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.     

Unique pattern of point mutations arising after gene transfer into mammalian cells.

We have used a simian virus 40 (SV40)-based shuttle vector, pZ189, to analyze the sequence specificity of spontaneous point mutations that arise after transfection of this vector into monkey cells. The majority of the mutants which we studied had multiple base substitutions (mostly G-C----A-T transitions and G-C----T-A transversions) within the 160-bp region sequenced. Almost all of the mutations occurred in the right-hand G-C bp of one of the two following sequences, 5'-TC-3':3'-AG-5' or 5'-CC-3':3'-GG-5'. We postulate that these mutations result from DNA replication infidelity occurring during repair of the transfected DNA which has been damaged by cellular nucleases. The sequence specificity of the mutations suggests an effect of the following nucleotide on misincorporation wherein A (or less frequently T) is preferentially misincorporated opposite C when the next nucleotide inserted is A (or less frequently G). Our results support the utility of the shuttle vector as a model in studies on gene transfer and document the extreme plasticity of DNA transfected into mammalian cells.     

Similarity of mutation spectra of the mitochondrial DNA hypervariable segment 1 in Homo and Pan species

The mutation spectrum of mtDNA hypervariable segment 1 (HVS1) was compared for east chimpanzee Pan troglodytes schweigfurthi and human. The two HVS1 had much the same nucleotide composition, and their mutation spectra were similar in major characteristics (substantial prevalence of transitions over transversions, pyrimidine transitions over purine ones, and C --> T over T --> C). DNA strand displacement (dislocation) during replication was identified as a major mechanism of context-dependent mutagenesis in human and chimpanzee mtDNAs. Nucleotide positions with mutations fitting the model of dislocation mutagenesis accounted for 21% of all variable positions in the chimpanzee HVS1. Variable motifs proved to be similar in the chimpanzee and human HVS1. Comparison of the Neanderthal and modern human HVS1 nucleotide sequences showed that most variable nucleotides are in DNA sites allowing context-dependent mutagenesis.     

Transitions,transversions, and the molecular evolutionary clock

Summary Nucleotide substitutions in the form of transitions (purine-purine or pyrimidine-pyrimidine interchanges) and transversions (purine-pyrimidine interchanges) occur during evolution and may be complied by aligning the sequences of homologous genes. Referring to the genetic code tables, silent transitions take place in third positions of codons in family boxes and two-codon sets. Silent transversions in third positions occur only in family boxes, except for AC transversions between AGR and CGR arginine codons (R=A or G). Comparisons of several protein genes have been made, and various subclasses of transitional and transversional nucleotide substitutions have been compiled. Considerable variations occur among the relative proportions of transitions and transversions. Such variations could possibly be caused by mutator genes, favoring either transitions or, conversely, transversions, during DNA replication. At earlier stages of evolutionary divergence, transitions are usually more frequent, but there are exceptions. No indication was found that transversions usually originate from multiple substitutions in transitions.     

UV-induced hprt mutations in a UV-sensitive hamster cell line from complementation group 3 are biased towards the transcribed strand.

The molecular nature of 254 nm ultraviolet light (UV)-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in UV24 Chinese hamster ovary (CHO) cells, which are defective in nucleotide excision repair, was determined. Sequence analysis of 19 hprt mutants showed that single base substitutions (9 mutants) and tandem base changes (7 mutants) dominated the UV mutation spectrum in this cell line. Sixty-five percent of the base substitutions were GC greater than AT transitions, whereas the rest consisted of transitions and transversions at AT base pairs. Analysis of the distribution of dipyrimidine sites over the two DNA strands, where the photoproducts causing these mutations presumably were formed, showed that 12 out of 14 mutations were located in the transcribed strand of the hprt gene. A similar strand distribution of mutagenic photoproducts as in UV24 has previously been found in two other UV-sensitive Chinese hamster cell lines (V-H1 and UV5), indicating that under defective nucleotide excision repair conditions the induction of mutations is strongly biased towards lesions in the transcribed strand of the hprt gene. A plausible explanation for this phenomenon is that during DNA replication large differences exist in the error rate with which DNA polymerase(s) bypass lesions in the templates for the leading and lagging strand, respectively.     

Similarity of Mutation Spectra of the Mitochondrial DNA Hypervariable Segment 1 in Homo and Pan Species

The mutation spectrum of mtDNA hypervariable segment 1 (HVS1) was compared for east chimpanzee Pan troglodytes schweigfurthi and human. The two HVS1 had much the same nucleotide composition, and their mutation spectra were similar in major characteristics (substantial prevalence of transitions over transversions, pyrimidine transitions over purine ones, and C T over T C). DNA strand displacement (dislocation) during replication was identified as a major mechanism of context-dependent mutagenesis in human and chimpanzee mtDNAs. Nucleotide positions with mutations fitting the model of dislocation mutagenesis accounted for 21% of all variable positions in the chimpanzee HVS1. Variable motifs proved to be similar in the chimpanzee and human HVS1. Comparison of the Neanderthal and modern human HVS1 nucleotide sequences showed that most variable nucleotides are in DNA sites allowing context-dependent mutagenesis.     

Characterization of mutational specificity within the lacI gene for a mutD5 mutator strain of Escherichia coli defective in 3''----5'' exonuclease (proofreading) activity.

The mutD (dnaQ) gene of Escherichia coli codes for the epsilon subunit of the DNA polymerase III holoenzyme which is involved in 3'----5' exonuclease proofreading activity. We determined the mutational specificity of the mutator allele, mutD5, in the lacI gene of E. coli. The mutD5 mutation preferentially produces single base substitutions as judged from the enhanced fraction of lacI nonsense mutations and the spectrum of sequenced dominant lacI (lacId) and constitutive lacO (lacOc) mutations which were predominantly (69/71) single nucleotide substitutions. The distribution of amber lacI and sequenced lacId mutations revealed that transitions occur more frequently than transversions. A . T----G . C and G . C----A . T transitions were equally frequent and, with one major exception, evenly distributed among numerous sites. Among the transversions, A . T----T . A events were the most common, A . T----C . G substitutions were rare, and G . C----C . G changes were not detected. Transversions were unequally distributed among a limited number of sites with obvious hotspots. All 11 sequenced transversions had a consensus neighboring sequence of 5'-C-C-(mutated G or A)-C-3'. Although no large deletions or complex mutational events were recovered, sequencing revealed that mutD5 induced single nucleotide deletions within consecutive G X C sequences. An extraordinary A . T----G . C transition hotspot occurred at nucleotide position +6 in the lac operator region; the mutD5 mutation frequency of this single base pair was calculated to be 1.2 X 10(-3).     

Mlh1-dependent suppression of specific mutations induced in vivo by the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP)

Disruption of the DNA mismatch repair (MMR) pathway results in elevated mutation rates, inappropriate survival of cells bearing DNA damage, and increased cancer risk. Relatively little is known about the potential impact of environmentally relevant carcinogens on cancer risk in individuals with MMR-deficiency. We determined the effect of MMR status (Mlh1+/+ versus Mlh1-/-) on mutagenesis induced by the cooked-meat mutagen, 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) within cII and supFG1 transgene reporters. Despite being a lymphomagen in mice, PhIP was not mutagenic in thymus. In colon, PhIP exposure induced 3-fold more mutations in Mlh1-deficient mice compared to their Mlh1+/+ littermates. Similar induction was seen in Mlh1-/- small intestine. Analysis of mutational spectra revealed that G/C to T/A transversions, the "signature PhIP mutation", were induced to similar levels regardless of Mlh1 status. In contrast, Mlh1-/- mice exhibited hypermutability to frameshifts, G/C to A/T transitions, and G/C to C/G transversions. Thus, both the level and types of mutation induced by PhIP are influenced by the activity of the MMR system. MMR may suppress PhIP-induced mutation through recognition and processing of specific mispairs (PhIP-G/T, PhIP-G/G, and PhIP-G/loop mispairs). In contrast, the PhIP-G/A mispair is unlikely to be a MMR substrate. In addition, the similar induction of both transversions and transitions in Mlh1-/- mice suggests that mutagenic bypass of PhIP-G is similarly efficient with dATP, dTTP, and dGTP, in contrast to previously published conclusions. Our data suggests that MMR-deficiency would increase the likelihood of PhIP-induced carcinogenic mutations. Further evaluation of the risk that consumption of heterocyclic amines may impart to MMR-deficient individuals therefore is warranted.     

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.     

Abnormal, Error-Prone Bypass of Photoproducts by Xeroderma Pigmentosum Variant Cell Extracts Results in Extreme Strand Bias for the Kinds of Mutations Induced by UV Light

Xeroderma pigmentosum (XP) is a rare genetic disease characterized by a greatly increased susceptibility to sunlight-induced skin cancer. Cells from the majority of patients are defective in nucleotide excision repair. However, cells from one set of patients, XP variants, exhibit normal repair but are abnormally slow in replicating DNA containing UV photoproducts. The frequency of UV radiation-induced mutations in the XP variant cells is significantly higher than that in normal human cells. Furthermore, the kinds of UV-induced mutations differ very significantly from normal. Instead of transitions, mainly C→T, 30% of the base substitutions consist of C→A transversions, all arising from photoproducts located in one strand. Mutations involving cytosine in the other strand are almost all C→T transitions. Forty-five percent of the substitutions involve thymine, and the majority are transversions. To test the hypothesis that the UV hypermutability and the abnormal spectrum of mutations result from abnormal bypass of photoproducts in DNA, we compared extracts from XP variant cells with those from HeLa cells and a fibroblast cell strain, MSU-1.2, for the ability to replicate a UV-irradiated form I M13 phage. The M13 template contains a simian virus 40 origin of replication located directly to the left or to the right of the target gene, lacZα, so that the template for the leading and lagging strands of DNA replication is defined. Reduction of replication to ~37% of the control value required only 1 photoproduct per template for XP variant cell extracts, but ~2.2 photoproducts for HeLa or MSU-1.2 cell extracts. The frequency of mutants induced was four times higher with XP variant cell extracts than with HeLa or MSU-1.2 cell extracts. With XP variant cell extracts, the proportion of C→A transversions reached as high as 43% with either M13 template and arose from photoproducts located in the template for leading-strand synthesis; with HeLa or MSU-1.2 cell extracts, this value was only 5%, and these arose from photoproducts in either strand. With the XP variant extracts, 26% of the substitutions involved thymine, and virtually all were T→A transversions. Sequence analysis of the coding region of the catalytic subunit of DNA polymerase delta in XP variant cell lines revealed two polymorphisms, but these do not account for the reduced bypass fidelity. Our data indicate that the UV hypermutability of XP variant cells results from reduced bypass fidelity and that unlike for normal cells, bypass of photoproducts involving cytosine in the template for the leading strand differs significantly from that of photoproducts in the lagging strand.     

In situ enzymology of DNA replication and ultraviolet-induced DNA repair synthesis in permeable human cells

Using permeable diploid human fibroblasts, we have studied the deoxyribonucleoside triphosphate concentration dependences of ultraviolet- (UV-) induced DNA repair synthesis and semiconservative DNA replication. In both cell types (AG1518 and IMR-90) examined, the apparent Km values for dCTP, dGTP, and dTTP for DNA replication were between 1.2 and 2.9 microM. For UV-induced DNA repair synthesis, the apparent Km values were substantially lower, ranging from 0.11 to 0.44 microM for AG1518 cells and from 0.06 to 0.24 microM for IMR-90 cells. Control experiments established that these values were not significantly influenced by nucleotide degradation during the permeable cell incubations or by the presence of residual endogenous nucleotides within the permeable cells. Recent data implicate DNA polymerase delta in UV-induced repair synthesis and suggest that DNA polymerases alpha and delta are both involved in semiconservative replication. We measured Km values for dGTP and dTTP for polymerases alpha and delta, for comparison with the values for replication and repair synthesis. Km values for polymerase alpha were 2.0 microM for dGTP and 5.0 microM for dTTP. For polymerase delta, the Km values were 2.0 microM for dGTP and 3.5 microM for dTTP. The deoxyribonucleotide Km values for DNA polymerase delta are much greater than the Km values for UV-induced repair synthesis, suggesting that when polymerase delta functions in DNA repair, its characteristics are altered substantially either by association with accessory proteins or by direct posttranslational modification. In contrast, the deoxyribonucleotide binding characteristics of the DNA replication machinery differ little from those of the isolated DNA polymerases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutagen-induced changes in cellular deoxycytidine triphosphate and thymidine triphosphate in Chinese hamster ovary cells

Deoxynucleoside triphosphate concentrations in Chinese hamster ovary cell lines, CHO-K1 and Mut 8–16, were examined following exposure of cells to UV or dimethylsulfate. Marked decreases in dCTP were observed 2 hr after exposure to both mutagens. In contrast, dTTP concentrations increased with increased cell killing after exposure to UV but not after exposure to dimethylsulfate. Examination of DNA synthesis in permeabilized cells in the presence of excess concentrations of dNTP substrates suggests that excess dCTP enhances replication while excess of dTTP inhibits replication. We therefore ask whether the increase in the $\text{dTTP}\text{dCTP}$ ration in mutagenized whole cells either contributes to or prolongs induced inhibition of replication. In addition we proposed that such an induced dNTP imbalance may also contribute to an increase in mutations by enhancing the probability for base-misincorporation.     

Major oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil, exhibit sequence context-dependent mispairing in vitro.

Two major stable oxidation products of 2'-deoxycytidine are 2'-deoxy-5-hydroxycytidine (5-OHdC) and 2'-deoxy-5-hydroxyuridine (5-OHdU). In order to study the in vitro incorporation of 5-OHdC and 5-OHdU into DNA by DNA polymerase, and to check the base pairing specificity of these modified bases, 5-OHdCTP and 5-OHdUTP were synthesized. Incorporation studies showed that 5-OHdCTP can replace dCTP, and to a much lesser extent dTTP, as a substrate for Escherichia coli DNA polymerase I Klenow fragment (exonuclease free). However, 5-OHdUTP can only be incorporated into DNA in place of dTTP. To study the specificity of nucleotide incorporation opposite 5-hydroxypyrimidines in template DNA, 18- and 45-member oligodeoxyribonucleotides, containing an internal 5-OHdC or 5-OHdU in two different sequence contexts, were used. Translesion synthesis past 5-OHdC and 5-OHdU in both oligonucleotides occurred, but pauses both opposite, and one nucleotide prior to, the modified base in the template were observed. The specificity of nucleotide incorporation opposite 5-OHdC and 5-OHdU in the template was sequence context dependent. In one sequence context, dG was the predominant nucleotide incorporated opposite 5-OHdC with dA incorporation also observed; in this sequence context, dA was the principal nucleotide incorporated opposite 5-OHdU. However in a second sequence context, dC was the predominant base incorporated opposite 5-OHdC. In that same sequence context, dC was also the predominant nucleotide incorporated opposite 5-OHdU. These data suggest that the 5-hydroxypyrimidines have the potential to be premutagenic lesions leading to C-->T transitions and C-->G transversions.     

Limited dCTP availability accounts for mitochondrial DNA depletion in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a severe human disease caused by mutations in TYMP, the gene encoding thymidine phosphorylase (TP). It belongs to a broader group of disorders characterized by a pronounced reduction in mitochondrial DNA (mtDNA) copy number in one or more tissues. In most cases, these disorders are caused by mutations in genes involved in deoxyribonucleoside triphosphate (dNTP) metabolism. It is generally accepted that imbalances in mitochondrial dNTP pools resulting from these mutations interfere with mtDNA replication. Nonetheless, the precise mechanistic details of this effect, in particular, how an excess of a given dNTP (e.g., imbalanced dTTP excess observed in TP deficiency) might lead to mtDNA depletion, remain largely unclear. Using an in organello replication experimental model with isolated murine liver mitochondria, we observed that overloads of dATP, dGTP, or dCTP did not reduce the mtDNA replication rate. In contrast, an excess of dTTP decreased mtDNA synthesis, but this effect was due to secondary dCTP depletion rather than to the dTTP excess in itself. This was confirmed in human cultured cells, demonstrating that our conclusions do not depend on the experimental model. Our results demonstrate that the mtDNA replication rate is unaffected by an excess of any of the 4 separate dNTPs and is limited by the availability of the dNTP present at the lowest concentration. Therefore, the availability of dNTP is the key factor that leads to mtDNA depletion rather than dNTP imbalances. These results provide the first test of the mechanism that accounts for mtDNA depletion in MNGIE and provide evidence that limited dNTP availability is the common cause of mtDNA depletion due to impaired anabolic or catabolic dNTP pathways. Thus, therapy approaches focusing on restoring the deficient substrates should be explored.     

Estimation of DNA Sequence Context-dependent Mutation Rates Using Primate Genomic Sequences

It is understood that DNA and amino acid substitution rates are highly sequence context-dependent, e.g., C --> T substitutions in vertebrates may occur much more frequently at CpG sites and that cysteine substitution rates may depend on support of the context for participation in a disulfide bond. Furthermore, many applications rely on quantitative models of nucleotide or amino acid substitution, including phylogenetic inference and identification of amino acid sequence positions involved in functional specificity. We describe quantification of the context dependence of nucleotide substitution rates using baboon, chimpanzee, and human genomic sequence data generated by the NISC Comparative Sequencing Program. Relative mutation rates are reported for the 96 classes of mutations of the form 5' alphabetagamma 3' --> 5' alphadeltagamma 3', where alpha, beta, gamma, and delta are nucleotides and beta not equal delta, based on maximum likelihood calculations. Our results confirm that C --> T substitutions are enhanced at CpG sites compared with other transitions, relatively independent of the identity of the preceding nucleotide. While, as expected, transitions generally occur more frequently than transversions, we find that the most frequent transversions involve the C at CpG sites (CpG transversions) and that their rate is comparable to the rate of transitions at non-CpG sites. A four-class model of the rates of context-dependent evolution of primate DNA sequences, CpG transitions > non-CpG transitions approximately CpG transversions > non-CpG transversions, captures qualitative features of the mutation spectrum. We find that despite qualitative similarity of mutation rates among different genomic regions, there are statistically significant differences.     

Effect of Ehrlich ascites cell chalone on nascent DNA synthesis in isolated nuclei

Ehrlich Ascites Tumor (EAT) chalone has been shown to inhibit nascent DNA synthesis by inhibiting DNA polymerase alpha and beta (Nakai, 1976), but one of the problems in studying eurkaryotic DNA replication has been the relative impermeability of the cell membrane to precursors and macromolecules; hence, to circumvent this restriction without sacrificing the integrity of the replication process, a broken cell system utilizing nuclei in aqueous media was investigated. Isolated nuclei appear to continue the process of DNA replication that was proceeding in vivo before their isolation and under optimal concitions are able to initiate new synthesis (Fraser & Huberman, 1977). The effects of partially purified EAT chalone on nascent DNA could be studied directly in this nuclear system, which excluded effects of the cell membrane, nucleotide pools and other cytosol elements. A concentration-related inhibition of [3H]thymidine triphosphate ([3H]dTTP) incorporation was noted over a chalone range of 50-200 micrograms/ml. It appears that chalone can inhibit DNA polymerase alpha directly within the nucleus without an intermediate step such as a cell membrane receptor.     

Ectopic expression of AID in a non-B cell line triggers A:T and G:C point mutations in non-replicating episomal vectors

Somatic hypermutation (SHM) of immunoglobulin genes is currently viewed as a two step process initiated by the deamination of deoxycytidine (C) to deoxyuridine (U), catalysed by the activation induced deaminase (AID). Phase 1 mutations arise from DNA replication across the uracil residue or the abasic site, generated by the uracil-DNA glycosylase, yielding transitions or transversions at G:C pairs. Phase 2 mutations result from the recognition of the U:G mismatch by the Msh2/Msh6 complex (MutS Homologue), followed by the excision of the mismatched nucleotide and the repair, by the low fidelity DNA polymerase eta, of the gap generated by the exonuclease I. These mutations are mainly focused at A:T pairs. Whereas in activated B cells both G:C and A:T pairs are equally targeted, ectopic expression of AID was shown to trigger only G:C mutations on a stably integrated reporter gene. Here we show that when using non-replicative episomal vectors containing a GFP gene, inactivated by the introduction of stop codons at various positions, a high level of EGFP positive cells was obtained after transient expression in Jurkat cells constitutively expressing AID. We show that mutations at G:C and A:T pairs are produced. EGFP positive cells are obtained in the absence of vector replication demonstrating that the mutations are dependent only on the mismatch repair (MMR) pathway. This implies that the generation of phase 1 mutations is not a prerequisite for the expression of phase 2 mutations.     

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.     

Molecular Analysis of Mutations Induced in the Vermilion Gene of Drosophila Melanogaster by Methyl Methanesulfonate

The nature of DNA sequence changes induced by methyl methanesulfonate (MMS) at the vermilion locus of Drosophila melanogaster was determined after exposure of postmeiotic male germ cell stages. MMS is a carcinogen with strong preference for base nitrogen alkylation (s = 0.86). The spectrum of 40 intralocus mutations was dominated by AT----GC transitions (23%), AT----TA transversions (54%) and deletions (14%). The small deletions were preferentially found among mutants isolated in the F1 (8/18), whereas the AT----GC transitions exclusively occurred in the F2 (6/22). The MMS-induced transversions and deletions are presumably caused by N-methyl DNA adducts, which may release apurinic intermediates, known to be a time-related process. Furthermore, MMS produces multilocus deletions, i.e., at least 30% of the F1 mutants analyzed were of this type. A comparison of the mutational spectra of MMS with that produced by ethylnitrosourea (ENU), also in the vermilion locus of Drosophila, reveals major differences: predominantly transition mutations (61% GC----AT and 18% AT----GC) were found in both the F1 and F2 spectrum induced by ENU. It is concluded that the mutational spectrum of MMS is dominated by nitrogen DNA adducts, whereas with ENU DNA sequence changes mainly arose from modified oxygen in DNA.     

Mitochondrial DNA somatic mutation burden and heteroplasmy are associated with chronological age,smoking, and HIV infection

The gradual accumulation of mitochondrial DNA (mtDNA) mutations is implicated in aging and may contribute to the accelerated aging phenotype seen with tobacco smoking and HIV infection. mtDNA mutations are thought to arise from oxidative damage; however, recent reports implicate polymerase γ errors during mtDNA replication. Investigations of somatic mtDNA mutations have been hampered by technical challenges in measuring low‐frequency mutations. We use primer ID‐based next‐generation sequencing to quantify both somatic and heteroplasmic blood mtDNA point mutations within the D‐loop, in 164 women and girls aged 2–72 years, of whom 35% were smokers and 56% were HIV‐positive. Somatic mutations and the occurrence of heteroplasmic mutations increased with age. While transitions are theorized to result from polymerase γ errors, transversions are believed to arise from DNA oxidative damage. In our study, both transition and transversion mutations were associated with age. However, transition somatic mutations were more prevalent than transversions, and no heteroplasmic transversions were observed. We also measured elevated somatic mutations, but not heteroplasmy, in association with high peak HIV viremia. Conversely, heteroplasmy was higher among smokers, but somatic mutations were not, suggesting that smoking promotes the expansion of preexisting mutations rather than de novo mutations. Taken together, our results are consistent with blood mtDNA mutations increasing with age, inferring a greater contribution of polymerase γ errors in mtDNA mutagenesis. We further suggest that smoking and HIV infection both contribute to the accumulation of mtDNA mutations, though in different ways.