Control of BRCA2-mediated Homologous Recombination

Institution: University of California, Davis
Investigator(s): Damon Meyer, Ph.D. -
Award Cycle: 2009 (Cycle 15) Grant #: 15FB-0109 Award: $90,000
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2009)

About 10% of individuals with breast cancer are genetically predisposed, and of these, 10-30% have a mutation in the BRCA2 gene. Of the women that have a mutation in the BRCA2 gene about 80% of them will develop breast cancer during their lifetime. In addition, they tend to develop breast cancer at an earlier age. Therefore, studying the function of BRCA2 and how it works within the cell will be vital in determining how a mutation in BRCA2 could lead to breast cancer. One proposed function of BRCA2 has been in the repair of damaged DNA by interacting with the other proteins that repair DNA. Recently, BRCA2 was shown to interact with one of the major DNA repair proteins and serves to stimulate the repair of DNA using a pathway known as “homologous recombination”, so named because the undamaged homologous DNA strand is used as a template to repair the damaged DNA strand. The study of these interactions and how they relate to the repair of DNA is relatively unexplored.

In this project we will study the role of BRCA2 in mediating DNA repair and how that may give insight into the development of breast cancer in women that have a BRCA2 mutation. Our experiments will use a reconstituted biochemical system to simulate the repair of DNA containing a double-strand break. This system uses small segments of DNA that mimic a broken DNA strand and the homologous sequence used to repair the broken DNA strand. BRCA2 and other DNA repair factors will be added to the DNA segments and then after defined amounts of time the DNA segments will be analyzed to determine if DNA repair via homologous recombination has occurred. Controls will be used to determine if the results are due to the proteins tested. This project will test various regions of the BRCA2 protein to determine which portion is required for proper function. In addition, the testing of interacting proteins such as DSS1, RAD54, polymerase Delta and polymerase Zeta in homologous recombination reactions has never been done in a defined biochemical setting.

The study of factors that influence BRCA2-mediated recombination and subsequent extension of strand invasion intermediates will aid in the understanding of the genetic and molecular events leading to breast cancer.




Final Report (2011)

Breast cancer affects about 1 in 8 women during their lifetime making it the most common form of cancer in women with the second highest incidence of cancer deaths. About 10% of individuals with breast cancer are genetically predisposed and about 10-30% of these have a mutation in the BRCA2 gene. Of the women that have a mutation in the BRCA2 gene about 80% of them will develop breast cancer during their lifetime. In addition, they tend to develop breast cancer at an earlier age. Therefore, the study of BRCA2 function and how it works within the cell will be vital in determining how a mutation in BRCA2 could lead to breast cancer. BRCA2 has been shown to be involved in the repair of DNA by interacting with the other proteins that repair DNA. Specifically, BRCA2 interacts with one of the major DNA repair proteins, RAD51, and this stimulates the repair of DNA. The study of these interactions and how they relate to the repair of DNA is relatively unexplored.

My work focused on the role of BRCA2 in mediating double-strand break (DSB) repair and how a know interacting protein, DSS1, modulates the function of BRCA2. I have been successful in obtaining full length BRCA2 and DSS1, but have not been successful in demonstrating a role for these proteins in a biochemical reconstituted (i.e. in viro, cell-free conditions) DSB repair assay. Current work is underway to define the optimal conditions, which will allow BRCA2 and/or DSS1 to function in promoting DSB repair biochemically. In addition to the BRCA2 work I am currently purifying DNA polymerase lambda, which was recently shown to be involved in DSB repair and could be a potential DNA polymerase involved in promoting BRCA2 mediated DSB repair.

Understanding the normal function of BRCA2 in DSB repair will give insight into the development of breast cancer in women that have a BRCA2 mutation. Specifically, the continuation of these studies will shed light on the function of BRCA2, its interacting proteins and DNA polymerases in DNA repair and how they work together to promote genome stability. Understanding BRCA2 function and its interaction with DNA repair proteins will lay the foundation for better prevention, diagnosis and potentially treatment by identifying molecular drug targets.

Publications:
DNA polymerases delta and lambda cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae




Symposium Abstract (2010)

Breast cancer affects about 1 in 8 women during their lifetime and of these about 10% of the individuals with breast cancer is genetically predisposed and about 10-30% of these have a mutation in the BRCA2 gene. BRCA2 has been shown to be involved in DNA repair by associating with the eukaryotic RecA homolog RAD51, which promotes homologous recombination and thereby maintain genome stability. Recently, a truncated BRCA2 protein was shown to stimulate RAD51-mediated strand invasion in vitro. Therefore, the examination of proteins that influence BRCA2/RAD51-mediated strand invasion and subsequent events could give insight into the development of breast cancer. BRCA2 is known to interact with a small acidic protein DSS1, which is required for BRCA2 stability and thought to have a significant role in BRCA2-dependent recombinational repair but has not been explicitly tested. In addition, RAD51 interacts with the motor protein RAD54, which is involved in stimulating RAD51-mediated strand invasion but is unknown whether it can stimulate BRCA2/RAD51-mediated strand exchange. Following BRCA2/RAD51-mediated strand invasion is the extension of heteroduplex DNA by a DNA polymerase that facilitates the recombinational repair by stabilizing heteroduplex DNA. Genetic evidence supports the involvement of DNA polymerase lambda in the extension of heteroduplex DNA following strand invasion, but this has not been confirmed in biochemical reactions. Therefore, I propose to examine the role of both DSS1 and RAD54 in BRCA2/RAD51-mediated strand invasion and the extension of the strand invasion intermediate in a reconstituted recombination reaction by DNA polymerase lambda.

Specifically, the role of DSS1 and RAD54 in BRCA2/RAD51-mediated strand invasion will be tested in vitro using a linear D-loop assay that measures a labeled ssDNA oligo invading an unlabeled dsDNA oligo to generate a D-loop product. Furthermore, the subsequent extension of D-loops by DNA polymerase lambda will be test using a D-loop extension assay, which measures extension of a labeled ssDNA oligo following D-loop formation. Particular attention will be paid to the requirement of PCNA/RFC in D-loop extension. Controls in both assays will be used by changing the amount or presence of the protein and/or oligos. The study of factors that influence BRCA2/RAD51-mediated recombination and subsequent extension of strand invasion intermediates will aid in the understanding of the genetic and molecular events leading to breast cancer. Understanding the proteins that interact with and help facilitate the function of BRCA2 may prove to be attractive targets in preventing or treating breast cancer since 80% of individuals possessing a BRCA2 mutation will develop breast cancer in their lifetime.