Specific interaction of DNA polymerase β and DNA ligase I in a multiprotein base excision repair complex from bovine testis

Abstract

Base excision repair (BER) is a cellular defense mechanism repairing modified bases in DNA. Recently, a G:U repair reaction has been reconstituted with several purified enzymes from Escherichia coli (Dianov, G., and Lindahl, T. (1994) Curr. Biol. 4, 1069-1076). Using bovine testis crude nuclear extract, we have shown that G:U is repaired efficiently in vitro, and DNA polymerase β (β-pol) is responsible for the single nucleotide gap-filling synthesis (Singhal, R. K., Prasad, R., and Wilson, S. H. (1995) J. Biol. Chem. 270, 949-957). To investigate potential interaction of β-pol with other BER protein(s), we developed affinity chromatography matrices by cross-linking purified rat β-pol or antibody against β-pol to solid supports. Crude nuclear extract 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 β-pol. This complex had a molecular mass of approximately 180 kDa and was able to conduct the complete uracil-initiated BER reaction. The BER complex contained both β-pol and DNA ligase I. An antibody to β-pol was able to shift the complex in sucrose gradients to a much larger molecular mass (>300 kDa) that again contained both β-pol and DNA ligase I. Furthermore, DNA ligase I and β-pol were co-immunoprecipitated from the testis nuclear extract with anti β-pol IgG. Thus, we conclude that β-pol and DNA ligase I are components of a multiprotein complex that performs BER.