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 certain types of base excision repair, This catalytic activity resides within the amino-terminal, 8-kDa domain of beta-pol and occurs via beta-elimination as opposed to hydrolysis (Matsumoto, Y., and Rim, K. (1995) Science 269, 699-702), The latter finding suggested 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-DNA and intact beta-pol-DNA complexes were detected and identified by autoradiography coupled to immunoblotting, Our results indicate that the chemical mechanism of the beta-pol dRpase reaction does proceed through an imine enzyme-DNA intermediate and that the active site residue responsible for dRP release must therefore contain a primary amine.