The active site of the rolling circle replication protein Rep75 is involved in site-specific nuclease, ligase and nucleotidyl transferase activities.
The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi replicates via a rolling circle mechanism. The protein Rep75, encoded by this plasmid, exhibits a nicking-closing (NC) activity in vitro on single-stranded oligonucleotides containing the pGT5 double-stranded origin sequence. In addition, Rep75 catalyses a site-specific nucleotidyl terminal transferase (NTT) activity, e.g. it can transfer one AMP or dAMP (from ATP or dATP) to the 3'-OH of an oligonucleotide corresponding to the left part of the nicking site. The Rep75 sequence contains a motif similar to the active-site motifs of Rep proteins from the PhiX174/pC194 superfamily. We show here that the tyrosine present in this motif is indeed essential for DNA cleavage by Rep75, but is dispensable for its NTT activity. However, a nearby arginine, which is not required for DNA cleavage, is involved in both NTT and closing, indicating that the same active site is involved in the NC and NTT activities of Rep75. For both NTT and NC, the G residue in 3' of the nicking site is essential, whereas the A residue in 5' is dispensable for NC, despite its conservation in RC plasmids of the PhiX174/pC194 superfamily. The NTT and closing activities have an optimal temperature lower than the nicking activity. These data indicate that the three reactions catalysed by Rep75 can be uncoupled, although they share part of their mechanisms. Finally, we show that NC is inhibited by ATP or dATP at concentrations that promote NTT. We propose a model in which the NTT activity of Rep75 plays a role in the regulation of pGT5 replication in vivo.
Marsin
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S
Institut de Génétique et Microbiologie, Bat. 409, CNRS UMR 8621, Université Paris-Sud, 91405 Orsay Cedex, France. marsin@biotech.jouy.inra.fr
Forterre
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D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D010584:Bacteriophage phi X 174 / Q000235:genetics; D001665:Binding Sites; D003001:Cloning, Molecular; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010957:Plasmids; D019711:Pyrococcus / Q000235:genetics*; D016415:Sequence Alignment; D013696:Temperature
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D003838:Deoxyadenine Nucleotides; D009838:Oligodeoxyribonucleotides; C121975:Rep75 protein, Pyrococcus abyssi; D009713:Nucleotidyltransferases; D003851:Deoxyribonucleases; D011088:DNA Ligases; C026600:2'-deoxyadenosine triphosphate