Certain human genetic disorders with extreme cancer risk, such as Bloom's syndrome and Werner syndrome, are associated with mutations in DNA helicases of the RecQ family. Mutants of the yeast Saccharomyces cerevisiae that lack Sgs1, the only RecQ- related DNA helicase in this yeast, have proven to be excellent model systems for some cellular phenotypes of the Bloom's and Werner syndromes, especially with respect to their hyperrecombination phenotype. Although rates of accumulating gross- chromosomal rearrangements are only moderately elevated in sgs1 mutants, it was recently shown that cells lacking Sgs1 are uniquely susceptible to undergoing complex, recurring translocations driven by small regions of homology in highly diverged genes (60-65 % identity). Although Sgs1 and mismatch repair proteins are inhibitors of recombination between similar but nonidentical (homeologous) sequences, only Sgs1 is required for the suppression of these complexes and recurring translocations. The objective of this proposal is to study the intra- and interchromosomal recombination mechanisms that cause homeology-driven translocations in the absence of Sgs1.
The specific aims of this study are to: (1) Determine the extent to which gene structure and chromosome environment control the rate and structure of homeology-driven translocations in sgs1 mutants lacking DNA damage checkpoint sensors or chromatin assembly factors. This will be achieved by modifying location, orientation and copy number of translocation targets as well as homology block distance and DNA sequence identity. (2) Elucidate the differential requirement of DNA-damage checkpoint components for the suppression and formation of recurring translocations in sgs1 mutants. (3) Examine the role of functional domains and known physical interactions of Sgs1 in the inhibition of homeology-driven translocations. (4) Determine the ability of the five human RecQ-like DNA helicases to substitute for Sgs1 in the suppression of homeology-driven translocations. SGS1 will be replaced with cDNAs of human RecQ homologs, some of which have been successfully expressed in yeast and suppress some aspects of the sgs1 mutant phenotype. These studies will provide mechanistic insights into the role of RecQ helicases in the maintenance of genome integrity and will shed light on the general mechanisms leading to gross-chromosomal rearrangements, especially gene translocations, which are often associated with human cancers.
The causes of genome instability, which is a hallmark of most cancers, are still unclear. The proposed studies will provide mechanistic insights into the role of DNA unwinding enzymes in the maintenance of genome integrity and will shed light on the general mechanisms leading to chromosomal rearrangements, especially gene translocations, which are often associated with human cancers and chromosome breakage syndromes.
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