| At this time in history the mysteries and beauties of the cell are unfolding at an exponentially increasing rate. The study of homologous recombination, which is the exchange of DNA strands within the cell, is necessary to understand the repair of certain types of DNA damage. This study has shed light on various protein machines that mediate this process within the cell. Different organisms maintain different protein machinery to mediate strand exchange; however, the overall mechanism of the exchange is the same. Recent interest in DNA damage repair has flourished since the Bloom's syndrome gene was identified to code for a DNA repair protein.; The mechanism of homologous pairing is the least understood step in homologous recombination. A spectroscopic assay is proposed that will allow the kinetic analysis of the details of homologous pairing. This assay will give us a clearer picture of the homologous recombination pathway. Helicase proteins, which unwind the double helix, are responsible for the catalysis of extensive DNA strand exchange in the homologous recombination pathway. A spectroscopic assay of protein mediated DNA unwinding was invented because the mechanism that helicase proteins employ to unwind DNA with the energy derived from ATP hydrolysis is unclear. The assay will provide a facile kinetic analysis of the helicase unwinding mechanism. The assay will also allow simple screening of inhibitors of helicases which are currently of interest as anticancer drug leads.; The eukaryotic recombination system can be modeled by the yeast, which is an easily manipulatible single-celled organism, and has many parallels in the prokaryotic system. Recently, in vitro DNA strand exchange reactions have been reconstituted with the purified yeast proteins Rad51, RPA, and Rad54. Many of the activities of these strand exchange proteins are the same with the yeast and with the human homologues suggesting a functional conservation in both systems. The bacterial and phage systems of recombination have been studied extensively over the past 20 years. However, there are no functional or amino acid sequence homologues for the Rad54 protein, which is required in vitro for efficient strand exchange.; In this study, the purification and mechanistic studies of the Rad54 protein are addressed. The Rad54 protein was partially purified by genetically attaching affinity tags to the protein and using metal chelate and antibody chromatography to purify the protein. In a collaboration with Patrick Sung who had subsequently purified large quantities of the protein, mechanistic studies of Rad54's biochemistry were performed. |
