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首页> 外文期刊>Biochemistry >Identification of DNA 3'-Phosphatase Active Site Residues and Their Differential Role in DNA Binding,Mg~(2+) Coordination,and Catalysis
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Identification of DNA 3'-Phosphatase Active Site Residues and Their Differential Role in DNA Binding,Mg~(2+) Coordination,and Catalysis

机译：DNA 3'-磷酸酶活性位点残基的鉴定及其在DNA结合，Mg〜（2+）配位和催化中的差异作用

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DNA 3'-phosphatase (Tppl) from Saccharomyces cerevisiae,a homologue of human polynucleotide kinase/3'-phosphatase,has been shown to participate in DNA damage repair by removing 3'-phosphate blocking lesions.Tppl shows similarity to the L-2-haloacid dehalogenase superfamily of enzymes.By comparison to phosphoserine phosphatase,a well-studied member of this family,we designed conservative and nonconservative substitutions of likely active site residues of Tppl and tested them in a variety of assays.From the loss or impairment of activity,we identified D35,D37,T39,S88,K170,D206,and D218 as being involved in Tpp1 catalysis.D35 and K170 were the most critical since maximum inactivation was seen with even conservative mutations.Tppl bound DNA through its active site in a Mg~(2+)-dependent manner and exhibited a preference for dsDNA.Although Tppl bound more strongly to DNA with a free 3' terminus,it also bound well to covalently closed DNA,suggesting a possible lesion scanning mechanism.DNA binding studies further indicated that Tppl coordinates Mg~(2+) through D35 and D206 and contacts the DNA 3' end through D37.The removal of 3'-phosphate involved a phospho-Tpp1l intermediate,and our results support D35 as being the point of covalent attachment.On the basis of these similarities in mutant phenotypes of Tppl and phosphoserine phosphatase,we propose a reaction mechanism for Tppl which explains its strict phosphate specificity.
机译：酿酒酵母的DNA 3'-磷酸酶（Tppl）是人类多核苷酸激酶/ 3'-磷酸酶的同源物，已被证明可通过去除3'-磷酸盐阻滞性损伤而参与DNA损伤修复。Tppl与L-2相似-卤代酸脱卤酶的超家族。与磷酸丝氨酸磷酸酶相比，该家族经过深入研究，我们设计了可能保守的Tppl活性位点残基的保守和非保守取代基，并在各种测定法中进行了测试。我们发现D35，D37，T39，S88，K170，D206和D218参与了Tpp1催化。D35和K170是最关键的，因为即使是保守突变也能看到最大的失活。 Mb〜（2+）依赖型且对dsDNA表现出优先性。尽管Tppl与带有3'末端的DNA结合更牢固，但它也与共价封闭的DNA结合得很好，提示可能存在病灶扫描机制。进一步的研究表明，Tppl通过D35和D206配位Mg〜（2+）并通过D37与DNA 3'末端接触。3'-磷酸的去除涉及到磷酸Tppl1中间体，我们的结果支持D35作为基于Tppl和磷酸丝氨酸磷酸酶突变表型的相似性，我们提出了Tppl的反应机理，解释了其严格的磷酸盐特异性。

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