The long-term goal of this continuing project is to use a combined theoretical-experimental collaborative approach to develop a molecular understanding of the principles of specific DNA damage and repair. In the proposed new project period, two mechanisms of thymine dimer (TD) repair will be studied. One mechanism is that of the enzyme endonuclease V (endoV) that specifically recognizes the TD and repairs it by excision. The other repair process is accomplished by an error-free translesion synthesis. EndoV specifically recognizes the TD and flips the complementary adenine to the 5'-thymine of the TD into a protein pocket. It appears that this mechanism of base flipping is universal to base excision repair. Thus the investigators will focus this project on gaining an understanding of the molecular, energetic and kinetic elements that differentiate base flipping in damaged DNA from that in undamaged DNA. The specificity of enzymes is determined not only by the recognition event, but also by the catalytic mechanism. The catalytic steps of endoV consist of a glycosylase step followed by a lyase step. The investigators propose to study the mechanism of enzymatic catalysis in endoV as a contribution to selectivity in DNA repair. Translesion synthesis is a newly discovered activity of several polymerases. Polymerase eta (poleta) is a particular example that successfully synthesizes the correct base sequence opposite the TD. Poleta has low processivity, suggesting a tolerant site for DNA and the incoming nucleoside triphosphate (NTP) binding. Low processivity is characteristic of several other polymerases (e.g., polymerase beta) engaged in DNA repair. The investigators propose that critical features of the mechanism of error-free translesion DNA synthesis by poleta are derived from its ability to recognize the special structural and dynamic properties of DNA containing a TD and that the site in the enzyme that preferentially binds deoxy-ATP over other nucleotides is in part formed by the interaction of the protein with the TD-containing template DNA. The investigators propose to identify the dATP binding site on the basis of homology with other low-processive polymerases, construct a model site by molecular modeling techniques and test the modeled predictions by spectroscopic and thermodynamic experiments. The investigators present these aims as a fully integrated collaborative approach in which they combine experimental and theoretical studies in a complementary way. They feel that the results of this integrated approach will lead to a better understanding of the factors that contribute to the specificity of repair enzymes and play an important role in developing a more general understanding of specific protein-DNA interactions.
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