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Imaging the Molecular Mechanisms of DNA Damage Repair
Homologous recombination (HR) is a DNA repair pathway that requires the coordinated action of a complex repertoire of proteins. The most common forms of familial breast cancer develop as a result of defects in Brca2, an essential component of the HR pathway. The exact role of Brca2 is unclear, but it is thought to direct Rad51 to the sites of DNA damage. Rad51 forms a filament on single-stranded DNA and catalyzes the DNA rearrangements that occur during HR. RPA and Rad52 are also essential components of the HR pathway and mediate the binding of Rad51 to damaged DNA. A growing body of evidence suggests defects in the HR repair pathway leading to genetic instability arise from improper assembly of the Rad51 nucleoprotein filament. To comprehend the steps leading to malignant transformation, it will be of paramount importance to understand how these different proteins interact with one another to ensure rapid and accurate repair of chromosomal damage. Our objective is to establish a novel experimental system for studying the assembly of the HR machinery by directly examining the dynamic behavior of the recombination proteins on single molecules of DNA. This work will be based on the overall hypothesis that interactions between Brca2, Rad51, RPA and Rad52 are all necessary to assemble the recombination machinery at the correct sites on damaged DNA. The following specific aims will help delineate the assembly mechanisms of the HR machinery: (1) Prepare fluorescently labeled RPA and Rad52 proteins, and make DNA substrates that mimic broken chromosome ends. (2) Directly observe the assembly of fluorescent RPA, Rad52, and Rad51 on DNA molecules that mimic natural DNA recombination intermediates. (3) Examine the influence of a precisely positioned Brca2 domain on the assembly mechanism. The primary experimental tool we use is a total internal reflection fluorescence microscope, which allows us to directly observe the behaviors of proteins on single DNA molecules in real time. We will use this system to dissect the assembly mechanisms of the recombination complexes responsible for the repair of broken DNA molecules. These experiments will help clarify how the HR machinery assembles onto broken DNA molecules and may reveal how errors in this process predispose some patients to inherited forms of cancer.
Genetic instability caused by chromosomal damage is often associated with the development of cancer. Chromosomal damage arises from exposure to a wide variety of environmental agents and as a normal consequence of cellular metabolism. To cope with this continual onslaught, our bodies have evolved specialized enzymes that detect and repair broken chromosomes before any damage can threaten genome integrity. Homologous recombination (HR) is an essential repair pathway cells use to repair broken strands of DNA, thus avoiding the dangers posed by the presence of broken chromosomes. This repair pathway requires several proteins including Brca2 and Rad51. Under normal circumstances, our cells repair DNA damage soon after it occurs without negative consequences. However, occasional errors are made during the repair process; these errors often lead to malignant transformation and cancer. For example, defects in Brca2 are associated with the most common inherited forms of breast cancer, and mutations in Rad51 have also been lined to the development of breast cancer. The importance of the HR proteins is clear, yet it is unknown how these different proteins interact with one another and with damaged DNA to prevent the development of cancer. Our research focuses on understanding the molecular nature of DNA repair mechanisms so that we can identify and fully understand the key steps involved in these critical cellular processes. To study the molecular mechanisms of DNA repair we use an ultra-sensitive microscope that can monitor the progress of a single DNA recombination reaction in vitro. This incredible sensitivity is achieved by taking advantage of a phenomenon called fluorescence, a process whereby a molecule illuminated with a particular color of light will in turn emit light of another color. We essentially turn proteins into miniature beacons and use the light emitted from these beacons to actually “see” how the proteins are behaving at the level of a single molecule. With this novel approach we will dissect the assembly pathways of the HR machinery by directly observing the behavior of the recombination proteins on individual DNA molecules. These state-of-the-art methods can be used to study biochemical processes in ways that are impossible with more standard biochemical techniques, and will lead to an entirely new way of studying the mechanisms of DNA repair with unmatched sensitivity.