Zhu C X, Qi H Y, Tse-Dinh Y C
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla 10595, USA.
J Mol Biol. 1995 Jul 28;250(5):609-16. doi: 10.1006/jmbi.1995.0402.
Cys662 is one of the 12 cysteine residues proposed to be co-ordination sites for binding of three Zn(II) in Escherichia coli DNA topoisomerase I. Oligonucleotide-directed mutagenesis was used to convert Cys662 to either serine or histidine. The mutant enzymes were overexpressed and purified to homogeneity. Analysis of the purified enzymes demonstrated that the mutations resulted in loss of one tightly bound Zn(II). In vivo complementation tests and in vitro relaxation activity assays at different temperatures showed that the partial relaxation activities retained in the two mutant enzymes were temperature sensitive. Fluorescence measurements indicated that the wild-type and mutant enzymes have structural differences. When DNA cleavage specificity was examined, the mutant enzymes were found to have altered cleavage site preferences. The preferred cleavage sites of the wild-type enzyme all had a cytosine residue four nucleotides to the minus side of the break. The cleavage sites produced by the mutant enzymes did not show a preference for cytosine at that position.
半胱氨酸662是大肠杆菌DNA拓扑异构酶I中被认为是结合三个锌离子（II）的配位位点的12个半胱氨酸残基之一。采用寡核苷酸定向诱变技术将半胱氨酸662分别转化为丝氨酸或组氨酸。突变酶经过量表达并纯化至同质。对纯化酶的分析表明，这些突变导致一个紧密结合的锌离子（II）丢失。在不同温度下进行的体内互补试验和体外松弛活性测定表明，两种突变酶中保留的部分松弛活性对温度敏感。荧光测量表明，野生型和突变型酶在结构上存在差异。当检测DNA切割特异性时，发现突变酶的切割位点偏好发生了改变。野生型酶的首选切割位点在断裂点负侧四个核苷酸处均有一个胞嘧啶残基。突变酶产生的切割位点在该位置对胞嘧啶没有偏好。
