Mutations that alter the charge of type I regulatory subunit and modify activation properties of cyclic AMP-dependent protein kinase from S49 mouse lymphoma cells.

Mutations in regulatory (R) subunit of cAMP-dependent protein kinase were analyzed from cAMP-resistant mutants of S49 mouse lymphoma cells by direct sequencing of amplified regions of mutant R subunit cDNAs. Eight distinct single base-change lesions were identified in 24 independent mutants that were hemizygous for expression of mutant R subunits with altered protein charge. CG----TA transitions predominated, but AT----GC transitions and GC----TA transversions were also observed. Four of five spontaneous mutants had identical C----T transitions at CG causing substitution of Trp for Arg-334. Sites mutated in isolates obtained after mutagenesis with ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine were more varied. Six of the lesions (two in binding site A and four in site B) were at amino acid residues that are highly conserved among cAMP-binding sites of R subunits and the Escherichia coli catabolite activator protein. These mutations all either prevented or strongly hindered binding of cyclic nucleotides to the mutated site. One of the remaining lesions (at Arg-242) also prevented cyclic nucleotide binding to the mutated binding site; the other (at Gly-170) had only minimal effects on binding of cyclic nucleotides but, nevertheless, increased the apparent constant for cAMP-dependent kinase activation. These results are discussed with reference to a model for the cAMP-binding sites of R subunit based on the crystal structure of the E. coli catabolite activator protein.     

The DeltauvrB mutations in the Ames strains of Salmonella span 15 to 119 genes

The lacI transgene used in the Big Blue (BB) mouse and rat mutation assays typically displays spontaneous mutation frequencies in the 5x10(-5) range. Recently, the bone marrow and bladder of the Big Blue rat were reported to have, by an order of magnitude, the lowest spontaneous mutation frequencies ever observed for lacI in a transgenic animal, approaching the value for endogenous targets such as hprt ( approximately 10(-6)). Since spontaneous mutations in transgenes have been attributed in part to deamination of 5-methylcytosine in CpG sequences, we have investigated the methylation status of the lacI transgene in bone marrow of BB rats and compared it to that present in other tissues including liver, spleen, and breast. The first 400 bases of the lacI gene were investigated using bisulfite genomic sequencing since this region contains the majority of both spontaneous and induced mutations. Surprisingly, all the CpG cytosines in the lacI sequence were fully methylated in all the tissues examined from both 2- and 14-week-old rats. Thus, there is no correlation between 5-methylcytosine content at CpG sites in lacI and the frequency of spontaneous mutation at this marker. We also investigated the methylation status of another widely used transgenic mutation target, the cII gene. The CpG sites in cII in BB rats were fully methylated while those in BB mice were partially methylated (each site approximately 50% methylated). Since spontaneous mutation frequency at cII is comparable in rat and mouse, the methylation status of CpG sequences in this gene also does not correlate with spontaneous frequency. We conclude that other mechanisms besides spontaneous deamination of 5-methylcytosine at CpG sites are driving spontaneous mutation at BB transgenic loci.     

Methylation levels at selected CpG sites in the factor VIII and FGFR3 genes, in mature female and male germ cells: implications for male-driven evolution.

Transitional mutations at CpG dinucleotides account for approximately a third of all point mutations. These mutations probably arise through spontaneous deamination of 5-methylcytosine. Studies of CpG mutation rates in disease-linked genes, such as factor VIII and FGFR3, have indicated that they more frequently originate in male than in female germ cells. It has been speculated that these sex-biased mutation rates might be a consequence of sex-specific methylation differences between the female and the male germ lines. Using the bisulfite-based genomic-sequencing method, we investigated the methylation status of the human factor VIII and FGFR3 genes in mature male and female germ cells. With the exception of a single CpG, both genes were found to be equally and highly methylated in oocytes and spermatocytes. Whereas these observations strongly support the notion that DNA methylation is the major determining factor for recurrent CpG germ-line mutations in patients with hemophilia and achondroplasia, the higher mutation rate in the male germ line is apparently not a simple reflection of sex-specific methylation differences.     

High frequency mutagenesis by a DNA methyltransferase.

HpaII methylase (M. HpaII), an example of a DNA (cytosine-5)-methyltransferase, was found to induce directly a high frequency of C-->U transition mutations in double-stranded DNA. A mutant pSV2-neo plasmid, constructed with an inactivating T-->C transition mutation creating a CCGG site, was incubated with M. HpaII in the absence of S-adenosylmethionine (SAM). This caused an approximately 10(4)-fold increase in the rate of reversion when the mutant neo plasmid was transformed into bacteria lacking uracil-DNA glycosylase. The mutation frequency was very sensitive to SAM concentration and was reduced to background when the concentration of the methyl donor exceeded 300 nM. The data support current models for the formation of a covalent complex between the methyltransferase and cytosine. They also suggest that the occurrence of mutational hot spots at CpG sites may not always be due to spontaneous deamination of 5-methylcytosine, but might also be initiated by enzymatic deamination of cytosine and proceed through a C-->U-->T pathway.     

Effect of target gene CpG content on spontaneous mutation in transgenic mice

Transgenic mutation assays utilizing bacterial target genes display a high frequency of spontaneous mutation at CpG sequences. This is believed to result from the fact that: (1) the prokaryotic genes currently being used as transgenic mutation targets have a high CpG content and (2) these sequences are methylated by mammalian cells to produce 5-methylcytosine (5MC), a known promutagenic base. To study the effect of CpG content on the frequency and type of spontaneous mutation, we have synthesized an analogue of the bacterial lacI target gene (mrkII) that contains a reduced number of CpG sequences. This gene was inserted into a lambda vector and used to construct trangenic mice that undergo vector rescue from genomic DNA upon in vitro packaging. Results on spontaneous mutation frequency and spectrum have been collected and compared to those observed at the lacI gene in Big Blue™ transgenic mice. Spontaneous mutations at the mrkII gene occurred at a frequency in the mid-10⁻⁵ range and were predominantly base pair substitutions, similar to results seen in Big Blue™. However, mrkII mutations were distributed toward the carboxyl end of the gene instead of the bias toward the amino terminus seen in lacI. Unexpectedly, 23% of the spontaneous mrkII mutations were GC → AT transitions at CpG sequences (compared to 32% in lacI), despite the reduction in CpG number from 95 in lacI to only 13 in mrkII. Nine of the CpG bases undergoing transition mutations in mrkII have not been recorded previously as spontaneous sites in Big Blue™. Therefore, substantial reduction of the number of CpG sequences in the lacI transgene did not significantly reduce the rate of spontaneous mutation or alter the contribution of CpG-related events. This suggests that other factors are also operating to establish frequency and composition of spontaneous mutations in transgenic targets.     

The rate of hydrolytic deamination of 5-methylcytosine in double-stranded DNA.

The modified base, 5-methylcytosine, constitutes approximately 1% of human DNA, but sites containing 5-methylcytosine account for at least 30% of all germline and somatic point mutations. A genetic assay with a sensitivity of 1 in 10(7), based on reversion to neomycin resistance of a mutant pSV2-neo plasmid, was utilized to determine and compare the deamination rates of 5-methylcytosine and cytosine in double-stranded DNA for the first time. The rate constants for spontaneous hydrolytic deamination of 5-methylcytosine and cytosine in double-stranded DNA at 37 degrees C were 5.8 x 10(-13) s-1 and 2.6 x 10(-13) s-1, respectively. These rates are more than sufficient to explain the observed frequency of mutation at sites containing 5-methylcytosine and emphasize the importance of hydrolytic deamination as a major source of human mutations.     

Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells.

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.     

Spontaneous Auxotrophic and Pigmented Mutants Occurring at High Frequency in Bacillus pumilus NRRL B-3275

Broth cultures of Bacillus pumilus NRRL B-3275 (BpB1) grown at 25, 30, or 37 C contain 1 to 2% spontaneous auxotrophic mutants in both the exponential and stationary phases of growth. Of 70 such mutants isolated from cultures grown at 37 C, approximately two-thirds reverted at such a high frequency as to preclude their study. Of the remaining 22 mutants, 18 required a single amino acid, 1 required adenine, and 1 required uracil. Two of the auxotrophs each required two unrelated amino acids resulting from two independent mutations. All of the mutations reverted spontaneously. Enhanced reversion of approximately one-third of the mutations was obtained with nitrosoguanidine, ethyl methane sulfonate, or diethyl sulfate, or with more than one of these mutagens. The reversion of one mutation was enhanced by 2-aminopurine. The reversion of the remaining mutations was not enhanced by the above mutagens, nor by mutagens known to induce (and revert) frameshift mutations in other bacterial systems. Nine of 10 mutants examined did not show a selective growth advantage over the parents. All but three of the mutations could be linked by PBS1 transduction to one of the previously described auxotrophic markers in strain BpB1. No evidence was obtained for clustering of the mutations on the BpB1 genome. Six of the mutations conferred a requirement for serine. One linked by transduction to trp-2, three linked to argA1, and two (ser-2, -3) linked to argO1. Pigmented mutants (containing a carotenoid-like pigment), which occur spontaneously in BpB1 cultures at a frequency on the order of 1 to 5 mutants per 10(4) cells, link by transduction to ser-2, -3. Spontaneous mutants of strain BpB1 resistant to rifampin, streptomycin, erythromycin, 5-fluorouracil, or 5-methyltryptophan occur at a frequency similar to that of strains of B. pumilus which do not exhibit a high rate of spontaneous mutation to auxotrophy. It is suggested that certain sites or regions of the BpB1 genome exhibit a high rate of spontaneous mutation.     

DNA mismatch-repair in Escherichia coli counteracting the hydrolytic deamination of 5-methyl-cytosine residues.

Derivatives of phage M13 were constructed and used for the in vitro preparation of heteroduplex DNA molecules containing base/base mismatches that mimick DNA lesions caused by hydrolytic deamination of 5-meC residues in Escherichia coli DNA (i.e. they carry a T/G mismatch in the special sequence context provided by the recognition site -CCA/TGG-of the Dcm-methyltransferase). Upon introduction of these heteroduplex DNAs into CaCl2-treated E. coli cells, the mismatches are efficiently repaired with high bias in favour of the DNA strand containing the mismatched guanine residue. This special DNA mismatch-repair operates on fully dam-methylated DNA and is independent of gene mutH. It thus fulfills the salient requirements of a repair pathway responsible for counteracting the spontaneous hydrolytic deamination of 5-meC in vivo. The repair efficiency is boosted by a 5-methyl group present on the cytosine residue at the next-nearest position to the 5' side of the mismatched guanine. The repair is severely impaired in host strains carrying a mutation in any of the three loci dcm, mutL and mutS.     

Strong and weak male mutation bias at different sites in the primate genomes: insights from the human-chimpanzee comparison

Male mutation bias is a higher mutation rate in males than in females thought to result from the greater number of germ line cell divisions in males. If errors in DNA replication cause most mutations, then the magnitude of male mutation bias, measured as the male-to-female mutation rate ratio (alpha), should reflect the relative excess of male versus female germ line cell divisions. Evolutionary rates averaged among all sites in a sequence and compared between mammalian sex chromosomes were shown to be indeed higher in males than in females. However, it is presently unknown whether individual classes of substitutions exhibit such bias. To address this issue, we investigated male mutation bias separately at non-CpG and CpG sites using human-chimpanzee whole-genome alignments. We observed strong male mutation bias at non-CpG sites: alpha in the X-autosome comparison was approximately 6-7, which was similar to the male-to-female ratio in the number of germ line cell divisions. In contrast, mutations at CpG sites exhibited weak male mutation bias: alpha in the X-autosome comparison was only approximately 2-3. This is consistent with the methylation-induced and replication-independent mechanism of CpG transitions, which constitute the majority of mutations at CpG sites. Interestingly, our study also indicated weak male mutation bias for transversions at CpG sites, implying a spontaneous mechanism largely not associated with replication. Male mutation bias was equally strong at CpG and non-CpG sites located within unmethylated "CpG islands," suggesting the replication-dependent origin of these mutations. Thus, we found that the strength of male mutation bias is nonuniform in the primate genomes. Importantly, we discovered that male mutation bias depends on the proportion of CpG sites in the loci compared. This might explain the differences in the magnitude of primate male mutation bias observed among studies.     

Role of the dinB gene product in spontaneous mutation in Escherichia coli with an impaired replicative polymerase

We isolated several new mutator mutations of the Escherichia coli replicative polymerase dnaE subunit alpha and used them and a previously reported dnaE mutation to study spontaneous frameshift and base substitution mutations. Two of these dnaE strains produce many more mutants when grown on rich (Luria-Bertani) than on minimal medium. A differential effect of the medium was not observed when these dnaE mutations were combined with a mismatch repair mutation. The selection scheme for the dnaE mutations required that they be able to complement a temperature-sensitive strain. However, the ability to complement is not related to the mutator effect for at least one of the mutants. Comparison of the mutation rates for frameshift and base substitution mutations in mutS and dnaE mutS strains suggests that the mismatch repair proteins respond differently to the two types of change. Deletion of dinB from both chromosome and plasmid resulted in a four- to fivefold decrease in the rate of frameshift and base substitution mutations in a dnaE mutS double mutant background. This reduction indicates that most mistakes in replication occur as a result of the action of the auxiliary rather than the replicative polymerase in this dnaE mutant. Deletion of dinB from strains carrying a wild-type dnaE had a measurable effect, suggesting that a fraction of spontaneous mutations occur as a result of dinB polymerase action even in cells with a normal replicative polymerase.     

Phosphorylation of regulatory subunit of type I cyclic AMP-dependent protein kinase: biphasic effects of cyclic AMP in intact S49 mouse lymphoma cells

Intact S49 mouse lymphoma cells were used as a model system to study the effects of cyclic AMP (cAMP) and its analogs on the phosphorylation of regulatory (R) subunit of type I cAMP-dependent protein kinase. Phosphorylation of R subunit was negligible in mutants deficient in adenylate cyclase; low levels of cAMP analogs, however, stimulated R subunit phosphorylation in these cells to rates comparable to those in wild-type cells. In both wild-type and adenylate cyclase-deficient cells, R subunit phosphorylation was inhibited by a variety of N6-substituted derivatives of cAMP; C-8-substituted derivatives were generally poor inhibitors. Two derivatives that were inactive as kinase activators (N6-carbamoylmethyl-5'-AMP and 2'-deoxy-N6-monobutyryl-cAMP) were also ineffective as inhibitors of R subunit phosphorylation. Preferential inhibition by N6-modified cAMP analogs could not be ascribed simply to selectivity for the more aminoterminal (site I) of the two cAMP-binding sites in R subunit: Analog concentrations required for inhibition of R subunit phosphorylation were always higher than those required for activation of endogenous kinase; 8-piperidino-cAMP, a C-8-substituted derivative that is selective for cAMP-binding site I, was relatively ineffective as in inhibitor; and, although thresholds for activation of endogenous kinase by site I-selective analogs could be reduced markedly by coincubation with low levels of site II-selective analogs, no such synergism was observed for the inhibitory effect. The uncoupling of cyclic nucleotide effects on R subunit phosphorylation from activation of endogenous protein kinase suggests that, in intact cells, activation of cAMP-dependent protein kinase requires more than one and fewer than four molecules of cyclic nucleotide.     

Methylation of CpG dinucleotides in the lacI gene of the Big Blue® transgenic mouse

Cytosine residues at CpG dinucleotides can be methylated by endogenous methyltransferases in mammalian cells. The resulting 5-methylcytosine base may undergo spontaneous deamination to form thymine causing G/C to A/T transition mutations. Methylated CpGs also can form preferential targets for environmental mutagens and carcinogens. The Big Blue® transgenic mouse has been used to investigate tissue and organ specificity of mutations and to deduce mutational mechanisms in a mammal in vivo. The transgenic mouse contains approximately 40 concatenated lambda-like shuttle vectors, each of which contains one copy of an Escherichia coli lacI gene as a mutational target. lacI mutations in lambda transgenic mice are characterized by a high frequency of spontaneous mutations targeted to CpG dinucleotides suggesting an important contribution from methylation-mediated events. To study the methylation status of CpGs in the lacI gene, we have mapped the distribution of 5-methylcytosines along the DNA-binding domain and flanking sequences of the lacI gene of transgenic mice. We analyzed genomic DNA from various tissues including thymus, liver, testis, and DNA derived from two thymic lymphomas. The mouse genomic DNAs and methylated and unmethylated control DNAs were chemically cleaved, then the positions of 5-methylcytosines were mapped by ligation-mediated PCR which can be used to distinguish methylated from unmethylated cytosines. Our data show that most CpG dinucleotides in the DNA binding domain of the lacI gene are methylated to a high extent (>98%) in all tissues tested; only a few sites are partially (70–90%) methylated. We conclude that tissue-specific methylation is unlikely to contribute significantly to tissue-specific mutational patterns, and that the occurrence of common mutation sites at specific CpGs in the lacI gene is not related to selective methylation of only these sequences. The data confirm previous suggestions that the high frequency of CpG mutations in lacI transgenes is related to the presence of 5-methylcytosine bases.     

Mutation probe of gene structure in E. coli: suppressor mutations in the seven-tRNA operon

Summary Cells defective in uracil-DNA glycosylase (ung:: Tn10) were used in two ways to reveal differences in select point mutations (GC to AT transitions) within the seven-tRNA operon of E. coli. The mutations were indicated as de novo or converted glutamine tRNA suppressor mutations in the genes glnU and/or glnV: (1) the kinetics of photoenzymatic monomerization of pyrimidine dimers quantitated by ung-dependent UV mutagenesis indicated more rapid repair of dimers at sites for converted suppressor mutation than of dimers at sites for de novo suppressor mutation, and (2) spontaneous deamination of cytosine was considerably more frequent at sites for converted suppressor mutation than at sites for de novo suppressor mutation. To explain these results we suggest the physical structure of the DNA in vivo is different at different sites in the seven-tRNA operon. The non-transcribed strand including specifically the anticodon region of the site for converted suppressor mutation may frequently be looped out in a single strand so that a T=C dimer is more accessible to DNA photolyase or a free cytosine residue of non-irradiated DNA is in an aqueous environment conducive to deamination. In addition, we analysed the spontaneous de novo suppressor mutation data to determine an estimate for the in vivo rate of cytosine deamination in double strand DNA of 3.2×10¹³/sec.     

Rapid Deamination of Cyclobutane Pyrimidine Dimer Photoproducts at TCG Sites in a Translationally and Rotationally Positioned Nucleosome in Vivo

Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C and 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by the DNA damage bypass polymerase η, thereby defining a probable mechanism for the origin of UV-induced C to T mutations. Deamination rates for T^mCG CPDs have been found to vary 12-fold with rotational position in a nucleosome in vitro. To determine the influence of nucleosome structure on deamination rates in vivo, we determined the deamination rates of CPDs at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and, together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared with two other sites located at outside positions, and all were much faster than for CPDs at non-TCG sites. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Thus, it was shown that CPDs of TCG sites deaminate the fastest in vivo and that nucleosomes can modulate both their formation and deamination, which could contribute to the UV mutation hot spots and cold spots.     

Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer.

Cytosine methylation at CpG dinucleotides is thought to cause more than one-third of all transition mutations responsible for human genetic diseases and cancer. We investigated the methylation status of the CpG dinucleotide at codon 248 in exon 7 of the p53 gene because this codon is a hot spot for inactivating mutations in the germ line and in most human somatic tissues examined. Codon 248 is contained within an HpaII site (CCGG), and the methylation status of this and flanking CpG sites was analyzed by using the methylation-sensitive enzymes CfoI (GCGC) and HpaII. Codon 248 and the CfoI and HpaII sites in the flanking introns were methylated in every tissue and cell line examined, indicating extensive methylation of this region in the p53 gene. Exhaustive treatment of an osteogenic sarcoma cell line, TE85, with the hypomethylating drug 5-aza-2'-deoxycytidine did not demethylate codon 248 or the CfoI sites in intron 6, although considerable global demethylation of the p53 gene was induced. Constructs containing either exon 7 alone or exon 7 and the flanking introns were transfected into TE85 cells to determine whether de novo methylation would occur. The presence of exon 7 alone caused some de novo methylation to occur at codon 248. More extensive de novo methylation of the CfoI sites in intron 6, which contains an Alu sequence, occurred in cells transfected with a vector containing exon 7 and flanking introns. With longer time in culture, there was increased methylation at the CfoI sites, and de novo methylation of codon 248 and its flanking HpaII sites was observed. These de novo-methylated sites were also resistant to 5-aza-2'-deoxycytidine-induced demethylation. The frequent methylation of codon 248 and adjacent Alu sequence may explain the enhanced mutability of this site as a result of the deamination of the 5-methylcytosine.     

Rotational position of a 5-methylcytosine-containing cyclobutane pyrimidine dimer in a nucleosome greatly affects its deamination rate

C to T mutation hotspots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. These mutations are proposed to arise from the insertion of A by DNA polymerase η opposite the T that results from deamination of the methylC ((m)C) within the CPD. Although the frequency of CPD formation and repair is modestly modulated by its rotational position within a nucleosome, the effect of position on the rate of (m)C deamination in a CPD has not been previously studied. We now report that deamination of a T(m)C CPD whose sugar phosphate backbone is positioned against the histone core surface decreases by a factor of 4.7, whereas that of a T(m)C CPD positioned away from the surface increases by a factor of 8.9 when compared with unbound DNA. Because the (m)Cs undergoing deamination are in similar steric environments, the difference in rate appears to be a consequence of a difference in the flexibility and compression of the two sites due to DNA bending. Considering that formation of the CPD positioned away from the surface is also enhanced by a factor of two, a T(m)CG site in this position might be expected to have up to an 84-fold higher probability of resulting in a UV-induced (m)C to T mutation than one positioned against the surface. These results indicate that rotational position may play an important role in the formation of UV-induced C to T mutation hotspots, as well as in the mutagenic mechanism of other DNA lesions.     

Spontaneous Mutation at a 5-Methylcytosine Hotspot Is Prevented by Very Short Patch (Vsp) Mismatch Repair

In many strains of Escherichia coli, the product of gene dcm methylates the internal cytosines in the sequence 5'CC(A or T)GG. Spontaneous deamination of 5-methylcytosine produces thymine which, if not corrected, can result in a transition mutation. 5-Methylcytosines in the lacI gene are hotspots for spontaneous C to T mutations. dcm is linked to vsr, a gene required for very short patch (VSP) repair. VSP repair corrects T.G mispairs in the following contexts:CTAAGGGGTCC, CTTGGGGACC, TAGGGTCC and CTAGGGTC. I have investigated the relationships between cytosine methylation, mutation, and VSP repair. Spontaneous mutations in the repressor (cI) gene of lambda prophage were isolated in wild-type and mutant lysogens. A hotspot for spontaneous mutation that corresponds with a 5-methylcytosine was observed in wild-type lysogens but was not present in bacteria lacking both methylase and VSP repair activity. Introduction of a plasmid containing dcm+ and vsr+ restored the mutation hotspot. If the added plasmid carried only dcm+, the frequency of spontaneous mutations at the 5-methylcytosine was over 10-fold higher than in Dcm+Vsr+ lysogens. The addition of vsr on a plasmid to a wild-type lysogen resulted in a 4-fold reduction in mutation at the hotspot. These findings support the previously untested hypothesis that VSP repair prevents mutations resulting from deamination of 5-methylcytosine.     

Differential enhancement of spontaneous transition mutations in the lacI gene of an Ung- strain of Escherichia coli

In this communication, the contribution of cytosine deamination to spontaneous mutagenesis in the lacI gene of E. coli was examined. In a wild-type strain, 75% of the amber mutations recovered were G:C----A:T transitions and 60% of these were at the 5-methylcytosine spontaneous hotspots Am6, Am15 and Am34. In a strain deficient for uracil-DNA glycosylase (Ung-), 96% of the amber mutations were G:C----A:T transitions while only 15% of these occurred at the hotspot sites. This shift in the mutational distribution demonstrates that cytosine deamination is a potent mutagenic process, which is enhanced in the absence of glycosylase. Moreover, some amber sites were greatly enhanced in the Ung- strain while others were only slightly enhanced. This result suggests that the rate of cytosine deamination at individual sites may be influenced by surrounding base composition. Therefore, we examined the neighboring sequences and found a strong correlation between the fold-increase in mutation and the A/T richness of the surrounding sequence. It is suggested that A/T-rich regions denature more often, forming transient single strands in which cytosine residues would be expected to deaminate more readily.