Functional analysis of the amino-terminal 8-kDa domain of DNA polymerase β 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 binding single-stranded DNA (ssDNA), recognition of a 5'-phosphate in gapped DNA structures, and as a 8'-deoxyribose phosphate (dRP) lyase, NMR and x-ray crystal structures of this domain have suggested several residues that may interact with ssDNA or play a role in the dRP lyase reaction. Nine of these residues were altered by site-directed mutagenesis. Each mutant was expressed in Escherichia coli, and the recombinant protein was purified to near homogeneity. CD spectra of these mutant proteins indicated that the alteration did not adversely affect the global protein structure, Single-stranded DNA binding was probed by photochemical cross-linking to oligo(dT)(16). Several mutants (F25W, K35A, K60A, and K68A) were impaired in ssDNA binding activity, whereas other mutants (H34G, E71Q, K72A, E75A, and K84A) retained near wild-type binding activity. The 5'-phosphate recognition activity of these mutants was examined by UV cross-linking to a 5-nucleotide gap DNA where the 5' terminus in the gap was either phosphorylated or unphosphorylated, The results indicate that Lys(35) is involved in 5'-phosphate recognition of DNA polymerase beta. Finally, the dRP lyase activity of these mutants was evaluated using a preincised apurinic/apyrimidinic DNA, Alanine mutants of Lys(35) and Lys(60) are significantly reduced in dRP lyase activity, consistent with the lower ssDNA binding activity. More importantly, alanine substitution for Lys(72) resulted in a greater than 90% loss of dRP lyase activity, without affecting DNA binding. Alanine 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 activity, suggesting that the effects of the alanine substitution at Lys(72) and Lys(35) were additive. The results suggest that Lys(72) is directly 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.