SPECIFIC INTERACTION OF DNA-POLYMERASE-BETA AND DNA-LIGASE-I IN A MULTIPROTEIN BASE EXCISION-REPAIR COMPLEX FROM BOVINE TESTIS

Base excision repair (EER) is a cellular defense mechanism repairing m odified bases in DNA. Recently, a G:U repair reaction has been reconst ituted with several purified enzymes from Escherichia coil (Dianov, G, , and Lindahl, T. (1994) Curr. Biol. 4, 1069-1076), Using bovine testi s crude nuclear extract, we have shown that G:U is repaired efficientl y in vitro, and DNA polymerase beta (beta-pol) is responsible for the single nucleotide gap-filling synthesis (Singhal, R. It., Prasad, R,, and Wilson, S, H. (1995) J, Biol, Chem, 270, 949-957), To investigate potential interaction of beta-pol with other BER protein(s), we develo ped affinity chromatography matrices by cross-linking purified rat bet a-pol or antibody against beta-pol to solid supports. Crude nuclear ex tract from bovine testis was applied to these affinity columns, which were then extensively washed, Proteins that bound specifically to the affinity columns were co-eluted in a complex with beta-pol, This compl ex had a molecular mass of approximately 180 kDa and was able to condu ct the complete uracil-initiated EER reaction, The EER complex contain ed both beta-pol. and DNA ligase I. An antibody to beta-pol was able t o shift the complex in sucrose gradients to a much larger molecular ma ss (>300 kDa) that again contained both beta-pol and DNA ligase I, Fur thermore, DNA ligase I and beta-pol were co-immunoprecipitated from th e testis nuclear extract with anti beta-pol IgG, Thus, we conclude tha t beta-pol and DNA ligase I are components of a multiprotein complex t hat performs BER.