Abstract
Breakage of chromosomal DNA caused by endonuclease enzymes such as EcoRI results in double-strand breaks (DSBs) within the chromosome. Eukaryotes have two main mechanisms for repair of DSBs. Nonhomologous end-joining (NHEJ) is a pathway that involves the direct rejoining of broken ends through the use of multiple enzymes. The alternative pathway is homologous recombination which involves an intricate, multi-step process in which DSBs are repaired virtually error-free using other homologous chromosomes within the same cell. Expression of EcoRI in non-bacterial cells has been performed, in vivo, to monitor the outcome of precise cohesive-ended DSBs and their repair. The initial focus of this study will be to create a new, highly regulatable expression system for EcoRI endonuclease in yeast (Saccharomyces cerevisiae) cells using the GAL1-V10 promoter that was created in this lab, which will be used to study pathways involved in repair of DSBs. The main focus of this study is to investigate a proposed third pathway of repair of cohesive-ended EcoRI-induced DSBs in vivo using the newly constructed EcoRI system and mutant cells that lack the proteins necessary for one or both known pathways. In addition, we will investigate the mechanism by which overexpression of several subunits of the telomerase DNA replication complex is able to enhance repair of EcoRI-induced DSBs.
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