DNA polymerases catalyze the replication of cellular DNA and play an integral role in DNA repair. Translesion synthesis, i.e. DNA synthesis across damaged DNA nucleotides, is a critical step in mutagenesis and the development of cancer. More than ten proteins capable of DNA synthesis have been identified in humans. These DNA polymerases differ in their fidelity and ability to perform translesion synthesis. The goal of this project is to solve the three dimensional structure of DNA polymerases from four different families poised to perform translesion synthesis. These structures will allow examination of the general hypothesis that interactions in the DNA polymerase active site modulate the miscoding and/or efficiency of translesion synthesis.
The specific aims i nvolve studies of structural and kinetic mechanisms of (a) error-prone translesion synthesis by high fidelity DNA polymerases ( Families A, B and X) (b) Iesion-blocked DNA synthesis and (c) accurate and error-prone synthesis by specialized translesion synthesis polymerases (Family Y). X-ray crystallography will be used to capture DNA polymerases complexed with oxidatively damaged DNA during various steps in this process. Coupled with steady-state kinetic analysis and site-directed mutagenesis, these structures should reveal structural and functional differences between Iow-(translesion) and high-fidelity DNA polymerases. Structural studies with high-fidelity polymerases will provide insight into the preferential incorporation of dCTP or dATP opposite 8-oxo-dG, establish a structural basis for the """"""""A-rule"""""""", and explain the miscoding potential of epsilon-dC and other exocyclic DNA adducts. The mechanism by which abasic sites and thymine glycol block the progression of DNA polymerases will also be examined. Structural analysis of two different Y-family (translesion) DNA polymerases, mouse pol Kappa and human pol eta, will provide insight into their substrate specificity. This project will analyze the mechanism of translesion synthesis at the atomic level and will complement studies of miscoding and mutagenesis (Project 1), the determination of thermodynamic parameters (Project 3) and NMR studies on DNA containing site-specifically placed lesions (Project 4). Overall, this project provides critical knowledge relevant to the structural biology of mutagenesis and human cancer.
|Minetti, Conceição A S A; Remeta, David P; Iden, Charles R et al. (2015) Impact of thymine glycol damage on DNA duplex energetics: Correlations with lesion-induced biochemical and structural consequences. Biopolymers 103:491-508|
|Völker, Jens; Plum, G Eric; Gindikin, Vera et al. (2014) Impact of bulge loop size on DNA triplet repeat domains: Implications for DNA repair and expansion. Biopolymers 101:1-12|
|Braunlin, William; Völker, Jens; Plum, G Eric et al. (2013) DNA meter: Energy tunable, quantitative hybridization assay. Biopolymers 99:408-17|
|Völker, Jens; Gindikin, Vera; Klump, Horst H et al. (2012) Energy landscapes of dynamic ensembles of rolling triplet repeat bulge loops: implications for DNA expansion associated with disease states. J Am Chem Soc 134:6033-44|
|Lukin, Mark; Minetti, Conceicao A S A; Remeta, David P et al. (2011) Novel post-synthetic generation, isomeric resolution, and characterization of Fapy-dG within oligodeoxynucleotides: differential anomeric impacts on DNA duplex properties. Nucleic Acids Res 39:5776-89|
|Völker, Jens; Plum, G Eric; Klump, Horst H et al. (2010) Energetic coupling between clustered lesions modulated by intervening triplet repeat bulge loops: allosteric implications for DNA repair and triplet repeat expansion. Biopolymers 93:355-69|
|Zaliznyak, Tanya; Lukin, Mark; El-khateeb, Mahmoud et al. (2010) NMR structure of duplex DNA containing the alpha-OH-PdG.dA base pair: a mutagenic intermediate of acrolein. Biopolymers 93:391-401|
|Minetti, Conceição A S A; Remeta, David P; Johnson, Francis et al. (2010) Impact of alpha-hydroxy-propanodeoxyguanine adducts on DNA duplex energetics: opposite base modulation and implications for mutagenicity and genotoxicity. Biopolymers 93:370-82|
|Minetti, Conceicao A S A; Remeta, David P; Dickstein, Rian et al. (2010) Energetic signatures of single base bulges: thermodynamic consequences and biological implications. Nucleic Acids Res 38:97-116|
|Völker, Jens; Plum, G Eric; Klump, Horst H et al. (2010) Energy crosstalk between DNA lesions: implications for allosteric coupling of DNA repair and triplet repeat expansion pathways. J Am Chem Soc 132:4095-7|
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