Incorporation Of dTTP Research Articles

Error-prone bypass of O6-methylguanine by DNA polymerase of Pseudomonas aeruginosa phage PaP1

O6-Methylguanine (O6-MeG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, generally leads to G:C to A:T mutagenesis. To study DNA replication encountering O6-MeG by the DNA polymerase (gp90) of P. aeruginosa phage PaP1, we analyzed steady-state and pre-steady-state kinetics of nucleotide incorporation opposite O6-MeG by gp90 exo−. O6-MeG partially inhibited full-length extension by gp90 exo−. O6-MeG greatly reduces dNTP incorporation efficiency, resulting in 67-fold preferential error-prone incorporation of dTTP than dCTP. Gp90 exo− extends beyond T:O6-MeG 2-fold more efficiently than C:O6-MeG. Incorporation of dCTP opposite G and incorporation of dCTP or dTTP opposite O6-MeG show fast burst phases. The pre-steady-state incorporation efficiency (kpol/Kd,dNTP) is decreased in the order of dCTP:G>dTTP:O6-MeG>dCTP:O6-MeG. The presence of O6-MeG at template does not affect the binding affinity of polymerase to DNA but it weakened their binding in the presence of dCTP and Mg2+. Misincorporation of dTTP opposite O6-MeG further weakens the binding affinity of polymerase to DNA. The priority of dTTP incorporation opposite O6-MeG is originated from the fact that dTTP can induce a faster conformational change step and a faster chemical step than dCTP. This study reveals that gp90 bypasses O6-MeG in an error-prone manner and provides further understanding in DNA replication encountering mutagenic alkylation DNA damage for P. aeruginosa phage PaP1.

Enzymatic Characterization of Human Immunodeficiency Virus Type 1 Reverse Transcriptase for Use in cDNA Synthesis

The aim of this study is to explore the advantages of using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in cDNA synthesis. Recombinant HIV-1 group M (HIV-1 M) RT and HIV-1 group O (HIV-1 O) RT were produced in an Escherichia coli expression system. In the incorporation of dTTP into poly(rA)-p(dT)(15) (T/P), the K (m) values for dTTP of HIV-1 M RT and HIV-1 O RT were 8 and 12 % of that of Moloney murine leukemia virus (MMLV) RT, respectively, and the K (m) values for T/P were 25 and 23 % of that of MMLV RT, respectively. Compared with MMLV RT, HIV-1 M RT and HIV-1 O RT were less susceptible to formamide, which is frequently used for cDNA synthesis with a G + C-rich RNA to improve specificity. The high substrate affinity and low susceptibility to formamide of HIV-1 RT might be advantageous for its use in cDNA synthesis.

Kinetic analysis of reverse transcriptase activity of bacterial family A DNA polymerases

Some bacterial thermostable, wild-type or genetically engineered family A DNA polymerases have reverse transcriptase activity. However, difference in reverse transcriptase activities of family A DNA polymerases and retroviral reverse transcriptases (RTs) is unclear. In this study, comparative kinetic analysis was performed for the reverse transcriptase activities of the wild-type enzyme of family A DNA polymerase (M1polWT) from Thermus thermophilus M1 and the variant enzyme of family A DNA polymerase (K4polL329A), in which the mutation of Leu329→Ala is undertaken, from Thermotoga petrophila K4. In the incorporation of dTTP into poly(rA)-p(dT)45, the reaction rates of K4polL329A and M1polWT exhibited a saturated profile of the Michaelis–Menten kinetics for dTTP concentrations but a substrate inhibition profile for poly(rA)-p(dT)45 concentrations. In contrast, the reaction rates of Moloney murine leukemia virus (MMLV) RT exhibited saturated profiles for both dTTP and poly(rA)-p(dT)45 concentrations. This suggests that high concentrations of DNA-primed RNA template decrease the efficiency of cDNA synthesis with bacterial family A DNA polymerases.

R964C Mutation of DNA Polymerase γ Imparts Increased Stavudine Toxicity by Decreasing Nucleoside Analog Discrimination and Impairing Polymerase Activity

The R964C mutation of human DNA polymerase gamma was recently linked to stavudine (d4T)-mediated mitochondrial toxicity. We utilized pre-steady-state kinetics to determine the effect of this mutation on incorporation of natural substrate dTTP and the active metabolite of d4T (d4TTP). The R964C polymerase gamma holoenzyme demonstrated a 33% decrease in dTTP incorporation efficiency and a threefold-lower d4TTP discrimination relative to that of the wild-type polymerase gamma, providing a mechanistic basis for genetic predisposition to nucleoside reverse transcriptase inhibitor toxicity.

Nuclear Magnetic Resonance Investigation of Primer−Template Models: Formation of a Pyrimidine Bulge upon Misincorporation

Our previous studies have shown that misaligned structures can occur upon misincorporation of a dNTP opposite thymine templates. The formation of misaligned structures during DNA replication, if not repaired properly, can be bypassed and extended by low-fidelity polymerases and ultimately lead to mutations. In this study, the base pair structures at the replicating sites of a set of primer-template models which mimic the situation upon misincorporation of a dNTP opposite cytosine templates have been determined. High-resolution NMR structural results show that misaligned structures with a C-bulge can be formed upon incorporation of dCTP, dTTP, and dATP opposite 5'-GC, 5'-AC, and 5'-TC templates, respectively. The stabilities of misaligned structures depend on the types of terminal base pairs at the replicating sites. Together with the structural findings in thymine templates, we conclude that terminal G.C and C.G base pairs always contribute a larger stabilizing effect to the misaligned structures containing a pyrimidine bulge than terminal A.T and T.A base pairs. Misalignment and thus deletion mutation are more likely to occur if misincorporation of a nucleotide opposite a pyrimidine template can cause template slippage to form a terminal G.C or C.G base pair. Although misalignment also occurs when the newly formed terminal base pair is an A.T base pair or a T.A base pair, both misaligned and mismatched conformers coexist, which can lead to deletion and substitution mutations, respectively.

Reverse transcriptases: Intron-encoded proteins found in thermophilic bacteria

A number of thermophilic bacteria have been surveyed for possessing reverse transcriptase genes using a degenerate primer approach derived from an alignment of known group II intron encoded reverse transcriptases (RT) from mesophilic prokaryotes and eukaryotes. Six out of 34 thermophilic isolates gave a PCR product that was indicative of an RT internal fragment on sequencing. A putative RT from Bacillus caldolyticus strain EA1 was isolated by genomic walking and cloned into an Escherichia coli expression vector. The recombinant protein proved to be insoluble and was unable to be recovered from the insoluble fraction of lysates of E. coli. The RT was successfully expressed in a baculovirus vector although yields remained low. We followed RT activity during purification using the poly(rC)•p(dG) 12–18, which specifically detects only RNA-dependent DNA polymerase activity. We could not detect incorporation of dTTP into poly(rC) )•p(dG) 12–18 when using uninfected Sf21 lysates and conclude that the substrate is not a template for DNA-dependent DNA polymerase. Although a high level of RT activity was detected in the total cell protein, when compared to the activity detected in the soluble fraction, only about 10% of the activity was soluble. Sequence comparisons showed significant differences between the EA1-IEP and a Geobacillus RT expressed by others. We conclude that it may be necessary to isolate the IEP RT as a ribonucleoprotein to obtain sufficient material for further analysis.

Translesion Synthesis Across 1,N2-Ethenoguanine by Human DNA Polymerases

1,N(2)-Etheno(epsilon)guanine (epsilon) is formed in DNA as a result of exposure to certain vinyl monomers (e.g., vinyl chloride) or from lipid peroxidation. This lesion has been shown to be mutagenic in bacteria and mammalian cells. 1,N(2)-epsilon-G has been shown to block several model replicative DNA polymerases (pols), with limited bypass. Recently, an archebacterial DNA pol, Sulfolobus solfataricus Dpo4, has been shown to copy past 1,N(2)-epsilon-G. In this study, we examined the abilities of recombinant, full-length human pol delta and three human translesion DNA pols to copy past 1,N(2)-epsilon-G. The replicative pol, pol delta, was completely blocked. Pols iota and kappa showed similar rates of incorporation of dTTP and dCTP. Pol eta was clearly the most active of these pols in copying past 1,N(2)-epsilon-G, incorporating in the order dGTP > dATP > dCTP, regardless of whether the base 5' of 1,N(2)-epsilon-G in the template was C or T. Pol eta also had the highest error frequency opposite 1,N(2)-epsilon-G. Analysis of the extended products of the pol eta reactions by mass spectrometry indicated only two products, both of which had G incorporated opposite 1,N(2)-epsilon-G and all other base pairing being normal (i.e., G:C and A:T). One-half of the products contained an additional A at the 3'-end, presumably arising from a noninformational blunt end addition or possibly a slipped insertion mechanism at the end of the primer-template replication process. In summary, the most efficient of the four human DNA pols was pol eta, which appeared to insert G opposite 1,N(2)-epsilon-G and then copy correctly. This pattern differs with the same oligonucleotide sequences and 1,N(2)-epsilon-G observed using Dpo4, emphasizing the importance of pols in mutagenesis events.

Pre-steady-state Kinetic Characterization of the DinB Homologue DNA Polymerase of Sulfolobus solfataricus

Equilibrium as well as pre-steady-state measurements were performed to characterize the molecular basis of DNA binding and nucleotide incorporation by the thermostable archaeal DinB homologue (Dbh) DNA polymerase of Sulfolobus solfataricus. Equilibrium titrations show a DNA binding affinity of about 60 nm, which is approximately 10-fold lower compared with other DNA polymerases. Investigations of the binding kinetics applying stopped-flow and pressure jump techniques confirm this weak binding affinity. Furthermore, these measurements suggest that the DNA binding occurs in a single step, diffusion-controlled manner. Single-turnover, single dNTP incorporation studies reveal maximal pre-steady-state burst rates of 0.64, 2.5, 3.7, and 5.6 s(-1) for dTTP, dATP, dGTP, and dCTP (at 25 degrees C), which is 10-100-fold slower than the corresponding rates of classical DNA polymerases. Another unique feature of the Dbh is the very low nucleotide binding affinity (K(d) approximately 600 mum), which again is 10-20-fold lower compared with classical DNA polymerases as well as other Y-family polymerases. Surprisingly, the rate-limiting step of nucleotide incorporation (correct and incorrect) is the chemical step (phosphoryl transfer) and not a conformational change of the enzyme. Thus, unlike replicative polymerases, an "induced fit" mechanism to select and incorporate nucleotides during DNA polymerization could not be detected for Dbh.

Mechanistic Insights into the Suppression of Drug Resistance by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Using α-Boranophosphate Nucleoside Analogs

A class of amino acid substitutions in drug-resistant HIV-1 reverse transcriptase (RT) is responsible for the selectively impaired incorporation of the nucleotide analog inhibitor into DNA. We have shown previously that alpha-boranophosphate nucleoside analogs suppress RT-mediated resistance when the catalytic rate is responsible for drug resistance such as in the case of K65R and dideoxy (dd)NTPs, and Q151M toward AZTTP and ddNTPs. Here, we extend this property to BH3-d4TTP and BH3-3TCTP toward their clinically relevant mutants Q151M and M184V, respectively. Pre-steady-state kinetics on mutants of the Q151M RT family reveal a 3-5-fold resistance to d4TTP. This resistance is suppressed using BH3-d4TTP. Likewise, resistance to 3TCTP by M184V RT (30-fold) and K65R/M184V RT (180-fold) is suppressed using BH3-3TCTP because of a 160-fold acceleration of the catalytic constant kpol. Mechanistic insights into the rate enhancement were obtained using various alpha-boranophosphate nucleotides. The presence of the BH3 group renders kpol independent of amino acid substitutions present in RT. Indeed, the approximately 100-fold decrease in polymerase activity caused by the R72A substitution is restored to wild-type levels using BH3-dTTP. Metal ion titration studies show that alpha-boranophosphate nucleoside analogs enhance 3-8-fold the binding of Mg2+ ions to the active site of the RT.DNA.dNTP complex and alleviate the requirement of critical amino acids involved in phosphodiester bond formation. To our knowledge, this is the first example of rescue of polymerase activity by means of a nucleotide analog.

Kinetics of Nucleotide Incorporation Opposite Polycyclic Aromatic Hydrocarbon−DNA Adducts by Processive Bacteriophage T7 DNA Polymerase

A series of six oligonucleotides with dihydrodiol epoxide metabolites of the polycyclic aromatic hydrocarbons (PAHs) benz[a]anthracene and benzo[a]pyrene attached to adenine N6 and guanine N2 atoms were prepared and studied with the processive bacteriophage DNA polymerase T7, exonuclease- (T7-). HIV-1 reverse transcriptase was much less efficient in polymerization than T7-. Benz[a]anthracene and benzo[a]pyrene adducts strongly blocked incorporation of dTTP and dCTP opposite the A and G derivatives, respectively. dATP was preferentially incorporated in all cases. Steady state kinetic analysis indicated that the low catalytic efficiency with adducted DNA was due to both increased K(m) and lowered k(cat) values. Some differences due to PAH stereochemistry were observed. Fluorescence estimates of K(d) and presteady state kinetic measurements of k(off) showed no major decrease in the affinity of T7- with damaged DNA substrates or with dNTPs. Presteady state kinetics showed a lack of the normal burst kinetics for dNTP incorporation with all PAH-DNA derivatives. These results indicate that the rate-limiting step is at or before the step of phosphodiester bond formation; release of the oligonucleotide is no longer the slowest step. Thio elemental effects (substitution of alpha-oxygen with sulfur) were relatively small, in contrast to previous work with T7- and 8-oxo-7,8-dihydroguanine. The effect of these bulky PAH adducts is either to attenuate rates of conformational changes or to introduce an additional conformation problem but not to alter the inherent affinity of the polymerase for DNA or dNTPs.

Expression of Deoxynucleotide Carrier Is Not Associated with the Mitochondrial DNA Depletion Caused by Anti-HIV Dideoxynucleoside Analogs and Mitochondrial dNTP Uptake

Our previous studies suggested that the dNTP/dNDP transporter systems that exist in mitochondria for transporting dNTP/dNDP from the cytoplasm to the mitochondria for mitochondrial DNA (mtDNA) synthesis play a critical role in delayed cytotoxicity of anti-human immunodeficiency virus (HIV) dideoxynucleoside analogs in mitochondria. A protein, termed mitochondrial deoxynucleotide carrier (DNC), based on its ability to transport dNTPs in reconstituted proteoliposomes, was recently isolated. Lacking cellular information to substantiate DNC's involvement in the delayed cytotoxicity of dideoxynucleoside analogs, we expressed DNC and reconstituted it into proteoliposomes. The K(m) values for dNTPs uptake by reconstituted DNC were in the millimolar range, which is a thousandfold higher than that of the physiological level. Furthermore, we found that overexpressing DNC (wt and G177A-mutated DNC) in RKO cells did not sensitize the cells to the mtDNA depletion caused by beta-d-2',3'-dideoxycytidine (ddC), 2',3'-didehydro-2',3'-dideoxythymidine, and 2',3'-dideoxyinosine or affect the mtDNA recovery rate after ddC treatment. Mitochondria isolated from DNC-overexpressing cells did not significantly differ from that isolated from RKO cells in terms of the rate of uptake or the incorporation of dTTP into mitochondria DNA. Down-regulation of DNC expression by small interfering RNA was also ineffective in changing the action of dideoxynucleoside analogs on the mtDNA depletion and the rate of dTTP uptake into isolated mitochondria. Down-regulation of both DNC and thymidine kinase-2 also did not cause mtDNA depletion. We conclude that DNC does not play an important role in the delayed cytotoxicity (mtDNA depletion) of anti-HIV dideoxynucleoside analogs and dNTPs uptake into mitochondria.

Altering DNA polymerase incorporation fidelity by distorting the dNTP binding pocket with a bulky carcinogen-damaged template.

Fidelity of DNA polymerases is predominantly governed by an induced fit mechanism in which the incoming dNTP in the ternary complex fits tightly into a binding pocket whose geometry is determined by the nature of the templating base. However, modification of the template with a bulky carcinogen may alter the dNTP binding pocket and thereby the polymerase incorporation fidelity. High fidelity DNA polymerases, such as bacteriophage T7 DNA polymerase, are predominantly blocked by bulky chemical lesions on the template strand during DNA replication. However, some mutagenic bypass can occur, which may lead to carcinogenesis. Experimental studies have shown that a DNA covalent adduct derived from (+)-anti-BPDE [(+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene], a carcinogenic metabolite of benzo[a]pyrene (BP), primarily blocks Sequenase 2.0, an exo(-) T7 DNA polymerase; however, a mismatched dATP can be preferentially inserted opposite the damaged adenine templating base within the active site of the polymerase [Chary, P., and Lloyd, R. S. (1995) Nucleic Acids Res. 23, 1398-1405]. The goal of this work is to elucidate structural features that contribute to DNA polymerase incorporation fidelity in the presence of this bulky covalent adduct and to interpret the experimental findings on a molecular level. We have carried out molecular modeling and molecular dynamics simulations with AMBER 6.0, investigating a T7 DNA polymerase primer-template closed ternary complex containing this 10S (+)-trans-anti-[BP]-N(6)-dA adduct in the templating position within the polymerase active site. All four incoming dNTPs were studied. The simulations show that the BP ring system fits well into an open pocket on the major groove side of the modified template adenine with anti glycosidic bond conformation, without disturbing critical polymerase-DNA interactions. However, steric hindrance between the BP ring system and the primer-template DNA causes displacement of the modified template adenine, so that the dNTP base binding pocket is enlarged. This alteration can explain the experimentally observed preference for incorporation of dATP opposite this lesion. These studies also rationalize the observed lower probabilities of incorporation of the other three nucleotides. Our results suggest that the differences in incorporation of dGTP, dCTP, and dTTP are due to the effects of imperfect geometric complementarity. Thus, the simulations suggest that altered DNA polymerase incorporation fidelity can result from adduct-induced changes in the dNTP base binding pocket geometry. Furthermore, plausible structural explanations for the observed effects of [BP]-N(6)-dA adduct stereochemistry on the observed stalling patterns are proposed.

Discovery of DNA Polymerase

Discovery of DNA Polymerase

Neighbouring bases in template influence base-pairing of isoguanine.

Assuming that the efficiency of the incorporation of 5-methyl-2'-deoxyisocytosine-5' triphosphate (dMiCTP) and dTTP opposite isoguanine (iG) is a measure of the proportion of the keto and enol tautomers of iG in the Thermus aquaticus DNA polymerase active centre, we studied the influence of temperature and iG-neighbouring bases in the template on base-pairing of iG. On the basis of experiments with four sequences (3'-TXT-5', 3'-GXG-5', 3'-CXC-5', 3'-CXT-5', where X=iG) at 37, 50, 65 and 80 degrees C, we found that 3'-neighbours decrease the fraction of the keto tautomer in the order C>G>or=T, whereas temperature apparently does not influence the tautomeric equilibrium of iG. The variability of the ratio of incorporation of dMiCTP versus dTTP (5-20) primarily reflects the variability of K (m) values, since V (max) values are roughly similar, which indicates that the iG.MiC and iG.T pairs fit the polymerase active centre equally well. The altering of the base-pairing of iG by sequence context is discussed in relation to tautomerism and miscoding of this oxidized adenine derivative. A key derivative for preparation oligodeoxynucleotides, O (2)-diphenylcarbamoyl- N (6)-[(dimethylamino)ethylidene]-2'-deoxyisoguanosine, is extremely labile (t (1/2)=3.5 min) in 3% trichloroacetic acid/dichloromethane, i.e. under the conditions of automated DNA synthesis, which results in low yield and length heterogeneity of templates.

Implication of the tRNA initiation step for human immunodeficiency virus type 1 reverse transcriptase in the mechanism of 3'-azido-3'-deoxythymidine (AZT) resistance.

There is a lack of correlation between biochemical studies and the observed clinical resistance of AIDS patients on long-term AZT therapy. Mutant HIV-1 reverse transcriptase in the viral isolates from these patients shows a 100-fold decrease in sensitivity to AZT whereas little or no difference is observed in kinetic parameters in vitro using steady-state kinetic analysis. A pre-steady-state kinetic analysis was used to examine the binding and incorporation of 2'-deoxythymidine 5'-triphosphate (dTTP) and 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP) by wild-type HIV-1 reverse transcriptase and a clinically important AZT-resistant mutant form of the enzyme (D67N, K70R, T215Y, K219Q) utilizing a physiologically relevant RNA 18-mer/RNA 36-mer primer-template substrate. It was determined that with this RNA/RNA substrate there is a 2.6-fold increase in the selection for incorporation of the natural nucleotide dTTP over the unnatural nucleoside analogue AZTTP by AZT-resistant reverse transcriptase as compared to its wild-type form. This observation indicates that the tRNALys initiation step plays an important role in the development of drug resistance. Furthermore, this result implies that the structural basis of AZT resistance in HIV-1 reverse transcriptase involves the conformation of the RNA-DNA junction (formed upon attachment of a deoxynucleotide to the RNA primer). Taken together, these observations suggest a new pharmacological basis for the development of more effective and novel AIDS drugs.

DNA polymerase beta: multiple conformational changes in the mechanism of catalysis.

Stopped-flow fluorescence assay was applied to identify conformational changes in the catalytic cycle of DNA polymerase beta (Pol beta), using a synthetic DNA primer/template containing 2-aminopurine (2-AP) at the template position opposite the incoming dNTP. Two phases of fluorescence change were observed in the stopped-flow fluorescence assay of the incorporation of the correct nucleotide dTTP. The rate of the slow phase corresponds to that of product formation. This slow phase was identified as the result of a rate-limiting conformational change step before chemistry because this slow phase was also observed with a dideoxynucleotide at the 3' end of the primer which prevents chemical bond formation. The fast phase was also attributed to a conformational change step since its dependence on [dTTP] is hyperbolic. The rates of the two phases and their dependence on [dTTP] and [Mg2+] suggest that the fast conformational change is induced by the binding of MgdNTP and the slow conformational change is induced by the binding of the catalytic Mg2+ ion. The same biphasic kinetics with different rates were also observed with the thio analog dTTPalphaS and incorrect nucleotides dATP, dGTP, and dCTP. The structural nature for the two conformational changes has been discussed in relation to the available structural information of this enzyme. The results could help to explain how a polymerase controls and achieves its fidelity with a multiple conformational change mechanism.

Structure of the hydrogen bonding complex of O6-methylguanine with cytosine and thymine during DNA replication.

During DNA replication, mutations occur when an incorrect dNTP is incorporated opposite a carcinogen-modified nucleotide. We have probed the structures of the interaction between O 6-methylguanine ( O 6mG) and cytosine and thymine during replication by kinetic means in order to examine the structure during the rate determining step. The kinetics of incorporation of dCTP and dTTP opposite O 6mG and three analogs, S 6-methyl-6-thioguanine, O 6-methyl-1-deazaguanine and O 6-methylhypoxanthine, have been measured with four polymerases, the Klenow fragment of DNA polymerase I, the Klenow fragment with the proof-reading exonuclease inactivated, Taq and Tth polymerases. In the insertion of dTTP opposite O 6mG, a large decrease in V max/ K m was observed only upon modification of the N1 position. This result is consistent with a Watson-Crick type configuration. For the incorporation of dCTP, the V max/ K m was significantly decreased only with removal of the exocyclic amino group at the 2 position. The pH dependence of the ratio of incorporation of dCTP and dTTP was independent of pH at physiological pH. This result suggests that dCTP is incorporated via an uncharged complex such as the wobble configuration.

Kinetic analysis of four HIV-1 reverse transcriptase enzymes mutated in the primer grip region of p66. Implications for DNA synthesis and dimerization.

The highly conserved primer grip region in the p66 subunit of HIV-1 reverse transcriptase (RT) is formed by the beta12-beta13 hairpin (residues 227-235). It has been proposed to play a role in aligning the 3'-OH end of the primer in a position for nucleophilic attack on an incoming dNTP. To analyze the importance of the primer grip for RT function, mutant RTs were used that contain single alanine substitutions of residues Trp229, Met230, Gly231, and Tyr232 in the p66 subunit of the heterodimeric p66/51 enzyme. Steady-state and pre-steady-state kinetic analyses of the enzymes were performed. All mutant enzymes revealed reduced polymerase activity. Mutation of Y232A showed the smallest effect on polymerase function. Equilibrium fluorescence titrations demonstrated that the affinity of the mutants for tRNA was only slightly affected. However, the affinity for primer-template DNA was reduced 27-fold for mutant p66(W229A)/51 and 23-fold for mutant p66(G231A)/51, and the maximal pre-steady-state rate of nucleotide incorporation, kpol, was reduced 27-fold for p66(W229A)/51 and 70-fold for p66(G231A)/51, respectively. Mutant p66(M230A)/51 revealed no reduced affinity for primer-template but showed a 71-fold reduced affinity for dTTP. Additionally, the mutations Trp229 and Gly231 affected the stability of the RT heterodimer.

Reactive oxygen species are involved in nickel inhibition of DNA repair

Nickel has been shown to inhibit DNA repair in a way that may play a role in its toxicity. Since nickel treatment increases cellular reactive oxygen species (ROS), we have investigated the involvement of ROS in nickel inhibition of DNA repair. Inhibition of glutathione synthesis or catalase activity increased the enhancing effect of nickel on the cytotoxicity of ultraviolet (UV) light. Inhibition of catalase and glutathione peroxidase activities also enhanced the retardation effect of nickel on the rejoining of DNA strand breaks accumulated by hydroxyurea plus cytosine-beta-D-arabinofuranoside in UV-irradiated cells. Since DNA polymerization and ligation are involved in the DNA-break rejoining, we have investigated the effect of ROS on these two steps in an extract of Chinese hamster ovary cells. Nickel inhibition of the incorporation of (3H)dTTP into the DNase I-activated calf thymus DNA was stronger than the ligation of poly(dA) x oligo(dT), whereas H2O2 was more potent in inhibiting DNA ligation than DNA polymerization. Nickel, in the presence of H2O2, exhibited a synergistic inhibition on both DNA polymerization and ligation and caused protein fragmentation. In addition, glutathione could completely recover the inhibition by nickel or H2O2 alone but only partially recover the inhibition by nickel plus H2O2. Therefore, nickel may bind to DNA-repair enzymes and generate oxygen-free radicals to cause protein degradation in situ. This irreversible damage to the proteins involved in DNA repair, replication, recombination, and transcription could be important for the toxic effects of nickel.

Replication across O6-Methylguanine by Human DNA Polymerase β in Vitro: INSIGHTS INTO THE FUTILE CYTOTOXIC REPAIR AND MUTAGENESIS OF O6-METHYLGUANINE

Replication in vivo across unrepaired O6-methylguanine (m6dG) lesions by mammalian DNA polymerase beta (pol beta) during short patch repair may contribute to the cytotoxicity and mutagenesis of m6dG. We have employed in vitro steady state kinetic analysis to investigate the replication of oligonucleotide templates containing site-specific m6dG by human pol beta. Our results show that m6dG is a strong but not absolute block to replication by pol beta. pol beta exhibits mixed kinetic discrimination during overall replication across dG and m6dG. pol beta preferentially inserts dTMP rather than dCMP opposite m6dG. However, pol beta extends from the dC-m6dG base pair more efficiently than from the dT-m6dG base pair. This is in strong contrast to other polymerases such as the exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (exo-KF) that preferentially extends dT-m6dG by a factor of 10 over dC-m6dG. When both insertion and extension are considered, pol beta has a 15-fold overall preference for incorporation of the mutagenic substrate dTTP rather than the nonmutagenic substrate dCTP during replication across m6dG. This suggests that pol beta, in concert with the T:G-specific thymine DNA glycosylase, may be intricately involved in the futile cytotoxic repair induced by m6dG. Our results also suggest that replication across m6dG by pol beta may contribute to m6dG-induced G --> A transition mutations.