title: Specific interaction of DNA polymerase beta 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 beta (beta-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 beta-pol with other BER protein(s), we developed affinity chromatography matrices by cross-linking purified rat beta-pol or antibody against beta-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 beta-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 beta-pol and DNA ligase I. An antibody to beta-pol was able to shift the complex in sucrose gradients to a much larger molecular mass (>300 kDa) that again contained both beta-pol and DNA ligase I. Furthermore, DNA ligase I and beta-pol were co-immunoprecipitated from the testis nuclear extract with anti beta-pol IgG. Thus, we conclude that beta-pol and DNA ligase I are components of a multiprotein complex that performs BER.
authors.affiliation: Sealy Center for Molecular Science, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1068, USA.
authors.forename: R
authors.initials: R
authors.lastname: Prasad
authors.forename: R K
authors.initials: RK
authors.lastname: Singhal
authors.forename: D K
authors.initials: DK
authors.lastname: Srivastava
authors.forename: J T
authors.initials: JT
authors.lastname: Molina
authors.forename: A E
authors.initials: AE
authors.lastname: Tomkinson
authors.forename: S H
authors.initials: SH
authors.lastname: Wilson
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism*; D002846:Chromatography, Affinity; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000302:isolation & purification / Q000378:metabolism*; D004256:DNA Polymerase I / Q000302:isolation & purification / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D006147:Guanine; D008190:Lyases / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D051381:Rats; D013379:Substrate Specificity; D013737:Testis / Q000378:metabolism*; D014498:Uracil
chemical_list: D029968:Escherichia coli Proteins; C000607895:Lig1 protein, rat; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D014498:Uracil; D006147:Guanine; D004256:DNA Polymerase I; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D011088:DNA Ligases; D000072481:DNA Ligase ATP