Carcinogenesis involves multiple genetic changes that result from errors in repair of DNA damage. Several highly error prone mechanisms for repair of DNA breaks exist which can serve as the source of mutations and drive formation and/or maintenance of a variety of cancers. It is thus of significant public health interest to define how and when cells rely on error prone mechanisms to repair DNA breaks and assess cellular and pathological consequences of genetic alterations associated with these processes. The goal of this proposal is to elucidate the molecular mechanisms of one of these error prone repair mechanisms of DNA breaks in eukaryotes and the newly identified recombination proteins crucial for such recombination reaction. Recombination between tandem repeat sequences is an evolutionary conserved error prone mechanism that repairs DNA breaks by producing sequence deletions. To define genetic component of this recombination process, we screened genes needed for efficient repair of breaks flanking direct repeats with an approach that combines yeast genetics and microarray technology. This screen uncovered two new recombination genes, SLX4 and SAW1, that function in removal of 3'flaps from recombination intermediates. Removal of a 3'flap also depends on the structure specific endonuclease complex, Rad1/Rad10, and dictates cellular tolerance to cancer chemotherapeutic agents, gene targeting, telomere integrity, repair of oxidative damage and suppression of aging. To decipher biochemical and molecular basis of error prone recombination, we plan to define the biochemical properties of Saw1 in recombination and recombination related biological processes including repair of DNA lesions blocking ongoing replication fork progression. We will reconstitute the 3'flap removal process during recombination using purified recombination proteins and a 3'flap DNA. The information will help devise clinical strategies to reduce or eliminate mutagenic repair germane to carcinogenesis and pave the way for improved cancer therapeutics.
The purpose of this application is to decipher molecular inner-workings of error prone mechanism for repairing DNA breaks that results in genetic changes pertinent to carcinogenesis. The information gleaned from the proposal will also provide a logical framework as to how cells tolerate cancer therapeutic agent-induced DNA lesions and shed lights on the prevention and improved therapeutic intervention of cancers.
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