Polymerase-specific differences in the DNA intermediates of frameshift mutagenesis. In vitro synthesis errors of Escherichia coli DNA polymerase I and its large fragment derivative

The sequences of more than 600 frameshift mutations produced as a consequence of in vitro DNA replication on an oligonucleotide-primed, single-stranded DNA template by the Escherichia coli polymerase I enzyme (PolI) or its large fragment derivative (PolLF) were compared. Four categories of mutants were found: (1) single-base deletions, (2) base substitutions, (3) multiple-base deletions and (4) complex frameshift mutations that change both the base sequence and the number of bases in a concerted mutational process. The template sequence 5'-Py-T-G-3', previously identified as a PolLF hotspot for single-base deletions opposite G, is also a hotspot for PolI. A PolI-specific warm spot for single-base deletions was identified. Among base substitutions, transitions were more frequent than transversions. Transversions were mediated by (template)G.G, (template)G.A, and (template)C.T mispairs. Multiple-base deletions were found only after PolI replication. Although each of these deletions can be explained by a misalignment mediated by directly repeated DNA sequences, deletion frequencies were often different for repeats of the same length. Both PolI and PolLF produced many complex frameshift mutants. The new sequences at the mutant sites are exactly complementary to nearby DNA sequences in the newly synthesized DNA strand. In each case, palindromic complementarity could mediate the misalignment needed to initiate the mutational process. The misaligned DNA synthesis accounts for the nucleotide changes at the mutant site and for homology that could direct realignment of the DNA onto the template. Most of the complex mutant sequences could be initiated by either intramolecular misalignments involving fold-back structures in newly synthesized DNA or by strand-switching during strand-displacement synthesis. The striking differences between the specificities of complex frameshift mutations and multiple-base deletions by PolI and PolLF identify the existence of polymerase-specific determinants that influence the frequency and specificity of misalignment-mediated frameshifts and deletions.     

Directionality of DNA replication fork movement strongly affects the generation of spontaneous mutations in Escherichia coli

Using a pair of plasmids carrying the rpsL target sequence in different orientations to the replication origin, we analyzed a large number of forward mutations generated in wild-type and mismatch-repair deficient (MMR(-)) Escherichia coli cells to assess the effects of directionality of replication-fork movement on spontaneous mutagenesis and the generation of replication error. All classes of the mutations found in wild-type cells but not MMR(-) cells were strongly affected by the directionality of replication fork movement. It also appeared that the directionality of replication-fork movement governs the directionality of sequence substitution mutagenesis, which occurred in wild-type cells at a frequency comparable to base substitutions and single-base frameshift mutations. A very strong orientation-dependent hot-spot site for single-base frameshift mutations was discovered and demonstrated to be caused by the same process involved in sequence substitution mutagenesis. It is surprising that dnaE173, a potent mutator mutation specific for sequence substitution as well as single-base frameshift, did not enhance the frequency of the hot-spot frameshift mutation. Furthermore, the frequency of the hot-spot frameshift mutation was unchanged in the MMR(-) strain, whereas the mutHLS-dependent mismatch repair system efficiently suppressed the generation of single-base frameshift mutations. These results suggested that the hot-spot frameshift mutagenesis might be initiated at a particular location containing a DNA lesion, and thereby produce a premutagenic replication intermediate resistant to MMR. Significant numbers of spontaneous single-base frameshift mutations are probably caused by similar mechanisms.     

The base substitution fidelity of eucaryotic DNA polymerases. Mispairing frequencies, site preferences, insertion preferences, and base substitution by dislocation

The base substitution fidelity of DNA polymerase-alpha, -beta, and -gamma (pol-alpha, -beta, and -gamma, respectively) has been determined in vitro, for all 12 possible mispairs at 96 sites in a forward mutational target. Averaging all errors over all known detectable sites, pol-gamma is the most accurate enzyme, producing one error for every 10,000 bases polymerized. Pol-beta is much less accurate, with an error rate of 1/1,500, while pol-alpha has an intermediate accuracy of 1/4,000. The relative differences in fidelity between the DNA polymerases are strongly influenced by the nature of the mispair. For example, G(template):dATP mispairs and G:dGTP mispairs are formed with about equal frequency by all three classes of DNA polymerases, yet pol-gamma produces T:dGTP mispairs at a 100-fold lower frequency than does pol-beta. The DNA polymerases exhibit distinct differences in template site preferences as well as substrate insertion preferences. The increase in accuracy apparent in proceeding from the least selective to the most accurate enzyme results primarily from a decrease in mispair formations at template A and T residues and a decrease in misinsertion of pyrimidine deoxynucleotides. These data clearly demonstrate a major role for eucaryotic DNA polymerases in modulating base mispair frequencies at the level of insertion. In addition to direct mispair formation due to an incorrect incorporation event, an examination of the errors produced by each of the three classes of DNA polymerases at two particular sites in the target sequence suggests that some base substitution errors result from transient misalignment of the primer-template. A model is presented to explain this phenomenon, termed "Dislocation Mutagenesis." The data are discussed in relation to the extensive literature on base substitution errors and to the origin of spontaneous base substitutions in animal cells.     

Frameshift mutagenesis by eucaryotic DNA polymerases in vitro

The frequency and specificity of frameshift errors produced during a single round of in vitro DNA synthesis by DNA polymerases-alpha, -beta, and -gamma (pol-alpha, -beta, and -gamma, respectively) have been determined. DNA polymerase-beta is the least accurate enzyme, producing frameshift errors at an average frequency of one error for each 1,000-3,000 nucleotides polymerized, a frequency similar to its average base substitution accuracy. DNA polymerase-alpha is approximately 10-fold more accurate, producing frameshifts at an average frequency of one error for every 10,000-30,000 nucleotides polymerized, a frequency which is about 2- to 6-fold lower than the average pol-alpha base substitution accuracy. DNA polymerase-gamma is highly accurate, producing on the average less than one frameshift error for every 200,000-400,000 nucleotides polymerized. This represents a more than 10-fold higher fidelity than for base substitutions. Among the collection of sequenced frameshifts produced by DNA polymerases-alpha and beta, both common features and distinct specificities are apparent. These specificities suggest a major role for eucaryotic DNA polymerases in modulating frameshift fidelity. Possible mechanisms for production of frameshifts are discussed in relation to the observed biases. One of these models has been experimentally supported using site-directed mutagenesis to change the primary DNA sequence of the template. Alteration of a pol-beta frameshift hotspot sequence TTTT to CTCT reduced the frequency of pol-beta-dependent minus-one-base errors at this site by more than 30-fold, suggesting that more than 97% of the errors at the TTTT run involve a slippage mechanism.     

Mechanisms of ultraviolet-induced mutation. Mutational spectra in the Escherichia coli lacI gene for a wild-type and an excision-repair-deficient strain

We have analyzed the DNA sequence changes in a total of 409 ultraviolet light-induced mutations in the lacI gene of Escherichia coli: 227 in a Uvr+ and 182 in a UvrB- strain. Both differences and similarities were observed. In both strains the mutations were predominantly (60 to 75%) base substitutions, followed by smaller contributions of single-base frameshifts, deletions and frameshift hotspot mutations. The base substitutions proved largely similar in the two strains but differences were observed among the single-base frameshifts, the deletions and the hotspot mutations. Among the base substitutions, both transitions (72.5%) and transversions (27.5%) were observed. The largest single group was G.C----A.T (60% of all base substitutions). The sites where G.C----A.T changes occurred were strongly correlated (97.5%) with sequences of adjacent pyrimidines, indicating mutation targeted ultraviolet photoproducts. Comparable amounts of mutation occurred at cytosine/cytosine and (mixed) cytosine/thymine sites. From an analysis of the prevalence of mutation at either the 5' or 3' side of a dipyrimidine, we conclude that both cyclobutane dimers and (6-4) lesions may contribute to mutation. Despite the general similarity of the base-substitution spectra between the wild-type and excision-defective strains, a number of sites were uniquely mutable in the UvrB- strain. Analysis of their surrounding DNA sequences suggested that, in addition to damage directly at the site of mutation, the potential for nearby opposite-strand damage may be important in determining the mutability of a site. The ultraviolet light-induced frameshift mutations were largely single-base losses. Inspection of the DNA sequences at which the frameshifts occurred suggested that they resulted from targeted mutagenesis, probably at cyclobutane pyrimidine dimers. The prevalence of frameshift mutations at homodimers (TT or CC) suggests that their formation involves local misalignment (slippage) and that base-pairing properties are partially retained in cyclobutane dimers. While the frameshift mutations in the Uvr+ strain were distributed over many different sites, more than half in the UvrB- strain were concentrated at a single site. Ultraviolet light-induced deletions as well as frameshift hotspot mutations (+/- TGGC at positions 620 to 632) are considered to be examples of untargeted or semitargeted mutagenesis. Hotspot mutations in the Uvr+ strain showed an increased contribution by (-)TGGC relative to (+)TGGC, indicating that ultraviolet light may specifically promote the loss of the four bases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mutational specificity of the dnaE173 mutator associated with a defect in the catalytic subunit of DNA polymerase III of Escherichia coli.

We developed a system to examine forward mutations that occurred in the rpsL gene of Escherichia coli placed on a multicopy plasmid. Using this system we determined the mutational specificity for a dnaE173 mutator strain in which the editing function of DNA polymerase III is impeded. The frequency of rpsL- mutations increased 32,000-fold, due to the dnaE173 mutator, and 87 independent rpsL- mutations in the mutator strain were analyzed by DNA sequencing, together with 100 mutants recovered from dnaE+ strain, as the control. While half the number of mutations that occurred in the wild-type strain were caused by insertion elements, no such mutations were recovered from the mutator strain. A novel class of mutation, named "sequence substitution" was present in mutants raised in the dnaE173 strain; seven sequence substitutions induced in the mutator strain occurred at six sites, and all were located in quasipalindromic sequences, carrying the GTG or CAC sequence at one or both endpoints. While other types of mutation were found in both strains, single-base frameshifts were the most frequent events in the mutator strain. Thus, the mutator effect on this class of mutation was 175,000-fold. A total of 95% of the single-base frameshifts in the mutator strain were additions, most of which occurred at runs of A or C bases so as to increase the number of identical residues. Base substitutions, the frequency of which was enhanced 25,000-fold by the mutator effect, occurred primarily at several hotspots in the mutator strain, whereas those induced in the wild-type strain were more randomly distributed throughout the rpsL sequence. The dnaE173 mutator also increased the frequency of duplications 28,000-fold. Of the three duplications recovered from the mutator strain, one was a simple duplication, the region of which was flanked by direct repeats. The other duplications were complex, one half part of which was in the inverted orientation of a region containing two sets of inverted repeats. The same duplications were also recovered from the wild-type strain. The present data suggest that dnaE173 is a novel class of mutator that sharply induces sequence-directed mutagenesis, yielding high frequencies of single base frameshifts, duplications with inversions, sequence substitutions and base substitutions at hotspots.     

The mutational specificity of DNA polymerases-alpha and -gamma during in vitro DNA synthesis

The frequency and specificity of mutations produced during in vitro DNA synthesis of the lacZ alpha gene in M13mp2 DNA by eucaryotic DNA polymerase-alpha (pol-alpha) and DNA polymerase-gamma (pol-gamma) have been determined. Pol-alpha, purified from five different sources, produces mutations resulting in loss of alpha-complementation at a frequency of 0.8-1.6%/single round of gap-filling DNA synthesis. DNA sequence analysis of 420 independent mutants produced by pol-alpha demonstrates three classes of errors. The majority of mutations result from single base substitutions, while single base frameshifts are detected at a lower but substantial frequency. Large deletions are also observed, with a frequency and specificity suggesting that they too are produced by pol-alpha in vitro. In contrast, pol-gamma is more accurate, producing mutants at a frequency of 0.3-0.5%. The specificity of pol-gamma errors is also different, since more than 90% of the mutants result from single base substitutions, while frameshift errors are not observed at a frequency significantly above background. The pol-gamma mutant spectrum also contains deletion mutations (10 of 179 mutants) presumably resulting from aberrant in vitro synthesis. When considered together with previous results using pol-beta (Kunkel, T. A. (1985) J. Biol. Chem. 260, 5787-5796) the relative accuracy of the three classes of purified vertebrate DNA polymerases for base substitutions, frameshifts, and deletions is in the order gamma greater than alpha greater than beta. These data demonstrate a correlation between the accuracy and processivity of DNA polymerization. Thus, the most accurate DNA polymerase (pol-gamma) also incorporates the most nucleotides per association with the primer-template, while the least accurate enzyme (pol-beta) is the least processive. This correlation exists both for base substitution mutations and for single base frameshifts, and is most obvious for minus-one-base frameshifts in runs of pyrimidines. In support of this correlation, increasing the processivity of pol-beta from 1 to 4-6 incorporations per association increases the accuracy of in vitro DNA synthesis by severalfold. The data imply that the processivity of DNA synthesis could be an important factor in controlling the levels of spontaneous and perhaps induced mutations.     

The Influence of Local DNA Sequence and DNA Repair Background on the Mutational Specificity of 1-Nitroso-8-Nitropyrene in Escherichia Coli: Inferences for Mutagenic Mechanisms

We have examined the mutational specificity of 1-nitroso-8-nitropyrene (1,8-NONP), an activated metabolite of the carcinogen 1,8-dinitropyrene, in the lacI gene of Escherichia coli strains which differ with respect to nucleotide excision repair (+/- delta uvrB) and MucA/B-mediated error-prone translesion synthesis (+/- pKM101). Several different classes of mutation were recovered, of which frameshifts, base substitutions, and deletions were clearly induced by 1,8-NONP treatment. The high proportion of point mutations (> 92%) which occurred at G.C sites correlates with the percentage of 1,8-NONP-DNA adducts which occur at the C(8) position of guanine. The most prominent frameshift mutations were -(G.C) events, which were induced by 1,8-NONP treatment in all strains, occurred preferentially in runs of guanine residues, and whose frequency increased markedly with the length of the reiterated sequence. Of the base substitution mutations G.C-->T.A transversions were induced to the greatest extent by 1,8-NONP. The distribution of the G.C-->T.A transversions was not influenced by the nature of flanking bases, nor was there a strand preference for these events. The presence of plasmid pKM101 specifically increased the frequency of G.C-->T.A transversions by a factor of 30-60. In contrast, the -(G.C) frameshift mutation frequency was increased only 2-4-fold in strains harboring pKM101 as compared to strains lacking this plasmid. There was, however, a marked influence of pKM101 on the strand specificity of frameshift mutation; a preference was observed for -G events on the transcribed strand. The ability of the bacteria to carry out nucleotide excision repair had a strong effect on the frequency of all classes of mutation but did not significantly influence either the overall distribution of mutational classes or the strand specificity of G.C-->T.A transversions and -(G.C) frameshifts. Deletion mutations were induced in the delta uvr, pKM101 strain. The endpoints of the majority of the deletion mutations were G.C rich and contained regions of considerable homology. The specificity of 1,8-NONP-induced mutation suggests that DNA containing 1,8-NONP adducts can be processed through different mutational pathways depending on the DNA sequence context of the adduct and the DNA repair background of the cell.     

Spontaneous Mutation in the Escherichia Coli Laci Gene

To gain more detailed insight into the nature and mechanisms of spontaneous mutations, we undertook a DNA sequence analysis of a large collection of spontaneous mutations in the N-terminal region of the Escherichia coli lacI gene. This region of circa 210 base pairs is the target for dominant lacI mutations (i-d) and is suitable for studies of mutational specificity since it contains a relatively high density of detectable mutable sites. Among 414 independent i-d mutants, 70.8% were base substitutions, 17.2% deletions, 7.7% additions and 4.3% single-base frameshifts. The base substitutions were both transitions (60%) and transversions (40%), the largest single group being G.C----A.T (47% of base substitutions). All four transversions were observed. Among the 71 deletions, a hotspot (37 mutants) was present: an 87-bp deletion presumably directed by an 8-bp repeated sequence at its endpoints. The remaining 34 deletions were distributed among 29 different mutations, either flanked (13/34) or not flanked (21/34) by repeated sequences. The 32 additions comprised 29 different events, with only two containing a direct repeat at the endpoints. The single-base frameshifts were the loss of a single base from either repeated (67%) or nonrepeated (33%) bases. A comparison with the spectrum obtained previously in strains defective in DNA mismatch correction (mutH, mutL, mutS strains) yielded information about the apparent efficiency of mismatch repair. The overall effect was 260-fold but varied substantially among different classes of mutations. An interesting asymmetry was uncovered for the two types of transitions, A.T----G.C and G.C----A.T being reduced by mismatch repair 1340- and 190-fold, respectively. Explanations for this asymmetry and its possible implications for the origins of spontaneous mutations are discussed.     

Specificity of mutations induced in transfected DNA by mammalian cells

DNA transfected into mammalian cells is subject to the high mutation frequency of approximately 1% per gene. We present data bearing on the derivation of the two main classes of mutations detected, base substitutions and deletions. The DNA sequence change is reported for nearly 100 independent base substitution mutations that occurred in shuttle vectors as a result of passage in simian cells. All of the mutations occur at G:C base pairs and involve either transition to A:T or transversion to T:A. To identify possible mutational intermediates, various topological forms of the vector DNA were introduced separately. Supercoiled and relaxed DNA are mutated at equal frequencies. However, linearized DNA leads to a greatly elevated frequency of deletions. Nicked and gapped templates stimulate both deletions and base substitutions. We discuss a model involving intracellular degradation of the transfected DNA which explains these observations.     

The effect of oxidative metabolism on spontaneous Pol zeta-dependent translesion synthesis in Saccharomyces cerevisiae

DNA lesions can stall or block high-fidelity polymerases, thus inhibiting replication. To bypass such lesions, low-fidelity translesion synthesis (TLS) polymerases can be used to insert a nucleotide across from the lesion or extend from a lesion:base mispair. When DNA repair is compromised in Saccharomyces cerevisiae, spontaneous DNA lesions can lead to a novel mutational event in which a frameshift is accompanied by one or more base pair substitutions. These "complex frameshifts" are dependent upon the TLS polymerase Pol zeta, and provide a mutational signature for mutagenic Pol zeta-dependent activity. In the current study, we have found that a specific subset of the Pol zeta-dependent mutational events requires oxidative metabolism. These results suggest that translesion bypass of spontaneously oxidized DNA bases can be a significant source of mutagenesis in repair compromised cells.     

Randomness of base substitution mutations induced in the lacI gene of Escherichia coli by ionizing radiation

The lacI system of Escherichia coli provides a method for monitoring mutational events at a large number of sites. Using this system, we have previously determined the mutational spectra for gamma-ray and beta-particle emissions resulting from the decay of tritium. Analysis of these mutational spectra reveals that base substitution mutations induced by ionizing radiation are distributed nearly randomly throughout the lacI gene and include all detectable substitution events. The distribution of ionizing radiation-induced mutagenesis is similar to the low frequency of occurrence mutational events induced by other SOS-dependent mutagens. The lack of an apparent nonrandom or high frequency of occurrence component seen with other SOS-dependent mutagens can be best explained as the result of the random interaction of ionizing radiation with the DNA bases leading to production of a variety of base substitutions.     

Fidelity of mammalian DNA replication and replicative DNA polymerases.

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectrum of spontaneous frameshift mutations. Sequences of bacteriophage T4 rII gene frameshifts

The DNA sequences of 185 independent spontaneous frameshift mutations in the rIIB gene of bacteriophage T4 are described. Approximately half of the frameshifts, including those at hot spot sites, are fully consistent with classical proposals that frameshift mutations are produced by a mechanism involving the misaligned pairing of repeated DNA sequences. However, the remaining frameshifts are inconsistent with this model. Correlations between the positions of two base-pair frameshifts and the bases of DNA hairpins suggest that local DNA topology might influence frameshift mutation. Warm spots for larger deletions share the property of having endpoints adjacent to DNA sequences whose complementarity to sequences a few base-pairs away suggest that non-classical DNA misalignments may participate in deletion mutation. A model for duplication mutation as a consequence of strand displacement synthesis is discussed. In all, 15 frameshifts were complex combinations of frameshifts and base substitutions. Three of these were identical, and have extended homology to a sequence 256 base-pairs away that is likely to participate in the mutational event; the remainder are unique combinations of frameshifts and transversions. The frequency and diversity of complex mutants suggest a challenge to the assumption that the molecular evolution of DNA must depend primarily upon the accumulation of single nucleotide changes.     

Suppressible base substitution mutations induced by angelicin (isopsoralen) in the Escherichia coli lacI gene: implications for the mechanism of SOS mutagenesis.

Angelicin- plus near-UV-induced mutations were umuC dependent in Escherichia coli K-12. Angelicin, a monofunctional psoralen derivative, is believed to damage DNA almost exclusively at pyrimidine bases. To broaden our knowledge about the mutagenic specificity of SOS-dependent mutagens, we determined the mutational specificity of 233 suppressible lacI mutations induced by angelicin. More than 90% of the nonsense mutations arose via transversion substitutions. The three most frequently mutated sites were at A-T base pairs and accounted for more than one-third of all induced nonsense mutations. The two hottest sites were at the only occurrences of the 5'-TATA-3' tetranucleotide in lacI, a sequence expected to be a preferred binding site for a psoralen. Both A-T-to-T-A and A-T-to-C-G transversions were well induced by angelicin treatment, but the frequency of each transversion depended on the particular site. We also detected significant induction of transversion mutations at G-C sites. The induction of transversions by an SOS-dependent mutagen that generates lesions at pyrimidines supports the idea that DNA lesions influence the selection of bases that are incorporated via the process of SOS repair.     

Mutations induced by DNA polymerase alpha upon in vitro replication of M13mp8(+) DNA.

The forward mutation of the lacZ part of the bacteriophage M13mp8 has been used to study the fidelity of the 9S DNA polymerase alpha from calf thymus during in vitro replication of single-stranded DNA. Errors leading to a loss of alpha-complementation were identified by DNA sequencing. The overall mutation rate of the lacZ target sequence was in the range of 1:300-1:1000 which is more than one order of magnitude higher than the spontaneous mutation rate. In a mutL host the mutation rate was nearly threefold higher as compared to the wildtype host. Base substitutions comprise 86% of the errors whereas base deletions amount to 12%. The addition of a base was detected only in one mutant out of 71 sequenced ones. The frameshift mutations occurred predominantly in runs of the same base. The frequencies of individual base substitution are in the order of 2 X 10(-4)-4 X 10(-4) for most of the mismatches. Mutations involving dCTP:T and dGTP:T mismatches are observed with a lower frequency, those involving dTTP:C mismatches with a higher frequency.     

Unique error signature of the four-subunit yeast DNA polymerase epsilon

We have purified wild type and exonuclease-deficient four-subunit DNA polymerase epsilon (Pol epsilon) complex from Saccharomyces cerevisiae and analyzed the fidelity of DNA synthesis by the two enzymes. Wild type Pol epsilon synthesizes DNA accurately, generating single-base substitutions and deletions at average error rates of 5' exonuclease activity is less accurate to a degree suggesting that wild type Pol epsilon proofreads at least 92% of base substitution errors and at least 99% of frameshift errors made by the polymerase. Surprisingly the base substitution fidelity of exonuclease-deficient Pol epsilon is severalfold lower than that of proofreading-deficient forms of other replicative polymerases. Moreover the spectrum of errors shows a feature not seen with other A, B, C, or X family polymerases: a high proportion of transversions resulting from T.dTTP, T.dCTP, and C.dTTP mispairs. This unique error specificity and amino acid sequence alignments suggest that the structure of the polymerase active site of Pol epsilon differs from those of other B family members. We observed both similarities and differences between the spectrum of substitutions generated by proofreading-deficient Pol epsilon in vitro and substitutions occurring in vivo in a yeast strain defective in Pol epsilon proofreading and DNA mismatch repair. We discuss the implications of these findings for the role of Pol epsilon polymerase activity in DNA replication.     

Mutational specificity of gamma-radiation-induced guanine-thymine and thymine-guanine intrastrand cross-links in mammalian cells and translesion synthesis past the guanine-thymine lesion by human DNA polymerase eta

Comparative mutagenesis of gamma- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links was investigated in simian (COS-7) and human embryonic (293T) kidney cells. For G[8,5-Me]T in 293T cells, 5.8% of progeny contained targeted base substitutions, whereas 10.0% showed semitargeted single-base substitutions. Of the targeted mutations, the G --> T mutation occurred with the highest frequency. The semitargeted mutations were detected up to two bases 5' and three bases 3' to the cross-link. The most prevalent semitargeted mutation was a C --> T transition immediately 5' to the G[8,5-Me]T cross-link. Frameshifts (4.6%) (mostly small deletions) and multiple-base substitutions (2.7%) also were detected. For the T[5-Me,8]G cross-link, a similar pattern of mutations was noted, but the mutational frequency was significantly higher than that of G[8,5-Me]T. Both targeted and semitargeted mutations occurred with a frequency of approximately 16%, and both included a dominant G --> T transversion. As in 293T cells, more than twice as many targeted mutations in COS cells occurred in T[5-Me,8]G (11.4%) as in G[8,5-Me]T (4.7%). Also, the level of semitargeted single-base substitutions 5' to the lesion was increased and 3' to the lesion decreased in T[5-Me,8]G relative to G[8,5-Me]T in COS cells. It appeared that the majority of the base substitutions at or near the cross-links resulted from incorporation of dAMP opposite the template base, in agreement with the so-called "A-rule". To determine if human polymerase eta (hpol eta) might be involved in the mutagenic bypass, an in vitro bypass study of G[8,5-Me]T in the same sequence was carried out, which showed that hpol eta can bypass the cross-link incorporating the correct dNMP opposite each cross-linked base. For G[8,5-Me]T, nucleotide incorporation by hpol eta was significantly different from that by yeast pol eta in that the latter was more error-prone opposite the cross-linked Gua. The incorporation of the correct nucleotide, dAMP, by hpol eta opposite cross-linked T was 3-5-fold more efficient than that of a wrong nucleotide, whereas incorporation of dCMP opposite the cross-linked G was 10-fold more efficient than that with dTMP. Therefore, the nucleotide incorporation pattern by hpol eta was not consistent with the observed cellular mutations. Nevertheless, at and near the lesion, hpol eta was more error-prone compared to a control template. The in vitro data suggest that translesion synthesis by another Y-family DNA polymerase and/or flawed participation of an accessory protein is a more likely scenario in the mutagenesis of these lesions in mammalian cells. However, hpol eta may play a role in correct bypass of the cross-links.