FUNCTIONAL-ANALYSIS OF THE AMINO-TERMINAL 8-KDA DOMAIN OF DNA-POLYMERASE-BETA AS REVEALED BY SITE-DIRECTED MUTAGENESIS - DNA-BINDING AND 5'-DEOXYRIBOSE PHOSPHATE LYASE ACTIVITIES

The amino-terminal 8-kDa domain of DNA polymerase beta functions in bi nding single-stranded DNA (ssDNA), recognition of a 5'-phosphate in ga pped DNA structures, and as a 8'-deoxyribose phosphate (dRP) lyase, NM R and x-ray crystal structures of this domain have suggested several r esidues that may interact with ssDNA or play a role in the dRP lyase r eaction. Nine of these residues were altered by site-directed mutagene sis. Each mutant was expressed in Escherichia coli, and the recombinan t protein was purified to near homogeneity. CD spectra of these mutant proteins indicated that the alteration did not adversely affect the g lobal protein structure, Single-stranded DNA binding was probed by pho tochemical cross-linking to oligo(dT)(16). Several mutants (F25W, K35A , K60A, and K68A) were impaired in ssDNA binding activity, whereas oth er mutants (H34G, E71Q, K72A, E75A, and K84A) retained near wild-type binding activity. The 5'-phosphate recognition activity of these mutan ts was examined by UV cross-linking to a 5-nucleotide gap DNA where th e 5' terminus in the gap was either phosphorylated or unphosphorylated , The results indicate that Lys(35) is involved in 5'-phosphate recogn ition of DNA polymerase beta. Finally, the dRP lyase activity of these mutants was evaluated using a preincised apurinic/apyrimidinic DNA, A lanine mutants of Lys(35) and Lys(60) are significantly reduced in dRP lyase activity, consistent with the lower ssDNA binding activity. Mor e importantly, alanine substitution for Lys(72) resulted in a greater than 90% loss of dRP lyase activity, without affecting DNA binding. Al anine mutants of Lys(68) and Lys(84) had wild-type dRP lyase activity. The triple alanine mutant, K35A/K68A/K72A, was devoid of dRP lyase ac tivity, suggesting that the effects of the alanine substitution at Lys (72) and Lys(35) were additive. The results suggest that Lys(72) is di rectly involved in formation of a covalent imino intermediate and are consistent with Lys(72) as the predominant Schiff base nucleophile in the dRP lyase beta-elimination catalytic reaction.