Asp34 of PvuII endonuclease is directly involved in DNA minor groove recognition and indirectly involved in catalysis
Asp34 of PvuII endonuclease is directly involved in DNA minor groove recognition and indirectly involved in catalysis
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DOI：
10.1006/jmbi.1998.2269
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发表时间：
1998-12-18
影响因子：
5.6
通讯作者：
Cheng, XD
中科院分区：
文献类型：
--
作者：
Horton, JR;Nastri, HG;Cheng, XD
PvuII限制性内切酶是一种同源二聚体，识别并切割双链DNA中的DNA序列5 '-CAGCTG-3'，并且该酶的结构已被报道。在野生型酶中，Asp 34与小沟侧识别序列的内部鸟嘌呤相互作用。Asp 34密码子被改变为Gly（D34 G），体外研究表明D34 G蛋白失去了对中心G. C碱基对的结合特异性，并且与野生型酶相比，它以10（-4）倍的活性降低来切割典型序列。我们现在已经确定了与含有同源序列的12 bp双链脱氧寡核苷酸复合的D34 G PvuII内切核酸酶在1.59埃分辨率下的结构。D34 G改变导致相对于野生型蛋白质/DNA复合物的几种结构变化。首先，内部鸟嘌呤的糖部分从C2 '-内折叠变为C3'-内折叠，而3'鸟嘌呤的糖部分从C3'-内折叠变为C2 '-内折叠。第二，内部G. C碱基对之间的轴向升高减少，而G. C和侧翼碱基对之间的轴向升高扩大。第三，观察到两个不同的单体活性位点，我们称之为“引发”和“未引发”的磷酸二酯键裂解。引发和未引发的网站不同的Asp 58侧链的构象，并在未引发的网站的情况下，四个网络化的水分子。这些水分子，存在于引发的网站，已经牵连在这个和其他核酸内切酶的催化机制;其中一些可以被替换为Mg 2+切割所必需的。两者合计，这些结构变化意味着Asp 34侧链从两个亚基保持其DNA底物的不同构象，正确定位的目标骨架磷酸盐和间接操纵的活性位点。这提供了一些见解如何识别特定的DNA序列连接到催化的高度特异性限制性内切酶，并揭示了一种方式，其中DNA的结构构象与PvuII蛋白的协调调节。(C)北京：科学出版社.
The PvuII restriction endonuclease is a homodimer that recognizes and cleaves the DNA sequence 5'-CAGCTG-3' in double-stranded DNA, and the structure of this enzyme has been reported. In the wild-type enzyme, Asp34 interacts with the internal guanine of the recognition sequence on the minor groove side. The Asp34 codon was altered to specify Gly (D34G), and in vitro studies have revealed that the D34G protein has lost binding specificity for the central G.C base-pairs, and that it cuts the canonical sequence with 10(-4)-fold reduced activity as compared to the wild-type enzyme. We have now determined the structure at 1.59 Angstrom resolution of the D34G PvuII endonuclease complexed with a 12 bp duplex deoxyoligonucleotide containing the cognate sequence. The D34G alteration results in several structural changes relative to wild-type protein/DNA complexes. First, the sugar moiety of the internal guanine changes from a C2'-endo to C3'-endo pucker while that of the 3' guanine changes from C3'-endo to C2'-endo pucker. Second, the axial rise between the internal G.C base-pairs is reduced while that between the G.C and flanking base-pairs is expanded. Third, two distinct monomeric active sites are observed that we refer to as being "primed" and "unprimed" for phosphodiester bond cleavage. The primed and unprimed sites differ in the conformation of the Asp58 side-chain, and in the absence from unprimed sites of four networked water molecules. These water molecules, present in the primed site, have been implicated in the catalytic mechanism of this and other endonucleases; some of them can be replaced by the Mg2+ necessary for cleavage. Taken together, these structural changes imply that the Asp34 side-chains from the two subunits maintain a distinct conformation of its DNA substrate, properly situating the target backbone phosphates and indirectly manipulating the active sites. This provides some insight into how recognition of the specific DNA sequence is Linked to catalysis by the highly specific restriction endonucleases, and reveals one way in which the structural conformation of the DNA is modulated coordinately with that of the PvuII protein. (C) 1998 Academic Press.