The long-term goal of the proposed research is to understand fully the mutagenic activity (SOS mutagenesis) induced in E. coli in response to radiations and carcinogenic chemicals as part of a more complex response (the SOS response) to genetic damage. LexA protein is the common repressor of at least 17 SOS genes, which are derepressed when RecA protein is activated by DNA damage to a form (RecA*) that can cause proteolytic cleavage of LexA. Activated RecA* has an additional essential role in SOS mutagenesis, distinct from cleavage of LexA or any other repressor. The additional activity could be either proteolytic processing of one or more other proteins or a mechanistic function possible only in the activated state. As an approach to identification of this unknown RecA* role, a mutant allele, recA730, that causes constitutive hyperactivity of RecA*, will be cloned, RecA730 protein purified, and its activity in an in vitro assay for cleavage of LexA compared with that of RecA+ and other mutant RecA proteins. Another mutant allele, recA718, which causes UV sensitivity and UV hypermutability, will also be cloned and its protein product purified and partially characterized. Both RecA730 and RecA718 will be made available to laboratories investigating RecA protein activities in various in vitro systems. The following additional investigations, all relating to aspects of SOS mutagenesis not well understood, are also proposed: 1) determination of the basis of the mutator activity caused by plasmid pKM101 that differs from SOS mutator activity in several ways, e.g., it is expressed in lexA(Ind-) strains and is enhanced by low temperatures; 2) testing of the hypothesis that SOS mutator activity involves inhibition of the proofreading activity of DNA polymerase III, by examining the interaction of mutD(dnaQ) alleles (which are known to inhibit Pol III proofreading) with recA730, which causes constitutive SOS mutator activity; 3) determination of the basis of the high frequency of novel recA alleles recovered in crosses of K12 recA441 donors x B/r recA+ recipients, and 4) reexamination of the phenomenon known as """"""""mutation frequency decline"""""""" (MFD) to test the hypothesis that it is caused by medium-dependent changes in DNA conformation that affect repair of UV photoproducts at specific sites in certain tRNA genes. A deeper understanding of the events leading from DNA damage to mutation in bacteria may provide new avenues of approach to human carcinogenesis, since at least some activities analogous to components of the SOS response have been demonstrated in human cells.
Showing the most recent 10 out of 11 publications
