Lavasani L S, Hiasa H
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Biochemistry. 2001 Jul 24;40(29):8438-43. doi: 10.1021/bi0155201.
Type II topoisomerases are responsible for DNA unlinking during DNA replication and chromosome segregation. Although eukaryotic enzymes are homodimers and prokaryotic enzymes are heterotetramers, both prokaryotic and eukaryotic type II topoisomerases belong to a single protein family. The amino- and carboxyl-terminal domains of eukaryotic enzymes are homologous to the ATP-binding and catalytic subunits of prokaryotic enzymes, respectively. Topoisomerase IV, a prokaryotic type II topoisomerase, consists of the ATP-binding subunit, ParE, and the catalytic subunit, ParC. We have joined the coding regions of parE and parC in frame and constructed a fusion protein of the two subunits of topoisomerase IV. This fusion protein, ParEC, can catalyze both decatenation and relaxation reactions. The ParEC protein is also capable of decatenating replicating daughter DNA molecules during oriC DNA replication in vitro. Furthermore, the fusion gene, parEC, complements the temperature-sensitive growth of both parC and parE strains, indicating that the ParEC protein can substitute for topoisomerase IV in vivo. These results demonstrate that a fusion protein of the two subunits of topoisomerase IV is a functional topoisomerase. Thus, a heterotetrameric type II topoisomerase can be converted into a homodimeric type II topoisomerase by gene fusion.
II型拓扑异构酶负责在DNA复制和染色体分离过程中解开DNA链。尽管真核生物的酶是同二聚体，原核生物的酶是异源四聚体，但原核生物和真核生物的II型拓扑异构酶都属于单一蛋白质家族。真核生物酶的氨基末端和羧基末端结构域分别与原核生物酶的ATP结合亚基和催化亚基同源。拓扑异构酶IV是一种原核II型拓扑异构酶，由ATP结合亚基ParE和催化亚基ParC组成。我们将parE和parC的编码区读框连接起来，构建了拓扑异构酶IV两个亚基的融合蛋白。这种融合蛋白ParEC能够催化解连环反应和松弛反应。ParEC蛋白在体外oriC DNA复制过程中也能够解开正在复制的子代DNA分子的连环。此外，融合基因parEC能够互补parC和parE菌株的温度敏感生长，表明ParEC蛋白在体内可以替代拓扑异构酶IV。这些结果表明，拓扑异构酶IV两个亚基的融合蛋白是一种功能性拓扑异构酶。因此，通过基因融合，异源四聚体II型拓扑异构酶可以转化为同二聚体II型拓扑异构酶。
