Loss of genome stability is associated with a number of human diseases that predispose patients to cancer. In particular, mutations in the genes encoding the RecQ family of DNA helicases have been shown to cause three distinct genetic diseases [Bloom syndrome (BLM), Werner syndrome (WRN), and a subset of Rothmund-Thomson syndrome (RTS)]. Cells from these patients display excessive DNA rearrangements suggesting that their primary defect is the failure to maintain genome stability. The only RecQ homolog in budding yeast, Sgs1, exists in a complex with DNA topoisomerase III (Top3) and serves as a model for human BLM which also bindsTop3. We have exploited the yeast system to identify genetic pathways that are functionally redundant with the RecQ-Top3 complex. Two of these pathways are defined by the Slx1-Slx4 and SIx5-SIx8 protein complexes. We have shown that Slx1-SIx4 is a 5'-flap endonuclease while SIx5-Slx8 has no known activity. Here we propose to explore the mechanism by which the RecQ-Top3 complex controls genome stability using biochemical and genetic analysis of these three complexes: Slxl-4, Slx5-8, and Sgsl-Top3.
In Aim1 we will purify and characterize the Slx1-SIx4 protein complex from yeast. New subunits will be identified and tested for their effect on 5'-flap endonuclease activity. A structure-function analysis of both subunits will be conducted and the role of these proteins in cell-cycle checkpoint control will be tested. Genome stability will be assayed in mutant cells by measuring recombination frequencies and by investigating the role of the Slx1-4 complex in controlling rDNA repeat structure. The proteins' nuclear localization will also be determined.
In Aim 2 we will similarly purify and characterize the Slx5-SIx8 complex. The Slx5 and Slx8 subunits are RING-finger proteins suggesting that the Slx5-8 complex may be involved in protein modification. Using purified recombinant protein we will test Slx5-8 for E3 ligase activity using Smt3 or ubiquitin as ligand. A positive result will be extended by searching for specific substrates of the modification.
In Aim 3 Sgsl-Top3 will be purified and tested for DNA helicase and topoisomerase activity using substrates that have been suggested by our genetic analysis.
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