Topoisomerase activity of the hyperthermophilic replication initiator protein Rep75.
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.
Marsin
S
S
Institut de Génétique et Microbiologie, Bâtiment 409, CNRS, UMR 8621, Université Paris-Sud, 91405 Orsay cedex, France. marsin@biotech.jouy.inra.fr
Marguet
E
E
Forterre
P
P
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics*; D001665:Binding Sites; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics*; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D019711:Pyrococcus / Q000201:enzymology*; D016415:Sequence Alignment; D013379:Substrate Specificity
D019843:Archaeal Proteins; D004278:DNA, Superhelical; C121975:Rep75 protein, Pyrococcus abyssi; D009713:Nucleotidyltransferases; D004264:DNA Topoisomerases, Type I; D011088:DNA Ligases