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首页> 外文期刊>Journal of Molecular Biology >THE ROLES OF ARGININE 41 AND TYROSINE 76 IN THE COUPLING OF DNA RECOGNITION TO PHOSPHODIESTER BOND CLEAVAGE BY DNASE I - A STUDY USING SITE-DIRECTED MUTAGENESIS
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THE ROLES OF ARGININE 41 AND TYROSINE 76 IN THE COUPLING OF DNA RECOGNITION TO PHOSPHODIESTER BOND CLEAVAGE BY DNASE I - A STUDY USING SITE-DIRECTED MUTAGENESIS

机译：精氨酸41和酪氨酸76在DNASE识别DNA与磷酸酯键的偶联中的作用-利用位点诱变的研究。

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Bovine pancreatic deoxyribonuclease I is an endonuclease of low specificity that interacts with the minor groove of DNA. Two amino acids, R41 and Y76, completely fill this groove, with R41 hydrogen bonding to the O-2/N-3 positions of pyrimidines and purines, and Y76 contacting a deoxyribose via an unusual hydrophobic ''stacking'' interaction. The roles of these amino acids in phosphodiester bond cleavage and in DNA hydrolysis selectivity have been studied by site-directed mutagenesis. Alterations have been made that are either conservative (R41K, Y76F) or more drastic (R41A, R41G, Y76A, Y76G). The surface loop (residues 73 to 76) that contains Y76 has also been deleted. Several double mutants in which both R41 and Y76 have been altered have also been prepared. The integrity of the catalytic site of the mutants has been investigated using the small, non-DNA, chromophoric substrate deoxythymidine-3',5'-di-(p-nitrophenyl)-phosphate. Hydrolysis of this compound was hardly changed, even by the most extreme alterations to R41 and Y76. In contrast, all the mutants bound DNA about ten times more weakly than the wild-type and, with the exception of R41K and Y76F, hydrolysed DNA much more slowly. This suggests that changes to R41 and Y76 have little effect on catalytic amino acids at the hydrolysis site, but are required to bind DNA and, more importantly, to correctly position the scissile phosphate for efficient hydrolysis. The selectivity of DNA hydrolysis for all the mutants has been tested using the 160 base-pair Escherichia coli Tyr T promoter DNA fragment. Very small differences were seen in global hydrolysis selectivity when either amino acid was altered. However, changes to R41 resulted in some differences to local cutting specificity that could be explained by the role of this amino acid in hydrogen bonding to particular bases relative to the scissile phosphate. [References: 45]
机译：牛胰腺脱氧核糖核酸酶I是低特异性的核酸内切酶，可与DNA的小沟相互作用。 R41和Y76这两个氨基酸完全填充了该凹槽，R41氢键合至嘧啶和嘌呤的O-2 / N-3位置，Y76通过异常的疏水性“堆积”相互作用与脱氧核糖接触。已经通过定点诱变研究了这些氨基酸在磷酸二酯键裂解和DNA水解选择性中的作用。所做的更改是保守的（R41K，Y76F）或更激进的（R41A，R41G，Y76A，Y76G）。包含Y76的表面环（残基73至76）也已删除。还制备了几个双突变体，其中R41和Y76都被改变了。已经使用小的非DNA发色底物脱氧胸苷-3'，5'-二-（对硝基苯基）-磷酸盐研究了突变体催化位点的完整性。即使对R41和Y76进行了最极端的改变，该化合物的水解也几乎没有改变。相反，所有的突变体结合DNA的能力比野生型弱约十倍，并且除R41K和Y76F外，它们水解DNA的速度要慢得多。这表明R41和Y76的变化对水解位点上的催化氨基酸影响很小，但是需要结合DNA，更重要的是，正确地将易裂磷酸定位以进行有效水解。已经使用160个碱基对的大肠杆菌Tyr T启动子DNA片段测试了所有突变体的DNA水解选择性。当任一氨基酸改变时，在整体水解选择性上观察到很小的差异。但是，R41的变化导致局部切割特异性产生一些差异，这可以通过该氨基酸在氢键合到特定碱基（相对于易磷酸酯）中的作用来解释。 [参考：45]

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