Purification of Escherichia coli DNA photolyase.
Purification of Escherichia coli DNA photolyase.
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发表时间：
1984-05
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通讯作者：
A. Sancar;F. W. Smith;G. Sancar
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作者：
A. Sancar;F. W. Smith;G. Sancar
大肠杆菌光裂合酶是一种DNA修复酶，其在光依赖性反应中将DNA中的主要UV光产物嘧啶二聚体单体化为嘧啶。我们最近描述了tac-phr质粒的构建，该质粒极大地过量产生该酶（Sancar，G. B.，Smith，F. W.，和Sancar，A.（1983）Nucleic Acids Res. 11，6667-6678）。使用携带过量生产质粒的菌株作为起始材料，我们已经开发了一种纯化程序，产生几毫克的明显均匀的酶。纯化的蛋白质是单一多肽，在变性和非变性条件下均具有49，000的表观Mr。该酶不需要二价阳离子，并且仅在光活化光的存在下才能恢复照射过的DNA的生物活性。纯化的光解酶的周转数为2.4二聚体/分子/分钟，这个值与E.杆菌
Escherichia coli photolyase is a DNA repair enzyme which monomerizes pyrimidine dimers, the major UV photoproducts in DNA, to pyrimidines in a light-dependent reaction. We recently described the construction of a tac-phr plasmid that greatly overproduces the enzyme (Sancar, G. B., Smith, F. W., and Sancar, A. (1983) Nucleic Acids Res. 11, 6667-6678). Using a strain carrying the overproducing plasmid as the starting material, we have developed a purification procedure that yields several milligrams of apparently homogeneous enzyme. The purified protein is a single polypeptide that has an apparent Mr of 49,000 under both denaturing and nondenaturing conditions. The enzyme has no requirement for divalent cations and it restores the biological activity of irradiated DNA only in the presence of photoreactivating light. The purified photolyase has a turnover number of 2.4 dimers/molecule/min; this value agrees well with the in vivo rate of photoreactivation in E. coli.