S-(2-chloroethyl)glutathione-generated p53 mutation spectra are influenced by differential repair rates more than sites of initial dna damage.
Several steps occur between the reaction of a chemical with DNA and a mutation, and each may influence the resulting mutation spectrum, i.e. nucleotides at which the mutations occur. The half-mustard S-(2-bro-moethyl)glutathione is the reactive conjugate implicated in ethylene dibromide-induced mutagenesis attributed to the glutathione-dependent pathway. A human p53-driven Ade reporter system in yeast was used to study the factors involved in producing mutations. The synthetic analog S-(2-chloroethyl)glutathione was used to produce DNA damage; the damage to the p53 exons was analyzed using a new fluorescence-based modification of ligation-mediated polymerase chain reaction and an automated sequencer. The mutation spectrum was strongly dominated by the G to A transition mutations seen in other organisms with S-(2-chloroethyl)glutathione or ethylene dibromide. The mutation spectrum clearly differed from the spontaneous spectrum or that derived from N-ethyl,N-nitrosourea. Distinct differences were seen between patterns of modification of p53 DNA exposed to the mutagen in vitro versus in vivo. In the four p53 exons in which mutants were analyzed, the major sites of mutation matched the sites with long half-lives of repair much better than the sites of initial damage. However, not all slowly repaired sites yielded mutations in part because of the lack of effect of mutations on phenotype. We conclude that the rate of DNA repair at individual nucleotides is a major factor in influencing the mutation spectra in this system. The results are consistent with a role of N(7)-guanyl adducts in mutagenesis.
Valadez
J Gerardo
JG
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Guengerich
F Peter
FP
D000477:Alkylating Agents / Q000494:pharmacology; D018736:DNA Adducts / Q000032:analysis / Q000235:genetics; D004260:DNA Repair / Q000502:physiology*; D017930:Genes, Reporter; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology*; D016296:Mutagenesis / Q000235:genetics*; D009153:Mutagens / Q000494:pharmacology*; D009711:Nucleotides / Q000235:genetics; D012441:Saccharomyces cerevisiae; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D000477:Alkylating Agents; D018736:DNA Adducts; D009153:Mutagens; D009711:Nucleotides; D016159:Tumor Suppressor Protein p53; D005978:Glutathione; C046570:S-(2-chloroethyl)glutathione