primer-terminal base. The R668A mutation of Klenow fragment abolished this requirement, identifying R668 as the probable hydrogen-bond donor. Detailed examination of the kinetic data suggested that Klenow fragment has an extremely low tolerance of even minor deviations of the analogue base pairs from ideal Watson-Crick geometry. Consistent with this idea, some analogue pairings were better tolerated by Klenow fragment mutants having more spacious active sites. In contrast, the Y-family polymerase Dbh was much less sensitive to changes in base pair dimensions and more dependent upon hydrogen bonding between base-paired partners. 相似文献
4.
The fidelity of base selection by the polymerase subunit of DNA polymerase III holoenzyme. 总被引:4,自引:0,他引:4
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In common with other DNA polymerases, DNA polymerase III holoenzyme of E. coli selects the biologically correct base pair with remarkable accuracy. DNA polymerase III is particularly useful for mechanistic studies because the polymerase and editing activities reside on separate subunits. To investigate the biochemical mechanism for base insertion fidelity, we have used a gel electrophoresis assay to measure kinetic parameters for the incorporation of correct and incorrect nucleotides by the polymerase (alpha) subunit of DNA polymerase III. As judged by this assay, base selection contributes a factor of roughly 10(4)-10(5) to the overall fidelity of genome duplication. The accuracy of base selection is determined mainly by the differential KM of the enzyme for correct vs. incorrect deoxynucleoside triphosphate. The misinsertion of G opposite template A is relatively efficient, comparable to that found for G opposite T. Based on a variety of other work, the G:A pair may require a special correction mechanism, possibly because of a syn-anti pairing approximating Watson-Crick geometry. We suggest that precise recognition of the equivalent geometry of the Watson-Crick base pairs may be the most critical feature for base selection. 相似文献
5.
cis-syn thymine dimers are not absolute blocks to replication by DNA polymerase I of Escherichia coli in vitro 总被引:8,自引:0,他引:8
Both Escherichia coli DNA polymerase I (pol I) and the large fragment of pol I (Klenow) were found to bypass a site-specific cis-syn thymine dimer, in vitro, under standard conditions. A template was constructed by ligating d(pCGTAT[c,s]TATGC), synthesized via a cis-syn thymine dimer phosphoramidite building block, to a 12-mer and 19-mer. The site and integrity of the dimer were verified by use of T4 denV endonuclease V. Extension of a 15-mer on the dimer-containing template by either pol I or Klenow led to dNTP and polymerase concentration dependent formation of termination and bypass products. At approximately 0.15 unit/microL and 1-10 microM in each dNTP, termination one prior to the 3'-T of the dimer predominated. At 100 microM in each dNTP termination opposite the 3'-T of the dimer predominated and bypass occurred. Bypass at 100 microM in each dNTP depended on polymerase concentration, reaching a maximum of 20% in 1 h at approximately 0.2 unit/microL, underscoring the importance of polymerase binding affinity for damaged primer-templates on bypass. Seven percent bypass in 1 h occurred under conditions of 100:10 microM dATP:dNTP bias, 1% under dTTP bias, and an undetectable amount under either dGTP or dCTP bias. At 100 microM in each dNTP, the ratio of pdA:pdG:pdC:pdT terminating opposite the 3'-T of the dimer was estimated to be 37:25:10:28. Sequencing of the bypass product produced under these conditions demonstrated that greater than 95% pdA was incorporated opposite both Ts of the dimer and that little or no frame shifting took place.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
Sampoli Benítez BA Arora K Balistreri L Schlick T 《Journal of molecular biology》2008,384(5):1086-1097
DNA polymerase X (pol X) from the African swine fever virus is a 174-amino-acid repair polymerase that likely participates in a viral base excision repair mechanism, characterized by low fidelity. Surprisingly, pol X's insertion rate of the G*G mispair is comparable to that of the four Watson-Crick base pairs. This behavior is in contrast with another X-family polymerase, DNA polymerase beta (pol beta), which inserts G*G mismatches poorly, and has higher DNA repair fidelity. Using molecular dynamics simulations, we previously provided support for an induced-fit mechanism for pol X in the presence of the correct incoming nucleotide. Here, we perform molecular dynamics simulations of pol X/DNA complexes with different incoming incorrect nucleotides in various orientations [C*C, A*G, and G*G (anti) and A*G and G*G (syn)] and compare the results to available kinetic data and prior modeling. Intriguingly, the simulations reveal that the G*G mispair with the incoming nucleotide in the syn configuration undergoes large-scale conformational changes similar to that observed in the presence of correct base pair (G*C). The base pairing in the G*G mispair is achieved via Hoogsteen hydrogen bonding with an overall geometry that is well poised for catalysis. Simulations for other mismatched base pairs show that an intermediate closed state is achieved for the A*G and G*G mispair with the incoming dGTP in anti conformation, while the protein remains near the open conformation for the C*C and the A*G syn mismatches. In addition, catalytic site geometry and base pairing at the nascent template-incoming nucleotide interaction reveal distortions and misalignments that range from moderate for A*G anti to worst for the C*C complex. These results agree well with kinetic data for pol X and provide a structural/dynamic basis to explain, at atomic level, the fidelity of this polymerase compared with other members of the X family. In particular, the more open and pliant active site of pol X, compared to pol beta, allows pol X to accommodate bulkier mismatches such as guanine opposite guanine, while the more structured and organized pol beta active site imposes higher discrimination, which results in higher fidelity. The possibility of syn conformers resonates with other low-fidelity enzymes such as Dpo4 (from the Y family), which readily accommodate oxidative lesions. 相似文献
7.
DNA damage that stalls replicative polymerases can be bypassed with the Y-family polymerases. These polymerases have more open active sites that can accommodate modified nucleotides. The lack of protein-DNA interactions that select for Watson-Crick base pairs correlate with the lowered fidelity of replication. Interstrand hydrogen bonds appear to play a larger role in dNTP selectivity. The mechanism by which purine-purine mispairs are formed and extended was examined with Solfolobus solfataricus DNA polymerase IV, a member of the RAD30A subfamily of the Y-family polymerases, as is pol eta. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 1-deaza- and 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. The time course of insertion of a single dNTP was examined with a polymerase concentration of 50 nM and a DNA concentration of 25 nM with various concentrations of dNTP. The time courses were fitted to a first-order equation, and the first-order rate constants were plotted against the dNTP concentration to produce k pol and K d (dNTP) values. A decrease in k pol/ K d (dNTP) associated with the deazapurine substitution would indicate that the position is involved in a crucial hydrogen bond. During correct base pair formation, the adenine to 1-deazaadenine substitution in both the incoming dNTP and template base resulted in a >1000-fold decrease in k pol/ K d (dNTP), indicating that interstrand hydrogen bonds are important in correcting base pair formation. During formation of purine-purine mispairs, the k pol/ K d (dNTP) values for the insertion of dATP and dGTP opposite 7-deazaadenine and 7-deazaguanine were decreased >10-fold with respect to those of the unmodified nucleotides. In addition, the rate of incorporation of 1-deaza-dATP opposite guanine was decreased 5-fold. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the anti conformation and the template base in the syn conformation. These results indicate that Dpo4 holds the incoming dNTP in the normal anti conformation while allowing the template nucleotide to change conformations to allow reaction to occur. This result may be functionally relevant in the replication of damaged DNA in that the polymerase may allow the template to adopt multiple configurations. 相似文献
8.
The SOS-induced DNA polymerases II and IV (pol II and pol IV, respectively) of Escherichia coli play important roles in processing lesions that occur in genomic DNA. Here we study how electrostatic and steric effects play different roles in influencing the efficiency and fidelity of DNA synthesis by these two enzymes. These effects were probed by the use of nonpolar shape analogues of thymidine, in which substituted toluenes replace the polar thymine base. We compared thymine with nonpolar analogues to evaluate the importance of hydrogen bonding in the polymerase active sites, while we used comparisons among a set of variably sized thymine analogues to measure the role of steric effects in the two enzymes. Steady-state kinetics measurements were carried out to evaluate activities for nucleotide insertion and extension. The results showed that both enzymes inserted nucleotides opposite nonpolar template bases with moderate to low efficiency, suggesting that both polymerases benefit from hydrogen bonding or other electrostatic effects involving the template base. Surprisingly, however, pol II inserted nonpolar nucleotide (dNTP) analogues into a primer strand with high (wild-type) efficiency, while pol IV handled them with an extremely low efficiency. Base pair extension studies showed that both enzymes bypass non-hydrogen-bonding template bases with moderately low efficiency, suggesting a possible beneficial role of minor groove hydrogen bonding interactions at the N-1 position. Measurement of the two polymerases' sensitivity to steric size changes showed that both enzymes were relatively flexible, yielding only small kinetic differences with increases or decreases in nucleotide size. Comparisons are made to recent data for DNA pol I (Klenow fragment), the archaeal polymerase Dpo4, and human pol kappa. 相似文献
9.
Thermodynamic and base-pairing studies of matched and mismatched DNA dodecamer duplexes containing cis-syn, (6-4) and Dewar photoproducts of TT. 总被引:2,自引:0,他引:2
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Cis-syn dimers, (6-4) products and their Dewar valence isomers are the major photoproducts of DNA and have different mutagenic properties and rates of repair. To begin to understand the physical basis for these differences, the thermal stability and base pairing properties of the corresponding photoproducts of the TT site in d(GAGTATTATGAG) were investigated. The (6-4) and Dewar products destabilize the duplex form by approximately 6 kcal/mol of free energy at 37 degreesC relative to the parent, whereas a cis-syn dimer only destabilizes the duplex form by 1.5 kcal/mol. Duplexes with G opposite the 3'-T of the (6-4) and Dewar products are more stable than those with A by approximately 0.4 kcal/mol, whereas the cis-syn dimer prefers A over G by 0.7 kcal/mol. Proton NMR suggests that wobble base pairing takes place between the 3'-T of the cis-syn dimer and an opposed G, whereas there is no evidence of significant H-bonding between these two bases in the (6-4) product. The thermodynamic and H-bonding data for the (6-4) product are consistent with a 4 nt interior loop structure which may facilitate flipping of the photoproduct in and out of the helix. 相似文献
10.
The XP-V (xeroderma pigmentosum variant) gene product, human DNA polymerase eta (pol eta), catalyzes efficient and accurate translesion synthesis (TLS) past cis-syn thymine-thymine dimers (TT dimer). In addition, recent reports suggest that pol eta is involved in TLS past various other types of lesion, including an oxidative DNA damage, 8-hydroxyguanine. Here, we compare the abilities of pol alpha and pol eta to replicate across thymine glycol (Tg) using purified synthetic oligomers containing a 5R- or 5S-Tg. DNA synthesis by pol alpha was inhibited at both steps of insertion of a nucleotide opposite the lesion and extension from the resulting product, indicating that pol alpha can weakly contribute to TLS past Tg lesions. In contrast, pol eta catalyzed insertion opposite the lesion as efficient as that opposite undamaged T, while extension was inhibited especially on the 5S-Tg template. Thus, pol eta catalyzed relatively efficient TLS past 5R-Tg than 5S-Tg. To compare the TLS abilities of pol eta for these lesions, we determined the kinetic parameters of pol eta for catalyzing TLS past a TT dimer, an N-2-acetylaminofluorene-modified guanine, and an abasic site analogue. The possible mechanisms of pol eta-catalyzed TLS are discussed on the basis of these results. 相似文献
11.
Vasquez-Del Carpio R Silverstein TD Lone S Johnson RE Prakash L Prakash S Aggarwal AK 《Journal of molecular biology》2011,408(2):252-261
Exposure of DNA to UV radiation causes covalent linkages between adjacent pyrimidines. The most common lesion found in DNA from these UV-induced linkages is the cis-syn cyclobutane pyrimidine dimer. Human DNA polymerase κ (Polκ), a member of the Y-family of DNA polymerases, is unable to insert nucleotides opposite the 3'T of a cis-syn T-T dimer, but it can efficiently extend from a nucleotide inserted opposite the 3'T of the dimer by another DNA polymerase. We present here the structure of human Polκ in the act of inserting a nucleotide opposite the 5'T of the cis-syn T-T dimer. The structure reveals a constrained active-site cleft that is unable to accommodate the 3'T of a cis-syn T-T dimer but is remarkably well adapted to accommodate the 5'T via Watson-Crick base pairing, in accord with a proposed role for Polκ in the extension reaction opposite from cyclobutane pyrimidine dimers in vivo. 相似文献
12.
Hypotheses on the origins of high fidelity in replicative DNA polymerases have recently focused on the importance of geometric or steric effects in this selectivity. Here we reported a systematic study of the effects of base pair size in T7 DNA polymerase (pol), the replicative enzyme for bacteriophage T7. We varied base pair size in very small (0.25 A) increments by use of a series of nonpolar thymidine shape mimics having gradually increasing size. Steady-state kinetics were evaluated for the 5A7A exonuclease-deficient mutant in a 1:1 complex with thioredoxin. For T7 pol, we studied insertion of natural nucleotides opposite variably sized T analogs in the template and, conversely, for variably sized dTTP analogs opposite natural template bases. The enzyme displayed extremely high selectivity for a specific base pair size, with drops in efficiency of as much as 280-fold for increases of 0.4 A beyond an optimum size approximating the size of a natural pair. The enzyme also strongly rejected pairs that were smaller than the optimum by as little as 0.3 A. The size preferences with T7 DNA pol were generally smaller, and the steric rejection was greater than DNA pol I Klenow fragment, correlating with the higher fidelity of the former. The hypothetical effects of varied active site size and rigidity are discussed. The data lend direct support to the concept that active site tightness is a chief determinant of high fidelity of replicative polymerases and that a less rigid (looser) and larger active site can lead to lower fidelity. 相似文献
13.
14.
In vivo formation and in vitro replication of a guanine-thymine intrastrand cross-link lesion 总被引:1,自引:0,他引:1
G[8-5m]T, a guanine-thymine intrastrand cross-link lesion where the C8 of guanine is covalently bonded to the neighboring 3'-thymine through its methyl carbon, was previously shown to form in an aqueous solution of duplex DNA upon exposure to gamma- or X-rays and in calf thymus DNA treated with Fenton reagents. Here, we employed LC-MS/MS and demonstrated for the first time that this lesion could be induced in a dose-dependent manner in human Hela-S3 cells upon exposure to gamma-rays. We further carried out in vitro replication studies on a substrate containing a site-specifically incorporated G[8-5m]T, and our results showed that the Klenow fragment of Escherichia coli DNA polymerase I stopped synthesis mostly after incorporating the correct nucleotide dAMP opposite the 3'-thymine moiety of the lesion. On the other hand, yeast Saccharomyces cerevisiae DNA polymerase eta (pol eta) was able to replicate past the cross-link lesion, but with markedly reduced efficiency in nucleotide incorporation opposite the 5'-guanine of the lesion. Steady-state kinetic analyses for nucleotide incorporation by yeast pol eta showed that the 5'-guanine portion of the lesion also decreased pronouncedly the fidelity of nucleotide incorporation; the insertion of dAMP and dGMP was favored over that of the correct nucleotide, dCMP. The above results support the conclusion that oxidative intrastrand cross-link lesions, if not repaired, can be cytotoxic and mutagenic. 相似文献
15.
The mechanism by which purine-purine mispairs are formed and extended was examined with the high-fidelity Klenow fragment of Escherichia coli DNA polymerase I with the proofreading exonuclease activity inactivated. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. A decrease in rate associated with a 7-deazapurine substitution would suggest that the nucleotide is in a syn conformation in a Hoogsteen base pair with the opposite base. During mispair formation, the k(pol)/K(d) values for the insertion of dATP opposite A (dATP/A) as well as dATP/G and dGTP/G were decreased greater than 10-fold with the deazapurine in the dNTP. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the syn conformation and the template base in the anti conformation. During mispair extension, the only decrease in k(pol)/K(d) was associated with the G/G base pair in which 7-deazaguanine was in the template strand. These results as well as previous results [McCain et al. (2005) Biochemistry 44, 5647-5659] in which a hydrogen bond was found between the 3-position of guanine at the primer terminus and Arg668 during G/A and G/G mispair extension indicate that the conformation of the purine at the primer terminus is in the anti conformation during mispair extension. These results suggest that purine-purine mispairs are formed via a Hoogsteen geometry in which the dNTP is in the syn conformation and the template is in the anti conformation. During extension, however, the conformation of the primer terminus changes to an anti configuration while the template base may be in either the syn or anti conformations. 相似文献
16.
Structural insights into the origins of DNA polymerase fidelity 总被引:5,自引:0,他引:5
DNA polymerases discriminate from a pool of structurally similar molecules to insert the correct nucleotide to preserve Watson-Crick base pairing rules. The ability to choose between "right and wrong" is highly dependent on the identity of the polymerase. Because naturally occurring polymerases with divergent fidelities insert incorrect nucleotides with comparable efficiencies, fidelity is primarily governed by the ability to insert the correct nucleotide. DNA polymerases generally bind the correct nucleotide with similar affinities, but low-fidelity polymerases insert correct nucleotides more slowly than higher fidelity enzymes. A comparison of crystallographic ternary substrate complexes of DNA polymerases from five families exhibiting a range of nucleotide insertion rates reveals possible structural features that lead to rapid, efficient, and faithful DNA synthesis. 相似文献
17.
The efficiency and fidelity of nucleotide incorporation and next-base extension by DNA polymerase (pol) κ past N(2)-ethyl-Gua were measured using steady-state and rapid kinetic analyses. DNA pol κ incorporated nucleotides and extended 3' termini opposite N(2)-ethyl-Gua with measured efficiencies and fidelities similar to that opposite Gua indicating a role for DNA pol κ at the insertion and extension steps of N(2)-ethyl-Gua bypass. The DNA pol κ was maximally activated to similar levels by a twenty-fold lower concentration of Mn(2+) compared to Mg(2+). In addition, the steady state analysis indicated that high fidelity DNA pol κ-catalyzed N(2)-ethyl-Gua bypass is Mg(2+)-dependent. Strikingly, Mn(2+) activation of DNA pol κ resulted in a dramatically lower efficiency of correct nucleotide incorporation opposite both N(2)-ethyl-Gua and Gua compared to that detected upon Mg(2+) activation. This effect is largely governed by diminished correct nucleotide binding as indicated by the high K(m) values for dCTP insertion opposite N(2)-ethyl-Gua and Gua with Mn(2+) activation. A rapid kinetic analysis showed diminished burst amplitudes in the presence of Mn(2+) compared to Mg(2+) indicating that DNA pol κ preferentially utilizes Mg(2+) activation. These kinetic data support a DNA pol κ wobble base pairing mechanism for dCTP incorporation opposite N(2)-ethyl-Gua. Furthermore, the dramatically different polymerization efficiencies of the Y-family DNA pols κ and ι in the presence of Mn(2+) suggest a metal ion-dependent regulation in coordinating the activities of these DNA pols during translesion synthesis. 相似文献
18.
We have recently challenged the widely held view that 2,4-difluorotoluene (dF) is a nonpolar isosteric analogue of the nucleotide dT, incapable of forming hydrogen bonds (HBs). To gain a further understanding for the kinetic preference that favors dAMP insertion opposite a templating dF, a result that mirrors the base selectivity that favors dAMP insertion opposite dT by RB69 DNA polymerase (RB69pol), we determined presteady-state kinetic parameters for incorporation of four dNMPs opposite dF by RB69pol and solved the structures of corresponding ternary complexes. We observed that both the F2 and F4 substituent of dF in these structures serve as HB acceptors forming HBs either directly with dTTP and dGTP or indirectly with dATP and dCTP via ordered water molecules. We have defined the shape and chemical features of each dF/dNTP pair in the RB69pol active site without the corresponding phosphodiester-linkage constraints of dF/dNs when they are embedded in isolated DNA duplexes. These features can explain the kinetic preferences exhibited by the templating dF when the nucleotide incorporation is catalyzed by wild type RB69pol or its mutants. We further show that the shapes of the dNTP/dF nascent base pair differ markedly from the corresponding dNTP/dT in the pol active site and that these differences have a profound effect on their incorporation efficiencies. 相似文献
19.
The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta (pol eta), which is involved in the replication of damaged DNA. Pol eta catalyzes efficient and accurate translesion synthesis past cis-syn cyclobutane di-thymine lesions. Here we show that human pol eta can catalyze translesion synthesis past an abasic (AP) site analog, N-2-acetylaminofluorene (AAF)-modified guanine, and a cisplatin-induced intrastrand cross-link between two guanines. Pol eta preferentially incorporated dAMP and dGMP opposite AP, and dCMP opposite AAF-G and cisplatin-GG, but other nucleotides were also incorporated opposite these lesions. However, after incorporating an incorrect nucleotide opposite a lesion, pol eta could not continue chain elongation. In contrast, after incorporating the correct nucleotide opposite a lesion, pol eta could continue chain elongation, whereas pol alpha could not. Thus, the fidelity of translesion synthesis by human pol eta relies not only on the ability of this enzyme to incorporate the correct nucleotide opposite a lesion, but also on its ability to elongate only DNA chains that have a correctly incorporated nucleotide opposite a lesion. 相似文献
20.
In the ternary substrate complex of DNA polymerase (pol) beta, the nascent base pair (templating and incoming nucleotides) is sandwiched between the duplex DNA terminus and polymerase. To probe molecular interactions in the dNTP-binding pocket, we analyzed the kinetic behavior of wild-type pol beta on modified DNA substrates that alter the structure of the DNA terminus and represent mutagenic intermediates. The DNA substrates were modified to 1) alter the sequence of the duplex terminus (matched and mismatched), 2) introduce abasic sites near the nascent base pair, and 3) insert extra bases in the primer or template strands to mimic frameshift intermediates. The results indicate that the nucleotide insertion efficiency (k(cat)/K(m), dGTP-dC) is highly dependent on the sequence identity of the matched (i.e. Watson-Crick base pair) DNA terminus (template/primer, G/C approximately A/T > T/A approximately C/G). Mismatches at the primer terminus strongly diminish correct nucleotide insertion efficiency but do not affect DNA binding affinity. Transition intermediates are generally extended more easily than transversions. Most mismatched primer termini decrease the rate of insertion and binding affinity of the incoming nucleotide. In contrast, the loss of catalytic efficiency with homopurine mismatches at the duplex DNA terminus is entirely due to the inability to insert the incoming nucleotide, since K(d)((dGTP)) is not affected. Abasic sites and extra nucleotides in and around the duplex terminus decrease catalytic efficiency and are more detrimental to the nascent base pair binding pocket when situated in the primer strand than the equivalent position in the template strand. 相似文献