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C W Lawrence

Publications and source records attributed to C W Lawrence.

At least 55 records · Page 3Linked to original sources
PubMed1990

SOS-dependent replication past a single trans-syn T-T cyclobutane dimer gives a different mutation spectrum and increased error rate compared with replication past this lesion in uninduced cells.

We have transfected SOS-induced and uninduced cells of a uvrA6 strain of Escherichia coli with single-stranded M13mp7-based vectors that carried a single trans-syn T-T cyclobutane dimer at a unique site. Unlike constructs carrying the cis-syn isomer of this lesion, these vectors could be replicated with modest efficiency (14%) in the absence of SOS induction and therefore provided an opportunity to measure directly the influence of such induction on error rate and mutation spectrum. We found that translesion synthesis in the absence of SOS induction was remarkably accurate; only 4% of the replicated bacteriophage contained mutations, which were exclusively targeted single T deletions. In SOS-induced cells, error frequency increased to 11% and the resulting mutations included targeted substitutions and near-targeted single base additions, as well as the T deletions. Replication efficiency was 29% in these conditions. SOS induction therefore leads not only to an enhanced capacity to replicate damaged DNA but also to a marked change in mutation frequency and spectrum.

Base Sequence↗
PubMed1989

REV3, a Saccharomyces cerevisiae gene whose function is required for induced mutagenesis, is predicted to encode a nonessential DNA polymerase.

We have cloned the REV3 gene of Saccharomyces cerevisiae by complementation of the rev3 defect in UV-induced mutagenesis. The nucleotide sequence of this gene encodes a predicted protein of Mr 172,956 showing significant sequence similarity to Epstein-Barr virus DNA polymerase and to other members of a class of DNA polymerases including human DNA polymerase alpha and yeast DNA polymerase I. REV3 protein shows less sequence identity, and presumably a more distant evolutionary relationship, to the latter two enzymes than they do to each other. Haploids carrying a complete deletion of REV3 are viable. We suggest that induced mutagenesis in S. cerevisiae depends on a specialized DNA polymerase that is not required for other replicative processes. REV3 is located 2.8 centimorgans from CDC60, on chromosome XVI.

Amino Acid Sequence↗
PubMed1988

Ultraviolet light induces different spectra of lacI sequence changes in vegetative and conjugating cells of Escherichia coli.

We have analyzed the nucleotide sequence changes responsible for mutations from lacIs to lacI- induced in ultraviolet light-irradiated, excision-deficient cells. Irradiated cells were either used as donors in the conjugational transfer of an F' lacIs plasmid to SOS-induced, excision-deficient recipients or allowed to continue vegetative growth. Although the types and proportions of premutagenic lesions are likely to have been very similar in these two circumstances, analysis of the sequence data shows that different spectra of mutations were induced. In vegetative cells there were about equal numbers of transitions and transversions, but transitions outnumbered transversions by about three to one in exconjugants. About 90% of the single nucleotide substitutions could be assigned to a bipyrimidine target sequence in both sets of data, but they differed with respect to the location of the substitution: more or less equal numbers were found at the 3' and 5' sites of the probable bipyrimidine target in vegetative cells, but in exconjugants over 80% were at the 3' site. It is also possible that mutations were targeted more commonly at T-C sequences in exconjugants than in vegetative cells, but the evidence for this is less secure. We conclude that these results reflect some dissimilarity between vegetative cells and exconjugants in the way damaged DNA is replicated or lesions tolerated, but the particular features of these processes responsible for the different mutational spectra have not yet been identified.

Base Sequence↗
PubMed1988

UmuC function is not essential for the production of all targeted lacI mutations induced by ultraviolet light.

Up to a quarter or more of the normal yield of lacI- mutations could be induced by ultraviolet light in a uvrA6 umuC122:: Tn5 strain if they were detected by plating on 5-bromo-4-chloro-3-indolyl-beta-D-galactoside medium, where all surviving cells can form colonies. Using phenyl beta-D-galactoside selection, which curtails post-irradiation growth, only low yields of mutations were induced. Nucleotide sequence analysis of 134 spontaneous and 145 ultraviolet light-induced mutations shows that broadly similar kinds of mutations were induced in the umuC mutant and its uvrA6 umuC+ counterpart. In particular, these data offer no reason for believing that most of the mutations induced in the umuC mutant were other than normal, targeted events. We conclude that UmuC function, rather than being essential, facilitates recovery and specifically, following the model of Bridges & Woodgate, that it facilitates the prompt resumption of chain elongation.

Base Sequence↗
PubMed1988

Frequency and spectrum of mutations produced by a single cis-syn thymine-thymine cyclobutane dimer in a single-stranded vector.

We have constructed a single-stranded vector that contains a uniquely located cis-syn T-T cyclobutane dimer by ligating a synthetic oligomer containing this UV photoproduct into M13mp7 viral DNA linearized with EcoRI. In the absence of SOS induction, transfection of a uvrA6 mutant of Escherichia coli with this vector gave very few progeny plaques, and the data imply that a single dimer blocks replication in at least 99.5% of the molecules. In vitro photoreactivation completely abolished this inhibition. Transfection of cells irradiated with UV at 4 J.m-2 to induce the SOS response gave 27% of the number of plaques found with a dimer-free control. Nucleotide sequence analysis of 529 progeny phage showed that translesion synthesis was usually accurate: the normal sequence was found in 93% of them. Where mutations occurred, all were targeted single-nucleotide substitutions, with approximately 90% being targeted at the 3' nucleotide of the lesion: of a total of 26 mutations, 15 were 3' T----A, 8 were 3' T----C, and 3 were 5' T----C. No T----G mutations were found. In addition to these results with the normal construct, data were also obtained from vectors in which the M13mp7 cloning site, which forms a hairpin in single-stranded DNA, was present 4 nucleotides on the 3' side of the T-T dimer. These hairpin-containing vectors gave a very similar mutation frequency (8% versus 7%) but altered mutation spectrum: all 12 mutations detected were 3' T----A transversions, a difference from the previous set of data that is significant (P = 0.03).

Base Sequence↗
PubMed1986

The isolation and characterization of ngm2, a mutation that affects nitrosoguanidine mutagenesis in yeast.

We have isolated and characterized a new mutant of Saccharomyces cerevisiae, carrying a single mutant allele that we designate ngm2-1, which is defective with respect to induced mutagenesis. This mutant was isolated by screening mutagenized clones for reduced frequencies of reversion of the his1-7 allele, induced by N-methyl-N-nitro-N-nitrosoguanidine. As judged by the reversion of his1-7 and ilv1-92, ngm2-1 mutant strains are also deficient with respect to mutability induced by methyl methane sulfonate, ethyl methane sulfonate and, at least partially, by UV. UV-induced reversion of the ochre mutation arg4-17 and the frameshift mutation his4-38 was not much affected by ngm2-1, however. Like rev3 and rev7 mutations, ngm2-1 also has little influence on the reversion of the proline missense allele, cyc1-115. Ngm2-1 mutants are only at best very slightly more sensitive to the toxicity of the four mutagens used, and homozygous diploids sporulate normally.

Alleles↗
PubMed1986

The isolation and characterization of an alkylating-agent-sensitive yeast mutant, ngs1.

We have isolated and characterized a mutant of baker's yeast, Saccharomyces cerevisiae, carrying the new mutation, ngs1, which is sensitive to the toxic effects of monofunctional alkylating agents, but normal with respect to 254-nm ultraviolet light sensitivity. ngs1 mutants exhibited more or less normal reversion frequencies for his1-7 and ilv1-92 induced by each of these mutagens. The various sensitivities associated with ngs1 cosegregated and have been shown to be the result of a lesion in a single nuclear gene. Extracts of ngs1 and NGS1+ strains contained approximately equal levels of an activity that removes 3-methyladenine (3MA) and 7-methylguanine (7MG) from DNA in vitro. The mutation also depressed sporulation.

Adenine↗
PubMed1985

REV7, a new gene concerned with UV mutagenesis in yeast.

Three allelic mutations of a new yeast gene, which we have named REV7, have been isolated by testing 313 methyl methane sulfonate sensitive mutants for UV-induced reversion of a lys2 allele. Rev7 mutants are markedly deficient with respect to UV-induced reversion of lys2, are slightly sensitive to UV and appear to be in the RAD6 epistasis group for UV survival. Rev7-1, which is probably an amber mutation, does not appear to affect sporulation in homozygous diploids. The REV7 gene is located about 12 cM distal to HIS5 on chromosome IX.

Chromosome Mapping↗
PubMed1985

UV and chemical mutagenesis in rev7 mutants of yeast.

We have examined induced mutagenesis in rev7-1 mutants of Baker's yeast' Saccharomyces cerevisiae, using a variety of contrasting test systems and several different mutagens. UV-induced reversion frequencies of the ochre allele arg4-17, the putative missense allele ilv1-92 and the frameshift allele his4-38 were 10 to 200 fold lower in haploid and diploid rev7-1 mutants compared with wild type strains, but UV-induced reversion frequencies of the frameshift allele leu2-3 and the proline missense allele cyc1-115 were reduced only a few fold. Ilv1-92 reversion frequencies induced by methyl methane sulfonate or by N-methyl-N'-nitro-N-nitrosoguanidine were 10 to 20 times lower in rev7-1 mutants, but normal frequencies of these revertants were induced with ethyl methane sulfonate, even though rev7-1 strains are slightly sensitive to this mutagen as well as to the others tested. We conclude that the rev7 mutants, like the rev3 mutants they closely resemble, have a substantial but not total deficiency concerning induced mutagenesis.

Alleles↗
PubMed1985

Untargeted mutagenesis induced by UV in the lacI gene of Escherichia coli.

Using a nonselective method, we have estimated the proportion of untargeted mutations in the lacI gene of E. coli by transferring either irradiated or unirradiated F' pro lac plasmids from an excision deficient donor to an excision deficient pro lac deleted recipient that had been irradiated and allowed to induce recA dependent functions for 30 min. We find that about 10 percent of the mutations induced by either 3.5 Jm-2 or 7 Jm-2 UV are untargeted.

Bacterial Proteins↗
PubMed1985

Conjugation-dependent enhancement of induced and spontaneous mutation in the lacI gene of E. coli.

The frequency of lac mutations induced in an F'lacIS plasmid, transferred by conjugation from UV-irradiated, excision-deficient donors to excision-deficient, delta pro lac recipients, is 2-3 fold higher than that typical of nonmating cells which contain the plasmid. These additional induced mutations can probably be ascribed to errors made during the first, or repliconation, synthesis that takes place in the recipient during the course of plasmid transfer. We also find that spontaneous mutation rates are enhanced in conjugating cells, indicating that fewer errors are corrected, or more made, during transfer replication.

Bacterial Proteins↗
PubMed1985

Identity of the photoproduct that causes lacI mutations in UV-irradiated Escherichia coli.

Estimates of the capacity of photoreactivation to act specifically on premutational lesions were obtained by conjugational transfer of an F' lac plasmid from a UV-irradiated, photoreactivated donor to a delta (pro-lac) recipient that had been UV irradiated and allowed to induce SOS functions for 30 min. This treatment reduced the frequency of induced lacI mutations by 70 to 80%, indicating that cyclobutane dimers cause most mutations in this system.

Escherichia coli↗
PubMed1984

UV-induced reversion of his4 frameshift mutations in rad6, rev1, and rev3 mutants of yeast.

The UV-induced reversion of two his4 frameshift alleles was much reduced in rad6 mutants of Saccharomyces cerevisiae, an observation that is consistent with the hypothesis that RAD6 function is required for the induction of all types of genetic alteration in misrepair mutagenesis. The reversion of these his4 alleles, together with two others of the same type, was also reduced in rev1 and rev3 mutant strains; in these, however, the extent of the reduction varied considerably with test allele used, in a manner analogous to the results in these strains for base repair substitution test alleles. The general features of UV-induced frameshift and substitution mutagenesis therefore appear quite similar, indicating that they may depend on related processes. If this conclusion is correct, greater attention must be given to integrating models which account for the production of nucleotide additions and deletions into those concerning misrepair mutagenesis.

Base Composition↗
PubMed1982

The mechanism of untargeted mutagenesis in UV-irradiated yeast.

The SOS error-prone repair hypothesis proposes that untargeted and targeted mutations in E. coli both result from the inhibition of polymerase functions that normally maintain fidelity, and that this is a necessary precondition for translesion synthesis. Using mating experiments with excision deficient strains of Bakers' yeast, Saccharomyces cerevisiae, we find that up to 40% of cycl-91 revertants induced by UV are untargeted, showing that a reduction in fidelity is also found in irradiated cells of this organism. We are, however, unable to detect the induction or activation of any diffusible factor capable of inhibiting fidelity, and therefore suggest that untargeted and targeted mutations are the consequence of largely different processes. We propose that these observations are best explained in terms of a limited fidelity model. Untargeted mutations are thought to result from the limited capacity of processes which normally maintain fidelity, which are active during replication on both irradiated and unirradiated templates. Even moderate UV fluences saturate this capacity, leading to competition for the limited resource. Targeted mutations are believed to result from the limited, though far from negligible, capacity of lesions like pyrimidine dimers to form Watson-Crick base pairs.

Crosses, Genetic↗
PubMed1981

Are pyrimidine dimers non-instructive lesions?

Published data from yeast and E. coli show that base substitution induced by UV in pyrimidine-pyrimidine sequences is not random, and suggest that fidelity of DNA replication is not entirely lost during transdimer synthesis. These observations question whether cyclobutane pyrimidine dimers are truly non-instructive lesions.

DNA Repair↗