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.

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics C Study Section (MGC)
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Hagan, Ann A
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University of South Florida
Schools of Arts and Sciences
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Arora, Sucheta; Deshpande, Rajashree A; Budd, Martin et al. (2017) Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast. Mol Cell Biol 37:
Shastri, Vivek M; Schmidt, Kristina H (2016) Cellular defects caused by hypomorphic variants of the Bloom syndrome helicase gene BLM. Mol Genet Genomic Med 4:106-19
Kennedy, Jessica A; Syed, Salahuddin; Schmidt, Kristina H (2015) Structural Motifs Critical for In Vivo Function and Stability of the RecQ-Mediated Genome Instability Protein Rmi1. PLoS One 10:e0145466
Bastos de Oliveira, Francisco Meirelles; Kim, Dongsung; Cussiol, José Renato et al. (2015) Phosphoproteomics reveals distinct modes of Mec1/ATR signaling during DNA replication. Mol Cell 57:1124-32
Doerfler, Lillian; Schmidt, Kristina H (2014) Exo1 phosphorylation status controls the hydroxyurea sensitivity of cells lacking the Pol32 subunit of DNA polymerases delta and zeta. DNA Repair (Amst) 24:26-36
Kennedy, Jessica A; Daughdrill, Gary W; Schmidt, Kristina H (2013) A transient ?-helical molecular recognition element in the disordered N-terminus of the Sgs1 helicase is critical for chromosome stability and binding of Top3/Rmi1. Nucleic Acids Res 41:10215-27
Mirzaei, Hamed; Schmidt, Kristina H (2012) Non-Bloom syndrome-associated partial and total loss-of-function variants of BLM helicase. Proc Natl Acad Sci U S A 109:19357-62
Doerfler, Lillian; Harris, Lorena; Viebranz, Emilie et al. (2011) Differential genetic interactions between Sgs1, DNA-damage checkpoint components and DNA repair factors in the maintenance of chromosome stability. Genome Integr 2:8
Mirzaei, Hamed; Syed, Salahuddin; Kennedy, Jessica et al. (2011) Sgs1 truncations induce genome rearrangements but suppress detrimental effects of BLM overexpression in Saccharomyces cerevisiae. J Mol Biol 405:877-91
Schmidt, Kristina H; Viebranz, Emilie B; Harris, Lorena B et al. (2010) Defects in DNA lesion bypass lead to spontaneous chromosomal rearrangements and increased cell death. Eukaryot Cell 9:315-24

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