Contacts between the 5′ nuclease of DNA polymerase I and its DNA substrate
Contacts between the 5′ nuclease of DNA polymerase I and its DNA substrate
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DOI：
10.1074/jbc.m100985200
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发表时间：
2001-08-10
影响因子：
4.8
通讯作者：
Joyce, CM
中科院分区：
文献类型：
--
作者：
Xu, Y;Potapova, O;Joyce, CM
大肠杆菌DNA聚合酶I（Pol I）的5 '核酸酶是一类重要的原核和真核核酸酶，参与DNA复制和修复，对单链和双链DNA之间的连接具有特异性。我们已经从蛋白质和DNA的角度研究了5 '核酸酶结构域与DNA底物的相互作用。磷酸乙基化干扰表明，核酸酶结合到周围的切割位点的核苷酸，也接触互补链的一半转身离开，表明接触的DNA底物的双链体部分的一个面。使用携带独特的甲基膦酸酯取代以及5 '核酸酶中的突变的DNA底物进一步研究磷酸二酯接触。这些实验表明，两个高度保守的碱性残基，Lys（78）和Arg（81），紧邻切割位点的5 '靠近磷酸二酯，而第三个高度保守的残基，Arg（20），可以与切割位点的3 '的磷酸二酯相互作用。我们的研究结果提供了强有力的支持DNA结合模型提出的相关核酸外切酶从噬菌体T5，其中上述保守的碱性残基定义的两个末端的螺旋弓，形成单链DNA结合区的一部分。在活性位点区域的9个高度保守的羧酸盐似乎在底物结合中发挥相对较小的作用，尽管它们对催化至关重要。除了结合切割点周围的DNA骨架外，5 '核酸酶还具有在上游引物链的3 '末端处的一个或两个磨损碱基的结合位点。与相关系统中的工作一致，5 '核酸酶切割被5 '标签中的双链体DNA阻断，但该酶对5 '尾内的脱碱基DNA或极性反转具有相当的耐受性。
The 5 ' nuclease of DNA polymerase I (Pol I) of Escherichia coli is a member of an important class of prokaryotic and eukaryotic nucleases, involved in DNA replication and repair, with specificity for the junction between single-stranded and duplex DNA. We have investigated the interaction of the 5 ' nuclease domain with DNA substrates from the standpoint of both the protein and the DNA. Phosphate ethylation interference showed that the nuclease binds to the nucleotides immediately surrounding the cleavage site and also contacts the complementary strand one-half turn away, indicating that contacts are made to one face only of the duplex portion of the DNA substrate. Phosphodiester contacts were investigated further using DNA substrates carrying unique methylphosphonate substitutions, together with mutations in the 5 ' nuclease. These experiments suggested that two highly conserved basic residues, Lys(78) and Arg(81), are close to the phosphodiester immediately 5 ' to the cleavage site, while a third highly conserved residue, Arg(20), may interact with the phosphodiester 3 ' to the cleavage site. Our results provide strong support for a DNA binding model proposed for the related exonuclease from bacteriophage T5, in which the conserved basic residues mentioned above define the two ends of a helical arch that forms part of the single-stranded DNA-binding region. The nine highly conserved carboxylates in the active site region appear to play a relatively minor role in substrate binding, although they are crucial for catalysis. In addition to binding the DNA backbone around the cleavage point, the 5 ' nuclease also has a binding site for one or two frayed bases at the 3 ' end of an upstream primer strand. In agreement with work in related systems, 5 ' nuclease cleavage is blocked by duplex DNA in the 5 ' tag, but the enzyme is quite tolerant of abasic DNA or polarity reversal within the 5 ' tail.