In my laboratory, we use the yeast Saccharomyces cerevisiae to study the genetics and biochemistry of recombination. Malfunctions of this important cellular process can cause loss of heterozygosity, a mechanism known to lead to tumorigenesis for some cancers. For example, one of the yeast genes that we isolated during our recombination studies has homologs implicated in two human disorders, Bloom and Werner syndromes, both of which cause an increased risk of cancer.
By searching for mutations that increase recombination, we have successfully identified new genes and functions involved in suppressing recombination between naturally occurring repetitive elements dispersed throughout the genome. The analysis of these hyper-recombination mutations has resulted in the identification of: TOP3, a novel DNA topoisomerase; SGS1, a helicase that defines the gene family for Bloom and Werner syndrome; POL12, a DNA polymerase alpha-associated factor and RFA1, the yeast single-stranded DNA binding protein. Each gene is evolutionarily conserved from bacteria to man, illustrating the importance of their function in all organisms.
Furthermore, to identify key eukaryotic proteins in recombination, we are studying genes whose functions are essential for recombination, repair and DNA checkpoints. We have found that the RAD52 gene product, a central recombination protein, functionally interacts with RAD51 protein, an evolutionarily conserved homolog of the bacterial RecA strand exchange protein. Homologs of these genes have been identified by us in mouse, maize and other fungi. We have shown that one biochemical function of Rad52 protein is to enhance DNA strand annealing. We are also studying MEC1 and MEC2 (RAD53), two essential yeast genes involved in multiple processes including recombination and cell cycle checkpoint control. MEC1 is a homolog of the human ATM gene, which is mutated in ataxia-telangiectasia patients. A genetic suppressor of mec1 and mec2 has been identified that separates the essential function from both recombination and checkpoint control.
Finally, using the entire genome sequence of an organism, we are developing methods to create complete gene disruption libraries as well as two-hybrid libraries to permit functional analyses on a genomic scale.
Selected Publications
Gangloff S, McDonald JP, Bendixen C, Arthur L and Rothstein R. The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase. Mol. Cell. Biol. 14: 8391-8398 (1994).
Smith J and Rothstein R. A mutation in the gene encoding the Saccharomyces cerevisiae single-stranded DNA binding protein Rfa1 stimulates a RAD52-independent pathway for direct-repeat recombination. Mol. Cell Biol 15:1632-1641(1995).
Rothstein R and Gangloff S. Hyper-recombination and Bloom's syndrome: microbes again provide clues about cancer. Genome Res 5:421-426 (1995).
Gangloff S, Zou H and Rothstein R. Gene conversion plays the major role in controlling the stability of large tandem repeats in yeast. EMBO J 15:1715-1725 (1996).
Mortensen UH, Bendixen C, Sunjevaric I and Rothstein R. DNA strand annealing is promoted by the yeast Rad52 protein. Proc Natl Acad Sci (USA) 93:10729-10734 (1996).
Zou H and Rothstein R. Holliday junctions accumulate in DNA replication mutants via a RecA homolog-independent mechanism. Cell.11;90(1):87-96 (1997).
Zhao X, Muller EG, Rothstein R. A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools. Mol Cell. 2(3):329-40 (19998).
Smith J, Rothstein R. An allele of RFA1 suppresses RAD52-dependent double-strand break repair in Saccharomyces cerevisiae. Genetics. 151(2):447-58.20 (1999).
Erdeniz N, Rothstein R. Rsp5, a ubiquitin-protein ligase, is involved in degradation of the single-stranded-DNA binding protein rfa1 in Saccharomyces cerevisiae. Mol Cell Biol. 20(1):224-32.10 (2000).
Zhao X, Georgieva B, Chabes A, Domkin V, Ippel JH, Schleucher J, Wijmenga S, Thelander L, Rothstein R. Mutational and structural analyses of the ribonucleotide reductase inhibitor Sml1 define its Rnr1 interaction domain whose inactivation allows suppression of mec1 and rad53 lethality. Mol Cell Biol. 20(23):9076-83 (2000).
Lisby M, Rothstein R, Mortensen UH. Rad52 forms DNA repair and recombination centers during S phase. Proc Natl Acad Sci (U S A) 98(15):8276-82 (2001).
Zhao X, Chabes A, Domkin V, Thelander L, Rothstein R. The ribonucleotide reductase inhibitor Sml1 is a new target of the Mec1/Rad53 kinase cascade during growth and in response to DNA damage. EMBO J. 20(13):3544-53 (2001).
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