BRCA1 and BRCA2 have established functions in homologous recombination (HR), particularly in the repair of replication-associated DNA breaks by sister chromatid recombination (SCR). Many rare BRCA1 missense mutant alleles, termed variants of uncertain significance (VUS), are difficult to classify as pathogenic or benign, due o their scarcity in the human population. Therefore, for a woman who carries a germ line BRCA1 VUS allele, the risk of developing breast or ovarian cancer is unknown. A major goal of this proposal is to study a large panel of ~150 BRCA1 variants, including ~60 BRCA1 VUS alleles in HR/SCR, using highly innovative SCR reporters that are unique to the Scully lab. Our work will reveal fundamental mechanisms of action of BRCA1 in HR. Further, it will advance human health by helping to predict cancer risk for individuals who carry a BRCA1 VUS allele in the germ line and, hence, make informed decisions regarding prophylactic measures.
AIM 1 : Perform a genetic analysis of a large panel of BRCA1 variants in homologous recombination and LTGC suppression. We developed novel flow cytometric reporters of SCR, triggered by a site-specific chromosomal double strand break (DSB). We discovered that, in addition to its known function in overall HR, BRCA1 also specifically suppresses aberrant long tract gene conversion (LTGC) between sister chromatids. Each of these defects is corrected by wild type human (h)BRCA1 but not by known pathogenic missense hBRCA1 alleles. In this Aim we will extend our analysis to include a large number of BRCA1 variants, including VUS alleles, with the goal of developing a robust predictor of the pathogenicity of specific BRCA1 VUS alleles.
This Aim will include analysis of BRCA1 SCR functions in normal development and during breast tumorigenesis in a mouse model.
AIM 2 : Define mechanisms by which BRCA1 controls hr at Tus/ter-arrested replication forks. A long-standing hypothesis proposes that BRCA1 controls HR at stalled replication forks. This has been difficult to test, due to the dearth of tractable tools for stalling the mammalian replication fork at a defined chromosomal locus. We recently solved this problem by adapting the Escherichia coli Tus/ter replication fork arrest complex for use in mammalian cells. We discovered that BRCA1 controls HR at Tus/ter-stalled replication forks, but that HR in this context is regulated differently from HR induced by a generic restriction endonuclease-induced chromosomal DSB. Remarkably, loss of wtBRCA1 causes an increase in the absolute frequency of LTGC events at Tus/ter-stalled replication forks. In this proposal, we will analyze how BRCA1 controls HR at stalled forks and will attempt to identify the specific mechanisms by which BRCA1 suppresses LTGC at Tus/ter-stalled mammalian chromosomal replication forks.

Public Health Relevance

If a woman inherits an error ('mutation') in the BRCA1 gene, she will have a ~10-fold increased risk of breast and ovarian cancer throughout her life. However, some BRCA1 mutations occur too rarely in the population to allow the associated risk of breast or ovarian cancer to be determined. Our goal here is to use new tools that the Scully lab has developed for studying the function of BRCA1 to help identify which of the rare BRCA1 variants are benign and which confer increased risk of cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA095175-13
Application #
9242585
Study Section
Cancer Genetics Study Section (CG)
Program Officer
Pelroy, Richard
Project Start
2002-05-01
Project End
2020-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
13
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02215
Willis, Nicholas A; Rass, Emilie; Scully, Ralph (2015) Deciphering the Code of the Cancer Genome: Mechanisms of Chromosome Rearrangement. Trends Cancer 1:217-230
Elia, Andrew E H; Wang, David C; Willis, Nicholas A et al. (2015) RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks. Mol Cell 60:280-93
Liu, Pengda; Gan, Wenjian; Guo, Chunguang et al. (2015) Akt-mediated phosphorylation of XLF impairs non-homologous end-joining DNA repair. Mol Cell 57:648-61
Liu, Xue-Song; Chandramouly, Gurushankar; Rass, Emilie et al. (2015) LRF maintains genome integrity by regulating the non-homologous end joining pathway of DNA repair. Nat Commun 6:8325
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
Liu, Yan; Marks, Kevin; Cowley, Glenn S et al. (2013) Metabolic and functional genomic studies identify deoxythymidylate kinase as a target in LKB1-mutant lung cancer. Cancer Discov 3:870-9
Somyajit, Kumar; Basavaraju, Shivakumar; Scully, Ralph et al. (2013) ATM- and ATR-mediated phosphorylation of XRCC3 regulates DNA double-strand break-induced checkpoint activation and repair. Mol Cell Biol 33:1830-44
Chandramouly, Gurushankar; Kwok, Amy; Huang, Bin et al. (2013) BRCA1 and CtIP suppress long-tract gene conversion between sister chromatids. Nat Commun 4:2404
Scully, Ralph; Xie, Anyong (2013) Double strand break repair functions of histone H2AX. Mutat Res 750:5-14
Hartlerode, Andrea J; Guan, Yinghua; Rajendran, Anbazhagan et al. (2012) Impact of histone H4 lysine 20 methylation on 53BP1 responses to chromosomal double strand breaks. PLoS One 7:e49211

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