In eukaryotes, recombination occurs in both meiosis and mitosis. Meiotic recombination ensures accurate chromosome disjunction, whereas mitotic recombination is important in the repair of DNA lesions, particularly double-stranded DNA breaks (DSBs). The importance of the homologous recombination (HR) pathway in the repair of DNA damage is indicated by the evolutionary conservation of many of the HR proteins from yeast to humans, and by the deleterious consequences of mutations in the pathway;for example, individuals lacking the HR protein BRCA1 are at high risk for developing breast cancer. Paradoxically, although HR repairs the genome, it can also have genome-destabilizing effects. In cells heterozygous for a tumor suppressor gene, mitotic recombination can lead to loss of heterozygosity (LOH), resulting in a cancer-prone homozygous cell. The long-term goal of the proposed research is to understand the mechanism of mitotic recombination, both spontaneous events and events induced by ultraviolet light (UV) and ? rays. Among the questions that will be examined are: 1) what types of DNA damage initiate spontaneous HR events?;2) are certain chromosome regions preferred sites for spontaneous recombination events?;3) are the HR events stimulated by UV light or ? rays similar to spontaneous HR events?;4) what are the consequences of DNA damage induced at different points in the cell cycle? All of these issues will be addressed in the yeast Saccharomyces cerevisiae using a novel method for selecting crossovers, and microarrays capable of high-resolution (<1 kb) mapping of LOH events.
Specific Aim I is to examine spontaneous mitotic crossovers and associated non-reciprocal recombination events (gene conversions), focusing on a 1 Mb region of chromosome IV that represents about 10% of the yeast genome. Hotspots for recombination previously identified in this interval will be analyzed in detail to determine their mechanism of action. The mechanism of mitotic gene conversion will be examined by analyzing yeast strains with mutations that eliminate DNA mismatch repair and the processing of broken DNA ends. Spontaneous crossovers will also be compared to recombination events induced by the site-specific HO endonuclease.
Specific Aim II is to characterize recombination events stimulated by UV or ? rays. Genome- wide mapping of LOH events will be performed in cells irradiated in G1 or G2 of the cell cycle. These studies will clarify whether UV introduces recombinogenic DSBs. In addition, these experiments will determine the fraction of ? ray induced-DSBs that produce LOH events.
Specific Aim III is to investigate a novel pattern of chromosome segregation in which one pair of sister chromatids segregates to one daughter cell, and the other pair segregates into the other daughter cell, mimicking a meiosis I segregation. This pattern of segregation produces two cells with uniparental disomy, a phenomenon associated with tumor cells.
The exchange of DNA sequences between pairs of chromosomes (recombination) can occur in either mitotic cells or meiotic cells. If an individual is heterozygous for a recessive mutation in a tumor suppressor gene, mitotic recombination can lead to the formation of a cell that is likely to develop into a cancer. Spontaneous mitotic recombination and recombination events induced by DNA damage (ultraviolet and y- radiation) will be analyzed throughout the genome in the budding yeast Saccharomyces cerevisiae.
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