Werner's Syndrome (WS) is a genetic disease characterized by premature aging and chromosomal instability. The gene responsible for WS (WRN) is similar to the SGS1 gene of yeast and, like WS, SGS1 mutants display premature aging and genomic instability. Both WRN and SGS1 encode 160 kDa proteins belonging to the RecQ family of DNA helicases. Because WRN and SGS1 are likely to function in identical genetic pathways, a search for mutations that interact with SGS1 should uncover genes that control cellular aging and genome stability. Genetic experiments are proposed to identify and characterize genes that interact directly and indirectly with SGS1, and biochemical experiments will be used to determine the function of the encoded gene products. An SGS1 synthetic-lethal mutant screen has been conducted to identify mutations that cause the cell to require SGS1 for viability. The genes responsible for these mutations (SLX genes) are likely to act in genetic pathways that are parallel to SGS1. Some of the SLX genes have been cloned and found to encode novel proteins with conserved human homologs. The complete set of SLX genes will be cloned and all SLX mutants will be characterized for effects on yeast aging and genomic stability. The expression pattern, cellular localization, and enzymatic activity of the SLX proteins will be determined. These results will be confirmed by similar studies of the SLX1 gene in human cells. To identify genes that interact directly with SGS1, we have isolated a set of novel conditional-lethal alleles of SGS1. Extragenic suppressors of these alleles are expected to act directly in the SGS1 pathway. Suppressor strains will be isolated and affected genes will be cloned. Additional genes in this pathway will be identified by searching for mutations that show SGS1-specific phenotypes and by performing a synthetic-lethal screen with SLX mutants. To determine the biochemical function of the SLX and SGS1 proteins, they will be purified from yeast in their native form. These purified proteins will be assayed for enzymatic activities involved in DNA metabolism. Stably-assoicated proteins that co-purify with the SLX and SGS1 proteins will be subjected to amino acid sequencing, molecular cloning and reverse genetics to identify their function in controlling cellular aging and genomic stability.
