Department of Biotechnology, Chosun University, Gwangju 501-759, Republic of Korea.
Arch Biochem Biophys. 2011 Apr 1;508(1):78-86. doi: 10.1016/j.abb.2011.01.016. Epub 2011 Jan 31.
DNA topoisomerases play critical roles in regulating DNA topology and are essential enzymes for cell survival. In this study, a gene encoding type IA DNA topoisomerase was cloned from Staphylococcus aureus (S. aureus) sp. strain C-66, and the biochemical properties of recombinant enzyme was characterized. The nucleotide sequence analysis showed that the cloned gene contained an open reading frame (2070 bp) that could encode a polypeptide of 689 amino acids. The cloned gene actually produced 79.1 kDa functional enzyme (named Sau-TopoI) in Escherichia coli (E. coli). Sau-TopoI enzyme purified from E. coli showed ATP-independent and Mg(2+)-dependent manners for relaxing negatively supercoiled DNA. The relaxation activity of Sau-TopoI was inhibited by camptothecin, but not by nalidixic acid and etoposide. Cleavage site mapping showed that the enzyme could preferentially bind to and cleave the sequence GGNN↓CAT (N and ↓ represent any nucleotide and cleavage site, respectively). All these results suggest that the purified enzyme is type IA DNA topoisomerase. In addition, domain mapping analysis showed that the enzyme was composed of conserved four domains (I through IV), together with a variable C-terminal region containing a unique domain V.
DNA 拓扑异构酶在调节 DNA 拓扑结构方面发挥着关键作用，是细胞生存所必需的酶。本研究从金黄色葡萄球菌（S. aureus）sp. 菌株 C-66 中克隆了一个编码 I 型 DNA 拓扑异构酶的基因，并对重组酶的生化特性进行了表征。核苷酸序列分析表明，克隆的基因包含一个开放阅读框（2070 bp），可编码 689 个氨基酸的多肽。该基因实际上在大肠杆菌（E. coli）中产生了 79.1 kDa 的功能性酶（命名为 Sau-TopoI）。从大肠杆菌中纯化的 Sau-TopoI 酶表现出 ATP 非依赖性和 Mg2+依赖性的方式来松弛负超螺旋 DNA。Camptothecin 抑制 Sau-TopoI 的松弛活性，但 nalidixic acid 和 etoposide 则没有抑制作用。切割位点图谱表明，该酶可以优先结合并切割序列 GGNN↓CAT（N 和 ↓ 分别代表任何核苷酸和切割位点）。所有这些结果表明，纯化的酶是 I 型 DNA 拓扑异构酶。此外，结构域映射分析表明，该酶由保守的四个结构域（I 至 IV）组成，同时包含一个独特的结构域 V 的可变 C 端区域。
