Backbone dynamics and refined solution structure of the N-terminal domain of DNA polymerase β. Correlation with DNA binding and dRP lyase activity

Abstract

Mammalian DNA polymerase β functions in the base excision DNA repair pathway filling in short patches (1–5 nt) in damaged DNA and removing deoxyribose 5′-phosphate from the 5′-side of damaged DNA. The backbone dynamics and the refined solution structure of the N-terminal domain of β-Pol have been characterized in order to establish the potential contribution(s) of backbone motion to the DNA binding and deoxyribose 5′-phosphate lyase function of this domain. The N-terminal domain is formed from four helices packed as two antiparallel pairs with a 60° crossing between the pairs. The RMSD of the NMR conformers (residues 13–80) is 0.37 Å for the backbone heavy atoms and 0.78 Å for all heavy atoms. NMR characterization of the binding site(s) for a ssDNA-5mer, ssDNA-8mer, ssDNA-9mer, and dsDNA-12mer shows a consensus surface for the binding of these various DNA oligomers, that surrounds and includes the deoxyribose 5′-phosphate lyase active site region. Connection segments between helices 1 and 2 and between helices 3 and 4 each contribute to DNA binding. Helix-3-turn-helix-4 forms a helix-hairpin-helix motif. The highly conserved hairpin sequence (LPGVG) displays a significant degree of picosecond time-scale motion within the backbone, that is possibly important for DNA binding at the phosphodiester backbone. An Ω-loop connecting helices 1 and 2 and helix-2 itself display significant exchange contributions (Rex) at the backbone amides due to apparent conformational type motion on a millisecond time-scale. This motion is likely important in allowing the Ω-loop and helix-2 to shift toward, and productively interact with, gapped DNA. The deoxyribose 5′-phosphate lyase catalytic residues that include K72 which forms the Schiff’s base, Y39 which is postulated to promote proton transfer to the aldehyde, and K35 which assists in phosphate elimination, show highly restricted backbone motion. H34, which apparently participates in detection of the abasic site hole and assists in the opening of the hemiacetal, shows conformational exchange.