At the DNA level, the biological consequences of individual DNA lesions are the product of the efficiency of repair of the lesion and the interaction of any unrepaired damages with the DNA replication apparatus. It is this latter interaction, by the process of translesion synthesis, that determines whether a DNA lesion will initiate the mutagenic/carcinogenic process. Thus, understanding the fundamental mechanisms underpinning translesion synthesis is critical to our understanding of the initial events in mutagenesis/carcinogenesis. The proposed studies address the hypotheses: (1) Surrounding DNA sequence positions a particular lesion in the active site of a DNA polymerase such that DNA lesion/amino acid contacts either facilitate or impede translesion synthesis and influence insertion of a particular base opposite the lesion ultimately affecting mutagenic outcome. (2) The observed DNA sequence context effects on translesion synthesis and thus mutational outcome are reflected in the sequences of genes and genomes. The first hypothesis will be tested by solving the crystal structures of a Pol alpha-type DNA polymerase complexed with DNA containing sites of base loss, an 8-oxoguanine lesion, or a 5-hydroxycytosine lesion together with the incoming deoxynucleoside triphosphate. The lesions will be embedded in sequence contexts shown to facilitate or impede translesion synthesis in the case of sites of base loss, or in sequence contexts that promote misinsertion or correct pairing in the cases of 8-oxoguanine and 5-hydroxycytosine. The second hypothesis will be tested using bioinformatics to determine sequence context-dependent rates for bacterial evolutionary base substitutions. Taken together, the proposed studies should delineate at the atomic level the roles that lesion structure, surrounding sequence, and the DNA polymerase active site play in the process of translesion synthesis and define the consequences of these events on an evolutionary scale.
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