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

Mammalian DNA polymerase beta functions in the base excision DNA repair pat hway filling in short patches (1-5 nt) in damaged DNA and removing deoxyrib ose 5'-phosphate from the 5'-side of damaged DNA. The backbone dynamics and the refined solution structure of the N-terminal domain of P-Pol have been characterized in order to establish the potential contribution(s) of backb one motion to the DNA binding and deoxyribose 5'-phosphate lyase function o f this domain. The N-terminal domain is formed from four helices packed as two antiparallel pairs with a 60 degrees crossing between the pairs. The RM SD of the NMR conformers (residues 13-80) is 0.37 Angstrom for the backbone heavy atoms and 0.78 Angstrom for all heavy atoms. NMR characterization of the binding site(s) for a ssDNA-5mer, ssDNA-8mer, ssDNA-9mer, and dsDNA-12 mer shows a consensus surface for the binding of these various DNA oligomer s, that surrounds and includes the deoxyribose 5'-phosphate lyase active si te 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 Omega-loop connecting helices 1 and 2 and helix-2 itself display signifi cant exchange contributions (R-ex) at the backbone amides due to apparent c onformational type motion on a millisecond time-scale. This motion is likel y important in allowing the Omega-loop and helix-2 to shift toward, and pro ductively interact with, gapped DNA. The deoxyribose 5'-phosphate lyase cat alytic residues that include K72 which forms the Schiff's base, Y39 which i s postulated to promote proton transfer to the aldehyde, and K35 which assi sts in phosphate elimination, show highly restricted backbone motion. H34, which apparently participates in detection of the abasic site hole and assi sts in the opening of the hemiacetal, shows conformational exchange. (C) 20 00 Academic Press.