uvrA and recA mutations inhibit a site-specific transition produced by a single O6-methylguanine in gene G of bacteriophage phi X174.

Abstract

Using site-specific mutagenesis, we have examined the mutagenic activity in vivo of O-methylguanine or 06-n-butylguanine located at a preselected site in gene G of bacteriophage OX174. The experiments were designed so that the phage mutant produced by a targeted transition from either of these alkylated derivatives would be recognizable by a simple plaque assay. Spheroplasts derived from normal and repairdeficient cells were transfected, and the lysates were screened for mutant virus. In cells with normal repair, DNA carrying the methylguanine produced the expected transition in 15% of the total phage; DNA carrying the butylguanine produced the same mutation in 0.3% of the phage4 In cells deficient in excision repair (uvrA) the transition frequency went up by a factor of 8 for O6-butylguanine and down by a factor of 40 for 06' methylguanine. In cells deficient in recombination (recA), the transition frequency increased 1.5-fold for butylguanine and decreased by a factor of 8 for methylguanine. The data show that (i) both methyl-and butylguanine produce site-directed transitions in OX174; (ii) the transition occurs in recA cells; (iii) the frequency of the transition is influenced by both recA and uvrA mutations; (iv) the recA and uvrA mutations alter the transition frequency for methylguanine and butylguanine in opposite directions.The gene products of uvrA, -B, and -C play a key role in removing a variety of damaged purine or pyrimidine nucleotide residues that deform the DNA helix (1-7). The ada gene product, on the other hand, appears to operate only on 06-methyl-or 06-ethylguanine in DNA, in an alkyl-transfer reaction that regenerates the guanine (8-12). In this paper we report our initial studies dealing with effects of DNA repair on the frequency of site-directed mutations produced in vivo by alkylated purine or pyrimidine residues located at preselected positions in an essential gene of bacteriophage OX174. The results show an unexpected effect of uvrA and recA mutations on the production of a specific transition from an 06-methylguanine or 06-n-butylguanine in the first position of the third codon in gene G of qX174.MATERIALS AND METHODS Bacterial Strains. Escherichia coli AB1157 and its isogeneic derivatives AB1886 (uvrA6), AB2463 (recA13), and AB2480 (uvrA6 recA13) were obtained from B.

Mutagenic frequencies of site-specifically located O6-methylguanine in wild-type Escherichia coli and in a strain deficient in ada-methyltransferase

“…The total mutation frequencies determined here for 06_ meGua in wild-type cells are in general agreement with the above-mentioned studies, except for that measured by Chambers et al (4). We selected for mismatch mutants, which represented that percentage of the population of bacteriophage that was replicated from the modified DNA strand and that, to some extent, escaped mismatch repair.…”

“…Chambers et al (4) attributed the high mutation frequency (15.8%) of 06-meGua in strain AB1157 found in their study to interference by uvrABC in repair of the lesion. We also found evidence for the ability of uvrABC to affect 06-meGua mutagenesis at some DNA positions (this study; 23), but to a much lower extent.…”

“…Hill-Perkins et al (10) reported a mutation frequency of 0.46% in E. coli JM101 (wild type for repair) for 06_ meGua synthesized into the a-complementing region of M13mp9 replicative form DNA. Chambers et al (4) reported a mutation frequency of 15.8% in E. coli AB1157 (wild type for repair) for 06-meGua synthesized to rescue a lethal mutation in the third codon of gene G of bacteriophage 4XX174. Bhanot and Ray (2), who used a selection system almost identical to that of Chambers et al (4), reported a mutation frequency of 8% in ada-5 cells (BS24) for 06_ meGua synthesized into gene G of bacteriophage 4~X174.…”

“…Chambers et al (4) reported a mutation frequency of 15.8% in E. coli AB1157 (wild type for repair) for 06-meGua synthesized to rescue a lethal mutation in the third codon of gene G of bacteriophage 4XX174. Bhanot and Ray (2), who used a selection system almost identical to that of Chambers et al (4), reported a mutation frequency of 8% in ada-5 cells (BS24) for 06_ meGua synthesized into gene G of bacteriophage 4~X174. In the wild type, they detected no mutants in 100 infective centers tested, which implies a mutation frequency of <1%; the strain used was C6100, not the true parent of BS24, which is AB1157.…”

DNA Repair Enzymes

“…It should be mentioned, however, that MNNG, which is not known to cause major structural changes in DNA, created substrate for the excinuclease even though the adduct responsible for incision was not identified (148). Similarly, based on mutation rates observed in uvr-and wild-type cells with a site specifically located Q 6 -mGua (which does not distort the helix), it has been proposed that the ABC excinuclease or some of its subunits bind to this adduct but fail to remove it and thus interfere with its repair and increase the mutation rate (149). Clearly, definition of the substrate �: tructure will be an important step toward understanding the action mechanism of the enzyme.…”