Proofreading of a mutagenic nucleotide, N4-aminodeoxycytidylic acid, by Escherichia coli DNA polymerase I

N4-Aminodeoxycytidine triphosphate, a putative metabolite of N4-aminocytidine which is a potent mutagen, is incorporated, in vitro, into polynucleotides in place of dCTP and at a much lesser extent, but significantly, in place of dTTP by E. coli DNA polymerase I large fragment. The activity of the polymerase to proofread this unnatural nucleotide has now been investigated. The results indicate that the 3'-5' exonuclease in the polymerase recognizes N4-aminocytosine as an incorrect base when N4-aminocytosine is incorporated opposite adenine but the enzyme cannot distinguish N4-aminocytosine from cytosine when it is incorporated opposite guanine.     

Ambiguous incorporation of N4-aminodeoxycytidine 5'-triphosphate to DNA synthesized in vitro

Molecular mechanism of the mutation induced by N4-aminocytidine was studied. The specificity of in vitro incorporation of N4-aminodeoxycytidine 5'-triphophate catalyzed by E. coli DNA polymerase large fragment was analyzed. The results have shown that this cytosine analog can be efficiently incorporated as a substitute of cytosine, and that it can also be incorporated with a low efficiency as a substitute of thymine. We have also shown that the N4-aminocytosine incorporated opposite adenine can be excised as its monophosphate at a high frequency. The N4-aminocytosine residues in the polynucleotide templates can be read by the enzyme as efficiently as cytosines, and guanines were incorporated opposite them.     

Mutagenesis by N4-aminocytidine: induction of AT to GC transition and its molecular mechanism

N4-Aminocytidine is a potent mutagen toward Escherichia coli and Salmonella typhimurium. It induced reversion of an amber mutant of phi X174 phage (am3) to the wild type. This reversion was shown to be exclusively due to the AT to GC transition. It is likely that N4-aminocytidine is metabolized within the bacterial cells into N4-aminodeoxycytidine 5'-triphosphate and this nucleotide is incorporated into DNA during the multiplication of the cells and the phages, thereby causing base-pair transitions. The molecular basis for this erroneous replication was obtained in studies of in vitro incorporation of N4-aminodeoxycytidine 5'-triphosphate into polynucleotides catalyzed by the E. coli DNA polymerase I large fragment. The results have shown that this cytosine analogue can be efficiently incorporated as a substitute of cytosine and that it can also be incorporated as a substitute of thymine. The ratio in the rate of the N4-aminocytosine nucleotide incorporation to that of natural nucleotide incorporation was 1/2 to cytosine and 1/30 to thymine. Furthermore, the N4-aminocytosine residues in the polynucleotide templates can be read by the enzyme as efficiently as cytosines, and guanines were incorporated opposite to them.     

Mutagenicity of N4-aminocytidine and its derivatives in Chinese hamster lung V79 cells. Incorporation of N4-aminocytosine into cellular DNA

N4-Aminocytidine induced mutation to 6-thioguanine resistance in Chinese hamster lung V79 cells in culture. Previous studies with experimental systems of in vitro DNA synthesis and of phage and bacterial mutagenesis have shown that this nucleoside analog induces base-pair transitions through its incorporation into DNA, with its erroneous base-pairing property. Incorporation of exogenously added [5-3H]N4-aminocytidine into the DNA of V79 cells was in fact observed in the present study. N4-Aminodeoxycytidine was not mutagenic for the V79 cells. Several alkylated N4-aminocytidine derivatives were tested for their mutagenicity in this system. Those with an alkyl group on the N'-nitrogen of the hydrazino group at position 4 of N4-aminocytidine were mutagenic, but those having an alkyl on the N4-nitrogen were not. These results are consistent with those previously observed in the bacterial mutagenesis systems, and agree with a mechanism of mutation in which a tautomerization of N4-aminocytosine is the necessary step for causing the erroneous base pairing.     

Mutagenic nucleoside analog N4-aminocytidine: metabolism, incorporation into DNA, and mutagenesis in Escherichia coli.

N4-Aminocytidine, a nucleoside analog, is strongly mutagenic to various organisms including Escherichia coli. Using E. coli WP2 (trp), we measured the incorporation of [5-3H]N4-aminocytidine into DNA and at the same time measured the frequency of reversion of the wild type, thereby attempting to correlate the incorporation with mutation induction. First, we observed that N4-aminocytidine uptake by the E. coli cells was as efficient as cytidine uptake. High-pressure liquid chromatographic analysis of nucleoside mixtures obtained by enzymatic digestion of isolated cellular DNA showed that the DNA contained [3H]N4-aminodeoxycytidine, corresponding to 0.01 to 0.07% of the total nucleoside; the content was dependent on the dose of N4-aminocytidine. There was a linear relationship between the N4-aminocytosine content in DNA and the mutation frequency observed. These results constitute strong evidence for the view that the N4-aminocytidine-induced mutation in E. coli is caused by the incorporation of this agent into DNA as N4-aminodeoxycytidine. We also found that the major portion of radioactivity in DNA of cells that had been treated with [5-3H]N4-aminocytidine was in the deoxycytidine fraction. We propose a metabolic pathway for N4-aminocytidine in cells of E. coli. This pathway involves the formation of both N4-aminodeoxycytidine 5'-triphosphate and deoxycytidine 5'-triphosphate; the deoxycytidine 5'-triphosphate formation is initiated by conversion of N4-aminocytidine into uridine. In support of this proposed scheme, a cytidine deaminase preparation obtained from E. coli catalyzed the decomposition of N4-aminocytidine into uridine and hydrazine.     

Structural basis for proficient incorporation of dTTP opposite O6-methylguanine by human DNA polymerase iota

O(6)-methylguanine (O(6)-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase ι core enzyme was determined for nucleoside triphosphate incorporation opposite O(6)-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol ι, which showed that dTTP incorporation occurs with high efficiency opposite O(6)-methylG. Misincorporation of dTTP opposite O(6)-methylG occurred with ～6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol ι with O(6)-methylG as the template base and incoming dCTP or dTTP were solved and showed that O(6)-methylG is rotated into the syn conformation in the pol ι active site and that dTTP misincorporation by pol ι is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O(6)-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O(6)-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O(6) atoms of O(6)-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O(6)-methylG by human pol ι, in contrast to the mispairing modes observed previously for O(6)-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.     

Chemical synthesis of novel base pairs and their enzymatic incorporation into DNA.

A novel base pair, 2-amino-6-(N,N-dimethylamino)purine (denoted x) and the counter part, pyridin-2-one (denoted y) were designed. The bulky 6-dimethylamino group of x is expected to eliminate base pairing with all natural bases. The phosphoramidite of x for DNA templates and the 2'-deoxyribonucleoside triphosphate of y (dyTP) for a substrate were synthesized, and the selectivity of the enzymatic incorporation of dyTP opposite x in the templates was examined. dyTP was preferentially incorporated opposite x than canonical dNTPs by Klenow fragment of Escherichia coli DNA polymerase I. While dyTP was also incorporated opposite A and G, the misincorporation was suppressed in the presence of dTTP and dCTP, respectively.     

Induction of mutation in vitro in phage phi X174 am3 by N4-aminodeoxycytidine triphosphate

When phi X174 am3-phage-infected E. coli is treated with N4-aminocytidine, reversion of the phage to the wild type is efficiently induced. The mechanism of this reversion is considered to consist of metabolic conversion of N4-aminocytidine into its deoxynucleoside 5'-triphosphate followed by incorporation of the nucleotide into the replicating phage DNA, thereby causing AT-to-GC transition at the am3 locus. The second half of this mechanism has now been experimentally proved, using an in vitro mutagenesis system. Thus, by nick-translation, N4-aminodeoxycytidine 5'-triphosphate was incorporated into the replicative form of phi X174 am3 DNA, and the DNA was used to transfect CA++-treated E. coli HF4714 (sup+). The reversion frequency of the phage produced was up to one-order of magnitude greater than that of the control in which the nick-translation had been done without the addition of N4-aminodeoxycytidine triphosphate. This nucleotide analog may be useful as a reagent for in vitro site-directed mutagenesis.     

DNA polymerases β and λ as potential participants of TLS during genomic DNA replication on the lagging strand

The main strategy used by pro-and eukaryotic cells for replication of damaged DNA is translesion synthesis (TLS). Here, we investigate the TLS process catalyzed by DNA polymerases β and λ on DNA substrates using mono-or dinucleotide gaps opposite damage located in the template strand. An analog of a natural apurinic/apyrimidinic site, the 3-hydroxy-2-hydroxymetylthetrahydrofuran residue (THF), was used as damage. DNA was synthesized in the presence of either Mg²⁺ or Mn²⁺. DNA polymerases β and λ were able to catalyze DNA synthesis across THF only in the presence of Mn²⁺. Moreover, strand displacement synthesis was not observed. The primer was elongated by only one nucleotide. Another unusual aspect of the synthesis is that dTTP could not serve as a substrate in all cases. dATP was a preferential substrate for synthesis catalyzed by DNA polymerase β. As for DNA polymerase λ, dGMP was the only incorporated nucleotide out of four investigated. Modified on heterocyclic base photoreactive analogs of dCTP and dUTP showed substrate specificity for DNA polymerase β. In contrast, the dCTP analog modified on the exocyclic amino group was a substrate for DNA polymerase λ. We also observed that human replication protein A inhibited polymerase incorporation by both DNA polymerases β and λ on DNA templates containing damage.     

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.     

Spectrum of N4-aminocytidine mutagenesis

N4-Aminocytidine, a nucleoside analog, is a potent mutagen towards phages, bacteria, Drosophila and mammalian cells in culture. In vitro, biochemical studies indicate that this reagent acts by being incorporated into DNA. To elucidate the mechanism of N4-aminocytidine mutagenesis, it is essential to identify the nature of DNA sequence alterations taking place during the mutagenesis. We have analyzed the nucleotide sequence changes in the lac promoter-lacZ alpha region of M13mp2 phage induced by treatment of phage-infected Escherichia coli with N4-aminocytidine. The sequence alterations of DNA samples from 89 mutants of the phage were determined. These mutants had single point mutations, except one mutant, in which a double point mutation was detected. Several hot spots were found: however, there are no apparent relations to particular DNA sequences regarding the locations of these spots. All the mutations are transitions; neither transversions nor deletions/insertions were found. A feature in these transitions is that the A/T to G/C and G/C to A/T changes occur at approximately equal rates. The overall picture of the mutagenesis is consistent with a scheme in which misincorporation and misreplication caused by the modified cytosine structure are the key steps in the DNA replication leading to transitions. Similar nucleotide alterations were found for the mutagenesis induced by an alkylated derivative, N'-methyl-N4-aminocytidine. N4-Aminocytidine also induced reversions of these mutants; both A/T to G/C and G/C to A/T transitions again took place.     

Toward understanding the mutagenicity of an environmental carcinogen: structural insights into nucleotide incorporation preferences

Bulky carcinogen-DNA adducts, including (+)-trans-anti-[BP]-N(2)-dG derived from the reaction of (+)-anti-benzo[a]pyrene diol epoxide with guanine, often block the progression of DNA polymerases. However, when rare bypass of the lesions does occur, they may be misreplicated. Experimental results have shown that nucleotides are inserted opposite the (+)-trans-anti-[BP]-N(2)-dG adduct by bacteriophage T7 DNA polymerase with the order of preference A>T>or=G>C. To gain structural insights into the effects of the bulky adduct on nucleotide incorporation within the polymerase active site, molecular modeling and molecular dynamics simulations were carried out using T7 DNA polymerase to permit the relation of function to structure. We modeled the (+)-trans-anti-[BP]-N(2)-dG adduct opposite incoming dGTP, dTTP and dCTP nucleotides, as well as unmodified guanine opposite its normal partner dCTP as a control, to compare with our previous simulation with dATP opposite the adduct. The modeling required that the (+)-trans-anti-[BP]-N(2)-dG adduct adopt the syn conformation in each case to avoid deranging essential protein-DNA interactions. While the dATP: (+)-trans-anti-[BP]-N(2)-dG pair was well accommodated within the active site of T7 DNA polymerase, dCTP fit poorly opposite the adduct, adopting an orientation perpendicular to the plane of the syn modified guanine during the simulation. Rotation about the glycosidic bond of the dCTP residue to this abnormal position was allowed because only one hydrogen bond between dCTP and the (+)-trans-anti-[BP]-N(2)-dG residue evolved during the simulation, and this hydrogen bond was directly across from the dCTP glycosidic bond. The dTTP and dGTP nucleotides, incorporated with an intermediate preference opposite (+)-trans-anti-[BP]-N(2)-dG, were accommodated reasonably well, but not as stably as the dATP nucleotide, due to a skewed primer-template alignment and more exposed BP moiety, respectively. In addition, the extent of stabilizing interactions between the nascent base-pair in each simulation was correlated positively with the incorporation preference of that particular nucleotide. The dATP nucleotide is accommodated most stably opposite the adduct, with protein-DNA hydrogen bonding interactions and an active-site pocket size that do not deviate significantly from those of the control simulation. The simulations of dTTP and dGTP opposite (+)-trans-anti-[BP]-N(2)-dG exhibited more instability in interactions between the protein and the nascent base-pair than the dATP system. However, the active-site pocket size of the dTTP and dGTP simulations remained stable. The dCTP: (+)-trans-anti-[BP]-N(2)-dG system had the least number of stabilizing interactions, and the active-site pocket of this system increased in size significantly compared to the control and other dNTPs opposite the adduct. These simulations elucidated why A is inserted opposite (+)-trans-anti-[BP]-N(2)-dG most frequently, while T and G are inserted opposite the adduct to an extent intermediate between A and C, and C is most rarely incorporated. Structural rationalization of the incorporation preference opposite (+)-trans-anti-[BP]-N(2)-dG by T7 DNA polymerase contributes to providing a molecular explanation for mutations caused by this carcinogen-DNA adduct in a model system.     

Quantitation of cellular deoxynucleoside triphosphates

Eukaryotic cells contain a delicate balance of minute amounts of the four deoxyribonucleoside triphosphates (dNTPs), sufficient only for a few minutes of DNA replication. Both a deficiency and a surplus of a single dNTP may result in increased mutation rates, faulty DNA repair or mitochondrial DNA depletion. dNTPs are usually quantified by an enzymatic assay in which incorporation of radioactive dATP (or radioactive dTTP in the assay for dATP) into specific synthetic oligonucleotides by a DNA polymerase is proportional to the concentration of the unknown dNTP. We find that the commonly used Klenow DNA polymerase may substitute the corresponding ribonucleotide for the unknown dNTP leading in some instances to a large overestimation of dNTPs. We now describe assay conditions for each dNTP that avoid ribonucleotide incorporation. For the dTTP and dATP assays it suffices to minimize the concentrations of the Klenow enzyme and of labeled dATP (or dTTP); for dCTP and dGTP we had to replace the Klenow enzyme with either the Taq DNA polymerase or Thermo Sequenase. We suggest that in some earlier reports ribonucleotide incorporation may have caused too high values for dGTP and dCTP.     

The E295K Cancer Variant of Human Polymerase β Favors the Mismatch Conformational Pathway during Nucleotide Selection

DNA polymerase β (pol β) is responsible for gap filling synthesis during repair of damaged DNA as part of the base excision repair pathway. Human pol β mutations were recently identified in a high percentage (∼30%) of tumors. Characterization of specific cancer variants is particularly useful to further the understanding of the general mechanism of pol β while providing context to disease contribution. We showed that expression of the carcinoma variant E295K induces cellular transformation. The poor polymerase activity exhibited by the variant was hypothesized to be caused by the destabilization of proper active site assembly by the glutamate to lysine mutation. Here, we show that this variant exhibits an unusual preference for binding dCTP opposite a templating adenine over the cognate dTTP. Biochemical studies indicate that the noncognate competes with the cognate nucleotide for binding to the polymerase active site with the noncognate incorporation a function of higher affinity and not increased activity. In the crystal structure of the variant bound to dA:dCTP, the fingers domain closes around the mismatched base pair. Nucleotide incorporation is hindered because key residues in the polymerase active site are not properly positioned for nucleotidyl transfer. In contrast to the noncognate dCTP, neither the cognate dTTP nor its nonhydrolyzable analog induced fingers closure, as isomorphous difference Fourier maps show that the cognate nucleotides are bound to the open state of the polymerase. Comparison with published structures provides insight into the structural rearrangements within pol β that occur during the process of nucleotide discrimination.     

Unusual aspects of human thymidylate synthase in mouse cells introduced by DNA-mediated gene transfer

Thymidylate synthase-negative mutants of mouse FM3A cells were transformed to thymidine prototrophs by human DNA. The stable transformants had only human thymidylate synthase and segments of human DNA. They grew normally but had unusually high levels of the human enzyme. In two transformants examined, however, neither was the dTTP pool elevated nor the dCTP pool decreased. DNA synthesis in permeabilized cells of a transformant was more efficient than that in the wild type with dATP, dGTP, dCTP, and dUMP as substrates, but this was not so when dUMP was replaced by dTTP. Unlike the mouse enzyme, the human enzyme in the transformants did not co-sediment with DNA polymerase alpha and thymidine kinase in a sucrose gradient, suggesting that the human enzyme is not incorporated into a multienzyme complex for DNA replication. The high levels of the human enzyme in the transformants were suppressed to various degrees by fusion with a wild type mouse line. No active hybrid dimer enzyme was found between the human and mouse enzymes, which each consist of two identical subunits. Thus, the human enzyme in the transformants seems to behave differently from the mouse enzyme and its overproduction seems to be necessary for supporting the normal growth of the transformants.     

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 (k_pol/K_d,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.     

Mutational specificity of DNA precursor pool imbalances in yeast arising from deoxycytidylate deaminase deficiency or treatment with thymidylate

Disruption of the dCMP deaminase (DCD1) gene, or provision of excess dTMP to a nucleotide-permeable strain, produced dramatic increases in the dCTP or dTTP pools, respectively, in growing cells of the yeast Saccharomyces cerevisiae. The mutation rate of the SUP4-o gene was enhanced 2-fold by the dCTP imbalance and 104-fold by the dTTP imbalance. 407 SUP4-o mutations that arose under these conditions, and 334 spontaneous mutations recovered in an isogenic strain having balanced DNA precursor levels, were characterized by DNA sequencing and the resulting mutational spectra were compared. Significantly more (greater than 98%) of the changes resulting from nucleotide pool imbalance were single base-pair events, the majority of which could have been due to misinsertion of the nucleotides present in excess. Unexpectedly, expanding the dCTP pool did not increase the fraction of A.T----G.C transitions relative to the spontaneous value nor did enlarging the dTTP pool enhance the proportion of G.C----A.T transitions. Instead, the elevated levels of dCTP or dTTP were associated primarily with increases in the fractions of G.C----C.G or A.T----T.A. transversions, respectively. Furthermore, T----C, and possibly A----C, events occurred preferentially in the dcd1 strain at sites where dCTP was to be inserted next. C----T and A----T events were induced most often by dTMP treatment at sites where the next correct nucleotide was dTTP or dGTP (dGTP levels were also elevated by dTMP treatment). Finally, misinsertion of dCTP or dTTP did not exhibit a strand bias. Collectively, our data suggest that increased levels of dCTP and dTTP induced mutations in yeast via nucleotide misinsertion and inhibition of proofreading but indicate that other factors must also be involved. We consider several possibilities, including potential roles for the regulation and specificity of proofreading and for mismatch correction.     

Substrate and mispairing properties of 5-formyl-2'-deoxyuridine 5'-triphosphate assessed by in vitro DNA polymerase reactions.

5-Formyluracil (fU) is one of the thymine lesions produced by reactive oxygen radicals in DNA and its constituents. In this work, 5-formyl-2'-deoxyuridine 5'-triphosphate (fdUTP) was chemically synthesized and extensively purified by HPLC. The electron withdrawing 5-formyl group facilitated ionization of fU. Thus, p K a of the base unit of fdUTP was 8.6, significantly lower than that of parent thymine (p K a = 10.0 as dTMP). fdUTP efficiently replaced dTTP during DNA replication catalyzed by Escherichia coli DNA polymerase I (Klenow fragment), T7 DNA polymerase (3'-5'exonuclease free) and Taq DNA polymerase. fU-specific cleavage of the replication products by piperidine revealed that when incorporated as T, incorporation of fU was virtually uniform, suggesting minor sequence context effects on the incorporation frequency of fdUTP. fdUTP also replaced dCTP, but with much lower efficiency than that for dTTP. The substitution efficiency for dCTP increased with increasing pH from 7.2 to 9.0. The parallel correlation between ionization of the base unit of fdUTP (p K a = 8.6) and the substitution efficiency for dCTP strongly suggests that the base-ionized form of fdUTP is involved in mispairing with template G. These data indicate that fU can be specifically introduced into DNA as unique lesions by in vitro DNA polymerase reactions. In addition, fU is potentially mutagenic since this lesion is much more prone to form mispairing with G than parent thymine.     

Bypass of N²-ethylguanine by human DNA polymerase κ

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.     

Inhibition of mouse myeloma DNA polymerase alpha by 5-triphosphates of 1-beta-D-arabinofuranosylthymine and 1-beta-D-arabinofuranosylcytosine

This is the first report dealing with the effect of 1-beta-D-arabinofuranosylthymine 5'-triphosphate (araTTP), synthesized by a new method, on eukaryotic DNA polymerase [EC 2.7.7.7]. AraTTP was tested for the inhibition of DNA synthesis in vitro using highly purified mouse myeloma DNA polymerase alpha in comparison with 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP). AraTTP was found to inhibit competitively the incorporation of [3H]dTTP into DNA and non-competitively the incorporation of [3H]dCTP, while the mode of the inhibition by araCTP was non-competitive with respect to dTTP and competitive with respect to dCTP. Neither araTTP nor araCTP was utilized as a substrate in place of dTTP or dCTP in DNA synthesis by DNA polymerase alpha.