Research Departments & Organizations
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
- 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.