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Nick recognition by DNA ligases

机译：DNA连接酶的尼克识别

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Phage T7 DNA ligase seals nicked DNA substrates and is a representative member of the ATP-dependent class of DNA ligases. Although the catalytic mechanism of DNA ligases has been delineated, Little is known about the nature of nick recognition by these enzymes. Here, we show that T7 ligase discriminates, at the nick-binding step, between nicks containing either a 5'-phosphate or a 5'-OH. T7 ligase binds preferentially to phosphorylated nicks and catalyses the sealing reaction. We also show using DNA footprinting studies, that T7 Ligase binds asymmetrically to nicks as a monomer, with the protein interface covering between 12 and 14 bp of DNA. Based on molecular modelling studies we propose a structural model of the ligase-DNA complex consistent with these and other data. Using photo-crosslinking and site-directed mutagenesis we have identified two residues, K238 and K240, critical for the transadenylation and nick-sealing reactions. Sequence conservation and structural analysis supports the premise that these two lysine residues are critical for both nucleotide binding and DNA nick recognition. The implications of these results on the ligation mechanism are discussed. (C) 2000 Academic Press. [References: 34]
机译：噬菌体T7 DNA连接酶密封有切口的DNA底物，并且是ATP依赖型DNA连接酶的代表成员。尽管已经描述了DNA连接酶的催化机理，但对于这些酶对缺口的识别性质知之甚少。在这里，我们显示在切口结合步骤中，T7连接酶可区分含有5'-磷酸或5'-OH的切口。 T7连接酶优先与磷酸化的切口结合，并催化封闭反应。我们还使用DNA足迹研究表明，T7连接酶不对称地与缺口作为单体结合，其蛋白质界面覆盖了12到14 bp的DNA。基于分子建模研究，我们提出了与这些数据和其他数据一致的连接酶-DNA复合物的结构模型。使用光交联和定点诱变，我们已经确定了两个残基，K238和K240，对转腺苷酸化和切口密封反应至关重要。序列保守和结构分析支持以下前提：这两个赖氨酸残基对于核苷酸结合和DNA缺口识别均至关重要。讨论了这些结果对结扎机制的影响。 （C）2000学术出版社。 [参考：34]

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