FUNCTION OF SGS1, A HOMOLOG OF BLM AND WRN

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The specific aim of this proposal is to study the function of Sgs1. This gene was first isolated as a slow growth suppressor of top3 mutants in Saccharomyces cerevisiae and found to be homologous to the E. coli RecQ helicase. Cells with Sgs1 mutations exhibit hyper- recombination between repeated sequences, show increased chromosome non-disjunction and sporulate poorly as homozygous diploids. Recently, the genes responsible for two human diseases, Bloom and Werner syndromes were cloned and found to be homologous with Sgs1. Thus, the study of Sgs1 in yeast may provide insights into the function of the members of this multigene family and may yield important clues to the etiology of cancer in these two syndromes. The specific approaches are: the PI will investigate both the physical and genetic interactions between Sgs1, topoisomerases, checkpoint genes and other yeast genes including helicases. He will develop a novel allele replacement technique to aid in the study of these interactions. (2) He will investigate the relationship between Sgs1 and its human counterparts by cross-complementation studies in both yeast and mammalian cells. Specifically, he will swap domains among these genes to define the units necessary for function. In addition, he will determine if the same physical interactions that occur in yeast can occur in mammalian cells. Furthermore, sensitivity to various inhibitors will be tested to characterized the human homologs. (3) The investigator will purify both Sgs1 and the components with which it interacts in order to define their biochemical function(s). In addition, DNA topology of both native sequences and introduced plasmids will be investigated by varying the gene dosage of Sgs1 and its interacting components. (4) He will determine the parameters that affect hyper-recombination between repeated sequences resulting from a Sgs1 deficiency. Specifically, he will examine sequences from two locations that exhibit hyper-recombination in the absence of Sgs1 -- the rDNA array and the SUP4 region. The PI will also investigate the relationship between replication fork pausing and hyper-recombination.