title: Evidence for an imino intermediate in the DNA polymerase beta deoxyribose phosphate excision reaction.
abstract: 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 Kim, 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.
authors.affiliation: Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1071, USA.
authors.forename: C E
authors.initials: CE
authors.lastname: Piersen
authors.forename: R
authors.initials: R
authors.lastname: Prasad
authors.forename: S H
authors.initials: SH
authors.lastname: Wilson
authors.forename: R S
authors.initials: RS
authors.lastname: Lloyd
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004256:DNA Polymerase I / Q000378:metabolism*; D004260:DNA Repair; D007097:Imines / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D012267:Ribosemonophosphates / Q000378:metabolism*
chemical_list: D007097:Imines; D012267:Ribosemonophosphates; C058245:2-deoxyribose 5-phosphate; D004256:DNA Polymerase I