Molecular Mechanisms Underlying Recombination at DNA Double-Strand Breaks and Stalled Replication Forks

Project Summary Genetic recombination is an essential biological process that is central to the repair of DNA damage that occurs during replication and other assaults on the genome. Unrepaired double-strand breaks (DSBs) are one of the most lethal kinds of DNA damage and their aberrant repair often leads to genome instability, one of the hallmarks of cancer. The damage can occur from endogenous sources, such as oxidation, or from external sources, such as gamma rays. Genetic recombination is also the mechanism for gene replacement and the repair of CRISPR-Cas-induced DSBs, processes that are commonly used to study all diseases in humans as well as human disease models in simpler systems. In this proposal, the mechanisms by which DSBs are repaired will be explored in vivo in yeast cells using a combination of cell biology, genetics and molecular biology. A system to study the repair of the two ends of a DSB has been designed to allow, for the first time, a view into the fate of these ends during the recombination process in diploid cells. This system will be used to assess chromosome end movement during the repair process in both wild type and mutant genetic backgrounds. The DSBs will be introduced at the same site using three different endonucleases that create 3’ overhangs, 5’ overhangs or blunt ends to determine how the repair system processes these different lesions. These same ends will be examined for repair using molecular biological techniques designed to measure (1) end resection, an important step in preparing the DSB end for recombination and (2) the repair synthesis stimulated at the end, which is a necessary step on the path to repair of the break. Another system will be designed to permit in vivo visualization of the first appearance of a successfully recombined chromosome using messenger RNA as surrogate. This system will be combined with various mutations in recombination genes to define when the timing of those gene products are important for the repair of a site-specific DSB. Broken DNA ends also arise during DNA replication, especially when the DNA polymerase is confronted with a nick. A system will be designed to induce a site-specific nick and will be used in conjunction with marked chromosome ends to define the fate of a DSB that is generated after replication of a nick. Finally, the function of the Rad5 protein, a homolog of the human HLTF that is often overexpressed in cancer cells, will be studied to determine its role in template switch recombination. It is known that both HLTF and Rad5 are necessary for recombination during template switch repair, but it is not known whether they are sufficient. By approaching the study of recombination in a genetically tractable system that is easy to manipulate, insights into the conserved mechanisms of recombination will be achieved.