Bloom’s Syndrome (BS) is a rare human disease characterized by genome instability and cancer predispostion. The gene mutated in BS, BLM, encodes a member of the RecQ family of DNA helicases. BLM is unique among this family because it is the only RecQ member conserved in unicellular eukaryotes. Apart from the DNA helicase domain, BLM/SGS1 orthologs contain a poorly characterized N-terminal domain of about 650 amino acids (aa). In yeast, this domain (Sgs1 1-652) is known to be physiologically important although it’s only known roles are to bind Top3 and Rmi1 through its N-terminal 100 aa, and in vitro single-strand DNA (ssDNA) binding, ssDNA annealing and strand exchange (SE) activities, through its SE domain (Sgs1 103-323) My research consisted of structure/function analyses of Sgs1 in its poorly characterized N-terminus of aa 323-652 and I found a domain that is important to Sgs1’s function. I interrogated this region by doing systematic deletions and tested these sgs1 mutants for complementation of synthetic lethality. The allele with the smallest deletion that did not complement sgs1slx4 synthetic lethality was found to be sgs1-∆386-621, and the allele with the smallest deletion that did not complement sgs1slx5, was found to be sgs1-∆500-621. I found that sgs1-∆386-621 is physiologically important also through the Top3 slow growth assay. Furthermore I believe that the difference in results between sgs1slx4 and sgs1slx5 will prove significant and warrants further investigation. I also used a BLM/GCN4 chimera where the SE domain was replaced with the coiled coil GCN4, and again tested alleles with systematic deletions within aa 323-652 in synthetic lethality assay. I found the smallest essential domain to be sgs1/gcn4-∆500-621 in the sgs1slx4 background, and interestingly the same deletion in sgs1-∆500-621 did grow weakly and complemented synthetic lethality. Therefore I postulate there is some interaction between the SE domain and aa 500-621 that could be physiologically important and again warrants further investigation.
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Cell and Developmental Biology
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Rutgers University Electronic Theses and Dissertations
Rutgers University. Graduate School - New Brunswick
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