Gaining an understanding of how a DNA polymerase interacts with the adducts formed by chemical carcinogens is an important goal since these interactions are the basis for many of the adduct-induced effects. The proposed research builds on our prior work that sought to understand the molecular interactions that contribute to the ability of a DNA polymerase to carry out synthesis on a template modified with a bulky carcinogenic adduct. In the current application, we propose to test the central hypothesis that the positioning of an adduct in the polymerase active site is dependent on the adduct structure and DNA sequence context and that specific structures promote specific mechanistic consequences. To accomplish this goal, we have designed a research plan that takes advantage of several novel experimental methods we have developed to measure these interactions between DNA polymerases and several well-defined DNA adducts. First, we will use the intrinsic fluorescent properties of the adducts we have studied in the past to allow us to use them as FRET donors to measure the position of these adducts in the polymerase active site. Second, we will use a single-molecule approach to measure the binding and dynamics of a polymerase during DNA synthesis with single base pair resolution on templates containing bulky DNA adducts in real time. This technique will be applied to both high-fidelity and bypass polymerases and adducts having very different structures in DNA. Third, we will continue to determine the effect of adduct structure and sequence context on polymerase-DNA binding using surface plasmon resonance, a technique that is more sensitive and accurate than the gel-based methods used in the past. Finally, we will continue the crystallographic studies of DNA polymerases bound to templates modified with carcinogenic DNA adducts. Taken together, these measurements should help to develop a molecular picture for how these various adducts are accommodated in the DNA polymerase active site and provide a better understanding of the molecular mechanism of mutagenesis and bypass synthesis that occurs during DNA replication.
In the broadest terms, the relevance of this work rests on the importance of the carcinogenic agents we propose to study in the initiation of cancer in eukaryotic cells. Gaining an understanding of the mutagenic response to the presence of these structures on the cell's DNA is of undeniable importance. More specifically with regards to the need to understand how these carcinogens cause mutations, it is important to study the response of a DNA polymerase to the presence of these adducts, interactions that are known to be dependent on a multitude of factors-most important among these being the adduct structure, the sequence context within which the adduct lies, and the particular structure of the DNA polymerase that must interact with these structures.
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