Breast Neoplasms; DNA Damage; DNA Repair; Radiation Oncology; BRCA2 Protein; Genomic Instability
Therapeutic Radiology: Radiobiology
Research in our lab is focused on the DNA Double Strand Break (DSB) repair response in mammalian cells. DNA DSB’s are critical lesions for a cell and can lead to cell death if left unrepaired. Alternatively, if repaired incorrectly, a DSB can result in mutagenic consequences leading normal cells down a path to tumorigenesis. We are currently investigating the role BRCA2 (breast cancer susceptibility gene 2) plays in the DSB response as well as its role in homologous recombination (HR). People who inherit a deleterious mutation in the BRCA2 gene are at an incredibly high lifetime risk for breast, ovarian, and other types of cancer. We are particularly interested in the molecular pathogenic events that lead to such a high risk for cancer in the absence of functional BRCA2.
Speciailzed Terms: Cancer biology; DNA repair; BRCA2; DNA damage signaling; Genomic instability
Extensive Research Description
A role for BRCA2 in DNA repair was
originally established based on its interaction with RAD51, a key player in HR.
RAD51 plays a central role in
recombination, assembling onto single-stranded DNA as a nucleoprotein filament,
and catalyzing the invasion and exchange of homologous DNA sequences. A major hurdle in any biochemical study
is to purify the protein of interest to homogeneity in an active state. At 384 kDa, BRCA2 is a large protein that has
proven difficult to purify. Recently, we
have successfully developed a method to purify the full-length protein from
human cells. We initially characterized BRCA2’s
ability to bind DNA substrates, protein partners involved in homologous
recombination, and its capability to catalyze DNA strand exchange, an in
vitro measure of recombination. Our
findings revealed that BRCA2 binds RAD51 with a defined stoichiometry, prevents
nucleation onto dsDNA, and enforces binding of RAD51 onto ssDNA thus stabilizing
the nascent nucleoprotein filament.
A major focus of the lab is to understand the role BRCA2 plays in the DSB response as well as its role in homologous recombination (HR). Homologous recombination is a high fidelity form of DSB repair utilizing substrates such as the sister chromatid after DNA replication to repair a break with restoration of the original DNA sequence. Other pathways for DSB repair, such as non-homologous end joining (NHEJ), operate in the G1 phase of the cell cycle and can be deleterious if sequence information is deleted or altered at the break.
In order to better understand how BRCA2 and other players in HR signal and catalyze repair reactions, we are using a multi-disciplinary approach encompassing biochemistry, genetics, cell biology, structural biology, and proteomics. We are currently using human cells to both express and purify many of the proteins involved in HR to further understand detailed mechanisms of action. We can then look at enzymatic activities biochemically such as DNA binding preferences, direct protein interactions, and DNA strand exchange activity. Several proteins participate in the HR process and many of the players, such as the RAD51 paralogs, remain ill-defined as to what exact role they are playing. BRCA2 itself interacts with several proteins: PALB2, BRCA1, FANCD2, EMSY, DMC1, DSS1 and it is unclear how these interacting partners influence HR. Our goal is to understand the functional consequences of these interactions and how disruptions in the HR pathway lead to cancer.
- Biochemical and genetic characterization of tumor associated mutations in the BRCA2 gene.
- Genetic screens to further understand the molecular pathogenesis leading to tumor formation in BRCA2 carriers.
- Proteomic strategies to identify novel proteins that interact with BRCA2 after DNA damage.
- Identifying mechanisms and proteins that govern the regulatory decision between HR and NHEJ after a DSB is generated in mammalian cells.
BRCA2 is epistatic to the RAD51 paralogs in response to DNA damage.
Jensen RB, Ozes A, Kim T, Estep A, Kowalczykowski SC. BRCA2 is epistatic to the RAD51 paralogs in response to DNA damage. DNA Repair (Amst). 2013 Feb 2. doi:pii: S1568-7864(13)00002-5.
Purified human BRCA2 stimulates RAD51-mediated recombination.
Jensen RB, Carreira A, Kowalczykowski SC. Purified human BRCA2 stimulates RAD51-mediated recombination. Nature. 2010 Oct 7;467(7316):678-83.
Cell-interdependent cisplatin killing by Ku/DNA-dependent protein kinase signaling transduced through gap junctions.
Jensen R, Glazer PM. Cell-interdependent cisplatin killing by Ku/DNA-dependent protein kinase signaling transduced through gap junctions. Proc Natl Acad Sci U SA. 2004 Apr 20;101(16):6134-9. Epub 2004 Apr 6.
ATM-dependent expression of the insulin-like growth factor-I receptor in a pathway regulating radiation response.
Peretz S, Jensen R, Baserga R, Glazer PM. ATM-dependent expression of the insulin-like growth factor-I receptor in a pathway regulating radiation response. Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1676-81. Epub 2001 Feb 6.
Full List of PubMed Publications
- Zhao W, Steinfeld JB, Liang F, Chen X, Maranon DG, Jian Ma C, Kwon Y, Rao T, Wang W, Sheng C, Song X, Deng Y, Jimenez-Sainz J, Lu L, Jensen RB, Xiong Y, Kupfer GM, Wiese C, Greene EC, Sung P: BRCA1-BARD1 promotes RAD51-mediated homologous DNA pairing. Nature. 2017 Oct 19; 2017 Oct 4. PMID: 28976962
- Daley JM, Jimenez-Sainz J, Wang W, Miller AS, Xue X, Nguyen KA, Jensen RB, Sung P: Enhancement of BLM-DNA2-Mediated Long-Range DNA End Resection by CtIP. Cell Rep. 2017 Oct 10. PMID: 29020620
- Turchick A, Hegan DC, Jensen RB, Glazer PM: A cell-penetrating antibody inhibits human RAD51 via direct binding. Nucleic Acids Res. 2017 Sep 28; 2017 Sep 28. PMID: 29036688
- Marsden CG, Jensen RB, Zagelbaum J, Rothenberg E, Morrical SW, Wallace SS, Sweasy JB: The Tumor-Associated Variant RAD51 G151D Induces a Hyper-Recombination Phenotype. PLoS Genet. 2016 Aug; 2016 Aug 11. PMID: 27513445
- Chatterjee G, Jimenez-Sainz J, Presti T, Nguyen T, Jensen RB: Distinct binding of BRCA2 BRC repeats to RAD51 generates differential DNA damage sensitivity. Nucleic Acids Res. 2016 Jun 20; 2016 Apr 15. PMID: 27084934
- Zhao W, Vaithiyalingam S, San Filippo J, Maranon DG, Jimenez-Sainz J, Fontenay GV, Kwon Y, Leung SG, Lu L, Jensen RB, Chazin WJ, Wiese C, Sung P: Promotion of BRCA2-Dependent Homologous Recombination by DSS1 via RPA Targeting and DNA Mimicry. Mol Cell. 2015 Jul 16; 2015 Jul 2. PMID: 26145171
- Jensen RB: BRCA2: one small step for DNA repair, one giant protein purified. Yale J Biol Med. 2013 Dec 13; 2013 Dec 13. PMID: 24348212