Date of Degree
PhD (Doctor of Philosophy)
Molecular and Cellular Biology
M. Todd Washington
DNA damage blocks replication by classical DNA polymerases, those that replicate nondamaged DNA during normal DNA replication and repair, by altering the geometry of the DNA. Consequently, translesion synthesis, the replication of damaged DNA, is catalyzed by non-classical DNA polymerases, which are capable of accommodating the inherent distorted geometry of damaged DNA.
The yeast Rev1 protein (Rev1p) specifically catalyzes the incorporation of cytosine opposite template guanine and several types of DNA damage utilizing a unique mechanism of nucleotide selection whereby the sidechain of Arg-324 acts as the template by forming hydrogen bonds with the incoming cytosine. To better understand the impact of this protein-template-directed mechanism on nucleotide incorporation, I carried out pre-steady-state kinetic studies with Rev1p. Interestingly, I found that Rev1p's specificity for incorporating cytosine is achieved solely at the initial nucleotide-binding step. In this respect, Rev1p differs from all previously investigated DNA polymerases. Based on these findings and on structures of another enzyme, MutM, I suggest possible structures for complexes of Rev1p with the other incoming nucleotides.
DNA polymerase ζ, encoded by the REV3 gene, functions in the error-prone replication of a wide range of DNA lesions by extending from nucleotides incorporated opposite template lesions by other polymerases. Here I describe genetic and biochemical studies of five yeast DNA polymerase ζ mutant proteins. Four mutant proteins do not complement the rev3Δ mutation, and these proteins have significantly reduced or no polymerase activity relative to the wild-type protein. However, the K1061A protein partially complements the rev3Δ mutation and has nearly normal polymerase activity. Interestingly, the K1061A protein has increased ability to distinguish between correct and incorrect substrates (increased fidelity and decreased misextension ability). These findings have important implications for the mechanism by which this enzyme accommodates distortions in the DNA caused by mismatches and lesions.
Additionally, I genetically characterized 21 mutant proteins, which may also affect the substrate specificity of this enzyme. The P962L, L1054A, T1063A, and G1215A mutant proteins were partially capable of complementing the rev3Δ mutation and are candidates for biochemical characterization, as they may have altered substrate specificity.
Copyright 2008 Craig A Howell
Howell, Craig A.. "Substrate recognition by the yeast Rev1 protein and DNA polymerase ζ." dissertation, University of Iowa, 2008.