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    Research Grants Awarded

    Single Molecule Studies of Homologous Recombination in Human Cells

    Study Section:
    Tumor Cell Biology V

    Scientific Abstract:
    The efficient repair of DNA double-strand breaks (DSB) is critical to cell survival. The repair of DSBs may represent the most dangerous form of DNA damage which if left unrepaired, are lethal events. Consequently, the inability to repair DNA damage correctly leads to various disorders and enhanced rates of tumour development in humans. In mammalian cells, damaged DNA can be repaired by two distinct pathways: non-homologous end joining or homologous recombination (HR). As HR uses an intact sister duplex as the template for repair, the reaction occurs with high fidelity. It is becomingly increasingly clear that due to the risks associated with inaccurate DSB repair, a DSB is met with accordingly large multi-protein complex es, whose temporal assembly is highly coordinated and corresponding activity tightly regulated. Disruption of the temporal assembly and activity of these complexes is potentially lethal. To understand how these large multi-protein and multi-faceted complexes put it all together, it is necessary to see them “live” in action. To this end, I use state-of-the-art single molecule techniques to probe these complexes in action, keeping in mind that a picture (or in my case, a movie) is worth a thousand words. Studies are currently aimed at the early events and protein complexes which assemble at and act on DSBs. These include the Mre11-Rad50-Nbs1 complex, the loading of Rad51 onto tailed duplexes by BRCA2 and the subsequent action of the Rad51-Rad54 nucleoprotein filament in the search for homology and DNA strand exchange. These studies are important from several perspectives. First, direct observation of HR in real time has the potential to provide significant, novel insights into the biochemical mechanism of this process. Second, the results will provide basic biochemical information on several nucleic acid enzymes that are essential to DNA metabolism, with particular emphasis on the biochemical mechanism of Rad51 loading at DSBs by the breast cancer susceptibility gene product, the BRCA2 protein. Finally, the collective results will provide detailed mechanistic insight into the molecular events responsible for aberrant DNA metabolic processes in mammalian cells.

    Lay Abstract:
    The efficient repair of DNA double-strand breaks (DSB) is critical to the maintenance of genome stability and cell survival. DSBs may represent the most dangerous form of DNA damage as they are the most difficult to correct. Consequently, the inability to repair them correctly leads to various disorders and enhanced rates of tumor development in humans. Mutations in BRCA 1 and BRCA 2 frequently leads to disastrous effects in cells and consequently to the cancerous state in many women. These cancer-susceptibility genes (i.e., BRCA 1 and BRCA 2) are required for normal levels of recombinational repair demonstrating a clear connection between efficient repair, maintenance of genome stability and the potential for tumor development. The main focus of the laboratory is to understand at the level of single DNA molecule, the biochemical events responsible for the timely and accurate repair of DSBs. As these events are complex and frequently require the interaction of large multi-protein complexes, they are studied in small stages and then assembled sequentially to gain a greater understanding of the whole. In order to understand how these large multi-protein put it all together, it is necessary to see them “live” in action. To this end, state-of-the-art single molecule techniques are used to probe these complexes in action, keeping in mind that a picture (or in this case, a movie) is worth a thousand words. Our work focuses on the early events following the formation of a lethal double stranded DNA break. Typically, the Rad50-Mrell-Nbs1 complex is loaded at the break so that the DNA can be processed to reveal single stranded DNA tails. The BRCA2 protein is then responsible for directing the loading of the Rad51 protein onto these tails and the action of Rad51 is to mediate the repair of the broken DNA by invading a neighboring intact DNA molecule. For Rad51 to function efficiently, it requires the assistance of accessory proteins, one of which is the Rad54 protein. Rad54 is thought to assist in the homology search where is clears the DNA of other proteins that could potentially inhibit the action of Rad51. The ability to directly visualize the action of these critical proteins in real time has the capacity to provide unparalleled insight into the process of DSB repair and consequently, will lead to a more detailed and mechanistic understanding of the molecular events responsible for aberrant DNA metabolic processes in mammalian cells.