Core 2: Chromosome Replication and Analysis (CRA) Core

ABSTRACT Failure to properly repair DNA double-strand breaks (DSBs) or damaged DNA replication forks leads to chromosome aberrations and neoplastic transformation of cells. Homology-directed repair (HDR) mediated by the recombinase RAD51 in conjunction with the tumor suppressors BRCA1-BARD1 and BRCA2 is a high fidelity mechanism of DSB and replication fork repair. In addition, these HDR factors also protect stressed DNA replication forks from nucleolytic attrition. The commitment step for HDR is 5’ strand resection of a DNA end to create a 3’-tailed ssDNA region for the assembly of HDR complexes that contain RAD51 as a central component. The three Research Projects of this Program will investigate the biochemical regulation of the DNA end resection and RAD51-ssDNA assembly steps of HDR and how these processes are negatively regulated by protein factors, such as DNYLL1 and the CTC1-STN1-TEN1 (CST) complex, of the 53BP1 regulatory axis. The Chromosomal and Replication Analysis (CRA) Core provides valuable shared resourses to help translate biochemical data into outcomes of DSB repair, replication fork repair and restart, chromosomal stability, mitotic success, and cellular survival. CRA will create specific mutations in HDR and 53BP1 axis proteins using CRISPR/Cas9 and generate cell models using RNA interference when gene mutations are incompatible with viability. The CRA will subject these modified cell lines to DNA fiber analysis, end resection analysis, cell cycle phase analysis, confocal immunofluorescent microscopy of foci, isolation of proteins on nascent DNA (iPOND), replication fork fusion/degradation analysis, cytogenetic and mitotic chromosomal structural analysis and correlate these with cell survival in replication stress. Our in-depth cellular analyses will permit the three Research Projects to construct epistatic pathways and, importantly, will help identify new targets for synthetic lethality in inherited disorders of HDR that lead to breast, ovarian, prostate and other cancers. Such targets are of great clinical relevance as the vast majority of these cancer patients eventually develop resistance to currently available synthetic lethal therapy, such as the PARP1 inhibitors. As such, the CRA will bind the Research Projects together by integrating biochemical data with replication fork, chromosomal, and cellular outcomes. 1