Abstract
Plasmids are extrachromosomal elements that can replicate autonomously in host cells. They are well known and studied in bacteria and have allowed the development of essential genetic tools in many bacterial species. Plasmids have also been very useful in studying fundamental cellular mechanisms, such as DNA replication. The plasmid pGT5 (3.4 kb), isolated from the euryarchaeon Pyrococcus abyssi , was the first plasmid studied in hyperthermophilic archaea.1 It was completely sequenced and shown to replicate via the rolling circle (RC) mechanism. RC replicons encode a replication initiator protein (usually named Rep) that exhibits a site-specific endonuclease/ligase activity. Several Rep protein encoded by bacterial or eukaryal RC replicons have been studied in vivo and in vitro , and they all share common characteristics. The purified Rep protein of pGT5 (Rep75) exhibits a highly thermophilic and specific nicking-closing (NC) activity on single-stranded oligonucleotides containing the pGT5 double-stranded origin (dso) Sequence. Rep75 exhibits an unusual site-specific nucleotidyl-terminal transferase (NTT) activity, never described before for a Rep protein. The protein Rep75 can be overproduced in Escherichia coli and purified to near homogeneity. The method used, as well as the activity tests, is described in detail here and could be relevant to the study of other hyperthermophilic Rep proteins.
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call