Molecular characterization of genes involved in DNA repair in Saccharomyces cerevisiae.

摘要

DNA is an inherently unstable molecule; it is also subject to inaccuracies of synthesis and modification through the action of naturally occurring radiation and chemicals and the byproducts of metabolism. There are multiple enzymatic pathways responsible for the detection, repair, and/or tolerance of potentially mutagenic alterations to DNA. Although many of the genes encoding proteins involved in DNA repair were initially characterized in microorganisms such as Escherichia coli and Saccharomyces cerevisiae , they have been shown to be conserved throughout phylogeny. Of the 125 genes known in humans to be directly or indirectly involved in DNA repair, mutations in several have been shown to be responsible for specific disorders ranging from premature aging to cancer.; We have studied three mutants of Saccharomyces cerevisiae that are sensitive to methylation of DNA, responsible for increased levels of mitotic recombination, and result in meiotic failure. Clearly these genes are involved in DNA metabolism at some level. One of these, MMS21 , was cloned and sequenced by our lab and found to be novel, although recent genome sequencing initiatives have revealed possible homologs in S. pombe, C. albicans, C. elegans, D. melanogaster, and humans. To elucidate the role of Mms21p in DNA repair, we have used the yeast two hybrid system to identify those proteins with which it interacts. GIN11 (g&barbelow;rowth i&barbelow;nhibitor) was cloned by this method and we have taken both biochemical and genetic approaches to confirm this interaction.; We have mapped mms13-1 to chromosome II and have been able to complement the MMS sensitivity with a genomic fragment containing APN2; while we have sequenced APN2 from mms13-1 strains we have not yet found a sequence change within the coding region. It is possible that the mms13-1 phenotype is due to a mutation within the extreme five prime end of the coding region or in the promoter region and we are currently sequencing the upstream region to that end. We have mapped mms9-1 to chromosome XI and have been able to complement its MMS sensitivity with a genomic fragment containing APN1. Preliminary sequence results have not revealed sequence changes from published sequence.