Evidence for an imino intermediate in the dna polymerase beta deoxyribose phosphate excision reaction

A recent study demonstrated that rat DNA polymerase beta (beta-pol) releases 5′-deoxyribose phosphate (dRP) termini from preincised apurinic/apyrimidinic DNA, a substrate generated during some types of base excision repair. This catalytic activity resides within the amino-terminal, 8-kDa domain of beta-pol and occurs via beta-elimination (Matsumoto, Y., and Kim, K. (1995) Science 269, 699-702). The last finding suggested to us that the dRP excision reaction might proceed via an imine intermediate. In order to test this hypothesis, we attempted to trap beta-pol on preincised apurinic/apyrimidinic DNA using NaBH4 as the reducing agent. Both 8-kDa domain- and intact beta-pol-DNA complexes were detected and identified by autoradiography coupled with immunoblotting. Helix-2 and its adjacent loops do not appear to be involved directly in catalysis; the corresponding 32-amino acid peptide (residues 27-58) could not be covalently trapped, and neither the peptide itself nor an antipeptide antibody could inhibit trapping of the 8-kDa domain (residues 1-87). Thus, the active site residue responsible for dRP release by beta-pol is proposed to be a primary amine, presumably one of the 9 lysines at the amino- or carboxyl-terminal end of the 8-kDa domain.