The aim of this proposal is to develop novel tools for studying the relationship between stalled or broken replication forks and sister chromatid recombination in mammalian cells. Although current models propose that recombination at sites of stalled DNA synthesis is critical for preventing genomic instability and cancer, the ability to model this process in mammalian cells is severely limited, due to the absence of recombination reporters that can accurately model this process in a quantitative and mechanistic fashion. Work proposed herein will provide such tools, by combining """"""""state-of-the-art"""""""" mammalian reporters of homologous recombination and sister chromatid recombination with novel strategies to cause site-specific replication fork stalling. This work will provide a much needed new tool to study this area of critical importance for human cancer predisposition.
Our specific aims are: 1. Adapt a known replication fork barrier for the induction of site-specific HR in mammalian cells. 2. Study HR and SCR at a replication fork barrier in mammalian cells. 3. Develop """"""""nickase"""""""" mutants of I-SceI to stimulate HR and SCR at a chromosomal I-SceI site in mammalian cells.
The accurate repair of chromosome breaks by a process termed """"""""sister chromatid recombination"""""""" (SCR) is critical for preventing premature aging and cancer. SCR is triggered when the DNA replication machine stalls on damaged DNA;however, no tools are available to study the relationship between replication arrest and SCR in mammalian cells. This proposal will address this need, by developing two new systems for provoking replication arrest at a specific chromosome site in living mammalian cells, and by measuring the stimulation of SCR in response to these events.
| Hwang, Yung; Futran, Melinda; Hidalgo, Daniel et al. (2017) Global increase in replication fork speed during a p57KIP2-regulated erythroid cell fate switch. Sci Adv 3:e1700298 |
| Willis, Nicholas A; Chandramouly, Gurushankar; Huang, Bin et al. (2014) BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks. Nature 510:556-9 |
| Chandramouly, Gurushankar; Willis, Nicholas A; Scully, Ralph (2011) A protective role for BRCA2 at stalled replication forks. Breast Cancer Res 13:314 |
| Pathania, Shailja; Nguyen, Jenna; Hill, Sarah J et al. (2011) BRCA1 is required for postreplication repair after UV-induced DNA damage. Mol Cell 44:235-51 |
| Hartlerode, Andrea J; Scully, Ralph (2009) Mechanisms of double-strand break repair in somatic mammalian cells. Biochem J 423:157-68 |
