Research Grants Awarded
Elucidating the Mechanism of Human DNA Repair: Direct Observations of the Interactions of Human Rad51 and BRCA2 Proteins with DNA.
Homologous recombination is a process whereby DNA damage in the form of double-strand breaks (DSBs) are repaired in an efficient and error-free manor. The human DNA recombination and repair protein, Rad51 (HsRad51), plays a central role in this process through its intrinsic activities of assembly onto DNA and homology searching and pairing between identical chromosomes. It is thought that the product of the BRCA2 tumor suppressor gene directs HsRad51 to the site of DSBs. This proposal contains two main objectives. The first is to directly visualize the process of protein filament formation of HsRad51 on single- and double-strand DNA substrates at the single-molecule level. With this study, the mechanism of HsRad51 polymerization on DNA will be determined. Data obtained from single-molecule experiments will provide an understanding of the nucleation process of HsRad51 on DNA, directionality of filament assembly on both single- and double-strand DNA substrates, and the role of ATP binding and hydrolysis in nucleoprotein filament formation and stability. Kinetic parameters for the rates of nucleation and assembly/disassembly will be obtained providing information on the time scales of the biochemical reactions and allowing us to determine what factors accelerate or disrupt the binding of HsRad51 to DNA. The second objective is to examine the role of the breast cancer associated protein, BRCA2, in directing HsRad51 filament formation on HsRPA-coated ssDNA. At the site of a DSB, the DNA is degraded to produce regions containing 3?-ssDNA, which is immediately protected by the HsRPA protein. The mechanism by which HsRad51 replaces RPA bound to ssDNA to form a complete nucleoprotein filament substrate viable for homologous recombination is not understood. However, emerging biochemical and structural data implicate the regulatory control of HsRad51 by BRCA2 at the site of double strand breaks. Direct observations of biochemical reactions involving HsRad51, BRCA2 or BRCA2 domains, and HsRPA, with single-strand DNA with lengths (~30-50 kb) suitable for visualization, will be obtained with single-molecule fluorescence microscopy utilizing an optical laser trap system. Our system provides control of reaction components and the ability to visualize their products, namely protein macromolecular assemblies on DNA. These biochemical reactions monitored by single-molecule microscopy and in conjunction with bulk phase methods will allow us to understand the events which occur during the initial stages of the repair of damaged DNA by homologous recombination.
The DNA of human cells (the genome) can be damaged by either external (ionizing radiation, chemical toxins) or internal (mechanical stress, DNA degrading enzymes, during DNA replication) agents. A specific type of damage, called a double-strand break (DSB), occurs when a DNA molecule (chromosome) is severed exactly in the same place on both strands. If DSBs are not repaired they can lead to chromosomal rearrangements, cell cycle arrest, and cell death. Such uncontrollable genomic instability can then result in carcinogenesis. The homologous recombinational repair pathway repairs DSBs without loss of genetic information. The human protein, Rad51, performs prominent functions in this pathway. Human Rad51 (HsRad51) protein must first, completely coat the regions of single-strand DNA (ssDNA) produced as a result of initial processing of the DSB. In doing so, Rad51 must compete with and ultimately displace the protective ssDNA binding protein, RPA. How this is accomplished is not yet understood, but it is proposed that accessory proteins such as the breast cancer associated protein, BRCA2, assist this process by recruiting Rad51 to the sites of DSBs. Genetic evidence has clearly shown that both HsRad51 and BRCA2 are essential for the repair of DSBs. RAD51 gene knockouts in mice produce inviable embryos indicating an inability to perform double strand break repair. Mutations in the BRCA2 gene have been associated with a high risk of breast (~85%) and ovarian (~15%) cancers in women, and familial, population-specific male and female breast cancers. Other specific BRCA2 mutations are linked to Fanconi anemia, a rare inherited disease linked to hematological disorders and susceptibility to acute myeloid leukemia as well as a characteristic syndrome associated with breast and brain tumors. Genetic, biochemical, and structural studies have validated the important biological functions of the HsRad51 and BRCA2 proteins, and have shown direct interactions between them. Nevertheless, direct observation, in real-time, of the individual reactions involving Rad51 and BRCA2, in cell-free systems, has not been realized. In this study, we aim to reconstitute the reactions of the homologous recombination pathway involving the HsRad51 and BRCA2 proteins and observe these events by a new method, single-molecule fluorescence microscopy. Collectively, the results obtained under this proposal will clearly detail, through direct visualization, the critical steps leading to the repair of DNA damage and the prevention of cancer. These results will provide a detailed description of the recombinational repair process and provide a cornerstone for this laboratory toward a total reconstruction of homologous recombination at the single molecule level. Furthermore, knowledge of the roles of HsRad51 and BRCA2 in DNA repair and their effects on cell survival, tumor prevention, and tumor development may also lead to improved diagnostic markers and therapies for cancer treatment.