Doherty A J, Worrall A F, Connolly B A
Department of Biochemistry, University of Southampton, UK.
J Mol Biol. 1995 Aug 18;251(3):366-77. doi: 10.1006/jmbi.1995.0440.
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 O2/N3 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.
牛胰脱氧核糖核酸酶I是一种特异性较低的核酸内切酶，它与DNA的小沟相互作用。两个氨基酸，R41和Y76，完全填充了这个沟，R41与嘧啶和嘌呤的O2/N3位置形成氢键，Y76通过一种不寻常的疏水“堆积”相互作用与脱氧核糖接触。通过定点诱变研究了这些氨基酸在磷酸二酯键断裂和DNA水解选择性中的作用。进行了保守性改变（R41K、Y76F）或更剧烈的改变（R41A、R41G、Y76A、Y76G）。包含Y76的表面环（残基73至76）也已被删除。还制备了几个R41和Y76都被改变的双突变体。使用小的非DNA发色底物脱氧胸苷-3',5'-二（对硝基苯基）-磷酸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变化。相比之下，所有突变体与DNA的结合力比野生型弱约十倍，除了R41K和Y76F外，它们水解DNA的速度要慢得多。这表明R41和Y76的变化对水解位点的催化氨基酸影响很小，但对于结合DNA以及更重要的是将可切割的磷酸正确定位以进行有效水解是必需的。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对DNA水解的选择性。当任何一个氨基酸发生改变时，在整体水解选择性上观察到非常小的差异。然而，R41的变化导致了局部切割特异性的一些差异，这可以通过该氨基酸与相对于可切割磷酸的特定碱基形成氢键的作用来解释。
