GENETIC AND CRYSTALLOGRAPHIC STUDIES OF THE 3',5'-EXONUCLEOLYTIC SITE OF DNA-POLYMERASE-I
GENETIC AND CRYSTALLOGRAPHIC STUDIES OF THE 3',5'-EXONUCLEOLYTIC SITE OF DNA-POLYMERASE-I
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DOI：
10.1126/science.2832946
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发表时间：
1988-04-08
期刊：
影响因子：
56.9
通讯作者：
STEITZ, TA
中科院分区：
文献类型：
--
作者：
DERBYSHIRE, V;FREEMONT, PS;STEITZ, TA
DNA 聚合酶 I 大片段（Klenow 片段）的定点诱变产生了两个缺乏 3'',5''-外切酶活性但具有正常聚合酶活性的突变蛋白。对突变蛋白的晶体分析表明，除了突变位点的预期变化之外，两者的蛋白结构都没有任何改变。这些结果证实了外切核酸酶活性位点在克列诺片段的小结构域上的推测位置及其与聚合酶活性位点的物理分离。野生型酶与二价锰离子和脱氧胸苷单磷酸的复合物的反常散射差傅里叶表明，外切核酸酶活性位点具有两个二价金属离子的结合位点。突变蛋白的特性表明，一种金属离子在底物结合中发挥作用，而另一种则参与核酸外切酶反应的催化。
Site-directed mutagenesis of the large fragment of DNA ploymerase I (Klenow fragment) yielded two mutant porteins lacking 3'',5''-exonoclease activity but having normal polymerase activity. Crystallographic analysis of the mutant proteins showed that neither had any alteration in protein structure other than the expected changes at the mutation sites. These results confirmed the presumed location of the exonuclease active site on the small domain of Klenow fragment and its physical separation from the polymerase active site. An anomalous scattering difference Fourier of a complex of the wild-type enzyme with divalent manganese ion and deoxythymidine monophosphate showed that the exonuclease active site has binding sites for two divalent metal ions. The properties of the mutant proteins suggest that one metal ion plays a role in substrate binding while the other is involved in catalysis of the exonuclease reaction.