Prasad R, Singhal R K, Srivastava D K, Molina J T, Tomkinson A E, Wilson S H
Sealy Center for Molecular Science, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1068, USA.
J Biol Chem. 1996 Jul 5;271(27):16000-7. doi: 10.1074/jbc.271.27.16000.
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.
碱基切除修复（BER）是一种修复DNA中修饰碱基的细胞防御机制。最近，利用从大肠杆菌中纯化的几种酶重建了G:U修复反应（迪亚诺夫，G.，和林达尔，T.（1994年）《当代生物学》4，1069 - 1076）。我们使用牛睾丸粗核提取物表明，G:U在体外能被高效修复，并且DNA聚合酶β（β - pol）负责单核苷酸缺口填充合成（辛哈尔，R. K.，普拉萨德，R.，和威尔逊，S. H.（1995年）《生物化学杂志》270，949 - 957）。为了研究β - pol与其他BER蛋白之间的潜在相互作用，我们通过将纯化的大鼠β - pol或抗β - pol抗体交联到固体支持物上，开发了亲和色谱基质。将牛睾丸粗核提取物应用于这些亲和柱，然后进行广泛洗涤。与亲和柱特异性结合的蛋白质与β - pol以复合物形式共洗脱。该复合物的分子量约为180 kDa，并且能够进行完整的尿嘧啶引发的BER反应。BER复合物同时包含β - pol和DNA连接酶I。抗β - pol抗体能够使该复合物在蔗糖梯度中迁移到更大的分子量（>300 kDa），该复合物仍然同时包含β - pol和DNA连接酶I。此外，DNA连接酶I和β - pol与抗β - pol IgG从睾丸核提取物中共免疫沉淀。因此，我们得出结论，β - pol和DNA连接酶I是执行BER的多蛋白复合物的组成成分。
