Morimatsu K, Horii T
Department of Molecular Protozoology, Osaka University, Japan.
Eur J Biochem. 1995 Mar 15;228(3):772-8.
To investigate the DNA-binding site in the Escherichia coli RecA protein, RecA was covalently cross-linked to oligodeoxythymidine [p(dT)14] by irradiation with ultraviolet light. We identified the site of cross-linking of the protein when the RecA.p(dT)14 complex was formed in the absence of nucleotide cofactor as well as in the presence of adenosine 5'-[gamma-thio]triphosphate. When RecA.p(dT)14 complex formed without nucleotide cofactor was irradiated with ultraviolet light, a cross-linked peptide was found after digestion with Achromobactor lyticus protease I. Amino acid composition of the peptide was determined. The results indicated that the site of cross-linking was in the region spanning amino acid residues 89-106. Further digestion of the cross-linked fragment with Staphylococcus aureus V8 protease indicated that Tyr103 was the site of cross-linking. When the complex formed with adenosine 5'-[gamma-thio]triphosphate was irradiated with ultraviolet light, two cross-linked sites were detected, which were in the region of residues 89-106 and residues 178-183. These regions are far from the two disordered loops in the crystal structure, which were suggested to be DNA-binding sites by Story et al. [Story, R. M., Weber, T. W. & Steitz, T. A. (1992) Nature 355, 318-325].
为了研究大肠杆菌RecA蛋白中的DNA结合位点，通过紫外线照射将RecA与寡聚脱氧胸苷酸[p(dT)14]共价交联。我们确定了在不存在核苷酸辅因子以及存在腺苷5'-[γ-硫代]三磷酸的情况下形成RecA.p(dT)14复合物时蛋白质的交联位点。当用紫外线照射在无核苷酸辅因子的情况下形成的RecA.p(dT)14复合物时，用溶菌无色杆菌蛋白酶I消化后发现了一个交联肽段。测定了该肽段的氨基酸组成。结果表明交联位点在跨越氨基酸残基89 - 106的区域。用金黄色葡萄球菌V8蛋白酶对交联片段进行进一步消化表明Tyr103是交联位点。当用紫外线照射与腺苷5'-[γ-硫代]三磷酸形成的复合物时，检测到两个交联位点，分别在残基89 - 106区域和残基178 - 183区域。这些区域远离晶体结构中被斯托里等人[斯托里，R.M.，韦伯，T.W.和斯蒂茨，T.A.（1992年）《自然》355，318 - 325]认为是DNA结合位点的两个无序环。
