Catalytic and DNA binding properties of P upsilon uII restriction endonuclease mutants
Catalytic and DNA binding properties of P upsilon uII restriction endonuclease mutants
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DOI：
10.1074/jbc.272.41.25761
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发表时间：
1997-10-10
影响因子：
4.8
通讯作者：
Riggs, PD
中科院分区：
文献类型：
--
作者：
Nastri, HG;Evans, PD;Riggs, PD
通过体内和体外的突变分析，研究了PvuII内切酶的特定残基在DNA结合和切割中的作用。虽然证实了通过晶体结构预测直接参与功能的残基的重要性，但分析导致了几个惊人的结果。天冬氨酸34通过小凹槽与PvuII位点(5‘-CAGCTG-3’)的中央碱基对接触，在结合特异性中发挥关键作用。D34G突变体与集合CANNTG中的任何序列都有高亲和力结合，尽管其低水平的切割活性只作用于野生型位点。此外，接触中心G的残基上His到Ala的突变被预测为被PvuII甲基化阻止，仍然需要PvuII甲基酶在体内保持，反对这一假设作为甲基化保护的唯一机制。最后，在凝胶位移分析中，五个降低切割活性但仍显示结合的突变中，有四个位于形成DNA或亚单位-亚单位接触的残基上，而不是在催化中心。这进一步证明了特定结合和催化之间存在很强的联系。
The role of particular residues of the PvuII endonuclease in DNA binding and cleavage was studied by mutational analysis using a number of in vivo and in vitro approaches. While confirming the importance of residues predicted to be involved directly in function by the crystal structure, the analysis led to several striking results. Aspartate 34, which contacts the central base pair of the PvuII site (5'-CAGCTG-3') through the minor groove, plays a critical role in binding specificity. A D34G mutant binds with high affinity to any of the sequences in the set CANNTG, although its low level of cleavage activity acts only on the wild-type site. In addition, a His to Ala mutation at the residue that contacts the central G and is predicted to be blocked by PvuII methylation still requires the PvuII methylase to be maintained in vivo, arguing against this hypothesis as the only mechanism for methylation protection. Finally, four of the five mutations that reduce cleavage activity while still exhibiting binding in the gel shift assay are at residues that form DNA- or subunit-subunit contacts rather than in the catalytic center. This provides further evidence for a strong linkage between specific binding and catalysis.