Marsin S, Marguet E, Forterre P
Institut de Génétique et Microbiologie, Bâtiment 409, CNRS, UMR 8621, Université Paris-Sud, 91405 Orsay cedex, France.
Nucleic Acids Res. 2000 Jun 1;28(11):2251-5. doi: 10.1093/nar/28.11.2251.
The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi replicates via the rolling circle mechanism. pGT5 encodes the replication initiator protein Rep75 that exhibits a nicking-closing (NC) activity in vitro on single-stranded oligonucleotides containing the pGT5 double-stranded origin (dso) sequence. Some mesophilic Rep proteins present site-specific DNA topo-isomerase-like activity on a negatively supercoiled plasmid harbouring the dso. We report here that Rep75 also exhibits topoisomerase activity on a negatively supercoiled DNA substrate. This DNA topoisomerase-like activity is dependent on the amino acids involved in NC activity of Rep75. However, in contrast with mesophilic Rep proteins, Rep75 topoisomerase activity is not dso dependent. Moreover, although pGT5 is known to be relaxed in vivo, Rep75 was not able to act on a relaxed plasmid in vitro, whether or not it contained the dso.
来自嗜热古菌深渊热球菌的质粒pGT5通过滚环机制进行复制。pGT5编码复制起始蛋白Rep75,该蛋白在体外对含有pGT5双链起源(dso)序列的单链寡核苷酸表现出切口-封闭(NC)活性。一些嗜温Rep蛋白对携带dso的负超螺旋质粒具有位点特异性DNA拓扑异构酶样活性。我们在此报告,Rep75在负超螺旋DNA底物上也表现出拓扑异构酶活性。这种DNA拓扑异构酶样活性依赖于Rep75的NC活性所涉及的氨基酸。然而,与嗜温Rep蛋白不同,Rep75拓扑异构酶活性不依赖于dso。此外,尽管已知pGT5在体内是松弛的,但Rep75在体外不能作用于松弛的质粒,无论其是否含有dso。