The goal of this proposal is to gain an understanding of 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. We plan to measure the interactions that occur between the polymerase and these modified templates so that we might understand the molecular mechanism that results in, on the one hand, bypass of a specific lesion (whether error-free or error-prone) or, on the other hand, a blockage of synthesis. Two specific hypotheses will be tested in this current application. The first is that there is a relationship between the conformation of an adduct that is present in DNA and the structure that exists inside the polymerase active site. Second that the adduct induces specific structures to form within the polymerase active site that block the nucleotide binding site, resulting in the inability of a polymerase to undergo a conformational change to the catalytically active ternary complex.
Four specific aims are proposed. First, we plan on continuing our studies with site-specifically positioned aromatic amine and polycyclic aromatic hydrocarbon adducts situated in the active site of DNA polymerase I (KF). We will make use of the techniques developed in the prior project period to construct oligonucleotides containing N-acetyl-2-aminofluorene (AAF), 2-aminofluorene (AF) and (+)-trans and (+)-cis-benzo[a]pyrene adducts and extend our models to include 4-amimobiphenyl and PhIP, both of which have structural and mutagenic characteristics that resemble the AF adduct. Second, we will determine how amino acid substitutions within the polymerase active site contribute the properties that effect polymerase mechanism and fidelity. Third, we will use the methods developed in the prior project period to measure the interactions that occur between these carcinogenic adducts and the bypass polymerase, human Pol h (eta). Fourth, we will determine the crystal structures of the T7 DNA polymerase bound to AAF, AF, and B[a]P-modified templates. Taken together, these measurements should help to develop a molecular picture for how these various adducts are accommodated in a polymerase's active site and provide a better understanding of the molecular mechanism of mutagenesis and bypass synthesis that occurs during DNA replication.
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