DNA polymerases perform a diverse repertoire of biological functions including genomic replication, DNA damage repair, DNA lesion bypass, immunoglobulin generation, and sister chromatid cohesion. These enzymes are phylogenetically grouped into six families (A, B, C, D, X, and Y). DNA polymerases from different families differ in their in vivo functions, primary sequences, DNA substrate preferences (non-gapped, gapped, or single-stranded DNA;damaged or undamaged DNA), nucleotide incorporation efficiency and fidelity, and polymerization processivity. Mechanistically, all kinetically characterized DNA polymerases share a minimal kinetic mechanism of nucleotide incorporation including several hypothetical protein conformational changes, one of them being rate-limiting. Although X-ray crystallographic studies of DNA polymerases have suggested that the rate-limiting conformational change involves a dramatic closing of the Finger domain alone, several recent stopped-flow fluorescence resonance energy transfer (FRET) studies of several DNA polymerases including Dpo4 indicate that the Finger domain closing is too fast to limit correct nucleotide incorporation. Dpo4 is from Sulfolobus solfataricus and belongs to the Y-family polymerases, which can bypass DNA lesions, thereby rescuing cells from apoptosis. The long term goal of the PIs is to understand how the structures and conformational dynamics of a DNA polymerase play a role in its enzymatic function. Recent stopped-flow FRET studies of Dpo4 by the Contact PI's group have revealed that all four structural domains of Dpo4, not just the Finger domain, undergo rapid conformational changes before and after phosphodiester bond formation. These observed motions were unfortunately invisible from numerous published X-ray crystallographic studies. More interestingly, when a DNA lesion is encountered, several domains of Dpo4 adopt different conformations as compared to those observed with a normal DNA substrate. To understand the exact nature of these domain motions in solution and their connection with the kinetic mechanism of nucleotide incorporation, we will employ solution NMR techniques to investigate this hyperthermostable and relatively small sized DNA polymerase, Dpo4 , with the following two specific aims: 1) Determine the solution structures of apo Dpo4 and its binary (Dpo4??DNA) and ternary (Dpo4??DNA??dNTP) complexes;2) Investigate conformational dynamics of Dpo4 in its apo form, binary (Dpo4??DNA) and ternary (Dpo4??DNA??dNTP) complexes. Once completed, our proposed studies will reveal detailed site-specific information on changes in protein structure and/or conformational dynamics that are important for substrate (DNA, correct or incorrect nucleotide) binding and catalysis. More importantly, our studies will unravel any structural and dynamic determinants responsible for the fidelity of a Y-family DNA polymerase during translesion DNA synthesis.
DNA polymerases are the proteins replicate genomic DNA in most organisms. Various solution structures of a model DNA polymerase will be solved through NMR for the first time. These structures will reveal how this class of proteins functions in solution.
|Zahurancik, Walter J; Baranovskiy, Andrey G; Tahirov, Tahir H et al. (2015) Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase É›. DNA Repair (Amst) 29:16-22|
|Vyas, Rajan; Reed, Andrew J; Tokarsky, E John et al. (2015) Viewing Human DNA Polymerase Î² Faithfully and Unfaithfully Bypass an Oxidative Lesion by Time-Dependent Crystallography. J Am Chem Soc 137:5225-30|