We pursue two areas of research, one focused on the mechanisms of homologous recombination and the other focused on mechanisms of gene regulation. These research topics evolved out of our studies of mating type in bakers yeast where cell type is changed by a programmed genetic rearrangement that allows expression of alternative genes encoding trans-acting regulatory proteins. The programmed recombination event is initiated by a site-specific DNA cleavage. Our studies of that process led us into the area of genetic recombination in general and double-strand-break (DSB) repair in particular. Currently, we are identifying functions involved in DSB repair by screening for strains defective in that process. We expect that such genes could add to the list of recombination and DNA damage repair defects known to be related to neoplastic disease. We recently demonstrated that the DNA synthesis associated with genetic recombination has substantially lower fidelity than that found in general genome duplication. Our results suggest that at least two different DNA polymerases have roles in this elevated mutation rate. The majority of the base substitutions associated with recombination are made by the translesion polymerase encoded by the REV3 gene. Our studies also identified a role for an exonuclease (EXO1) in double-strand-break repair. We are currently investigating the mitotic role of the protein encoded by SAE2, a gene previously defined by its role in meiotic recombination. We find that sae2 mutants generate high levels of inverted repeat gene amplification. We demonstrated that reverse transcription of cellular mRNAs can generate substrates for recombination, resulting in processed pseudogenes and RNA-mediated gene conversion. We continue to explore the role of reverse transcripts in genome evolution. In collaboration with David Garfinkel, we developed an in vivo assay; for HIV-1 reverse transcriptase (RT), based on hybrid Ty1/HIV-1 elements and a homologous recombination assay. This assay is sensitive to some known non-nucleoside inhibitors of HIV-1 RT. In collaboration with Christopher Michejda, we identified several new drugs that inhibit HIV-1 RT. We are initiating a new area of research into the fidelity of retrotransposition using tools similar to those that we used to study the fidelity of homologous recombination. This topic provides us with not only an opportunity to investigate the features of RT that govern its fidelity, but also an opportunity to investigate the properties that govern the fidelity of RNA polymerase, a project that we will pursue in collaboration with Mikhail Kashlev. Our interests in gene regulation are currently centered on the mechanism of gene silencing. This mechanism was first discovered as the means of inhibiting the transcription of donor loci used in the recombination event associated with mating-type switching in yeast. The source copies of the genes activated in this process are kept silent by the SIR genes (silent information regulators) and several other genes. A demonstrated role of histones, and an altered accessibility of the DNA in the silenced region, implicates chromatin modification within a defined domain as the means by which silencing occurs. The SIR2 gene has also been shown to have a role in silencing near the telomeres and in controlling recombination in the rDNA repeats. We showed that yeast have five genes closely related to SIR2 and that SIR2 homologs can be found in organisms ranging from prokaryotes to humans. We are continuing to define the roles of this gene family in genome regulation.
