Dr. Christopher Lawrence proposes to continue a long-term study on the mechanism of DNA-damage induced mutagenesis in S. cerevisiae. Mutations caused by certain types of DNA damage are thought to be a consequence of replication errors made during translesion synthesis. Dr. Lawrence and others have isolated yeast mutants that have a lower mutation rate when treated with certain DNA-damaging agents; such strains are also usually sensitive to various agents such as UV and MMS. Most of the experiments proposed by Dr. Lawrence involve analysis of the genes REV3, REV7 and REV1, REV (standing for defective mutant reversion). In the Progress Report, Dr. Lawrence reports the purification of Rev3p and Rev7p, and shows that they form a complex with DNA polymerase activity, which he calls DNA polymerase zeta. He shows that in vitro the enzyme can bypass a T-T cyclobutane dimer, a finding consistent with the postulated role of this enzyme in vivo. He also describes methods of preparing plasmids with specific photoproducts at specific sites, and plasmids with abasic lesions at specific sites. Such plasmids were transformed into either E. coli or yeast. Both the efficiency of bypass and the types of mutations generated during the bypass were examined. A particularly interesting result is that the nucleotide C is usually inserted opposite an abasic site in yeast. In most other systems, an A is inserted. Another area of research has been the identification of human proteins that are similar to Rev3p and Rev1p. Dr. Lawrence reports seven refereed publications during the last grant period, three reviews or invited papers, and two submitted manuscripts. The proposed experiments are logical extensions of the past research. The first series of experiments involve purification of presumptive mutagenesis proteins and refinement of the in vitro translesion DNA replication system. Using methods similar to those employed for Rev3p and Rev7p, Dr. Lawrence will purify Rev1p. He will look for a physical interaction between Rev1p and the other two proteins, as well as looking for the DNA-binding properties of Rev1p and Rev7p. He will also examine the effects of Rev1p on the translesion activity of DNA polymerase zeta. In addition, the effects of other proteins, such as RFA, PCNA, and RFC, on translesion DNA synthesis will be examined. Fractionated cell extracts may also be tried. In collaboration with Myron Goodman, he will examine whether DNA polymerase zeta is responsible for the insertion of dCMP opposite abasic sites. The second class of proposed experiments are to identify new genes associated with mutagenesis. One approach that will be taken is a two-hybrid screen using REV1, REV3 and REV7. In addition, he will clone the REV6 and NGM2 genes, two other genes that affect damage-induced mutagenesis. In addition, the cloned REV1, REV3 and REV7 genes will be mutagenized in vitro and screened for their effects on induced mutagenesis. The wild-type REV3 gene has an upstream ATG that is out of frame with the protein. Dr. Lawrence has found that mutating this ATG results in increased expression of the Rev3p. He plans to determine whether the mutated version of the gene affects growth rate or the frequency of spontaneous mutations. He will also find out if rev7 or rev1 mutants have altered levels of spontaneous mutations. The last series of experiments involve the use of plasmids with specifically-located UV photoproducts. Dr. Lawrence will examine the replication of plasmids with these lesions in wild-type and rev mutant strains. Both single-stranded and double-stranded vectors with lesions will be used. The products of the replicated plasmids will be examined by DNA sequence analysis.
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