Abstract
Several staphylococcal plasmids from different incompatibility (inc) groups which replicate by a rolling circle mechanism each specify a replication initiator protein (Rep) which is homologous with that of the inc3 tetracycline resistance plasmid pT181. The rep gene sequences of six pT181-like plasmids are known, each encoding proteins of molecular mass 38 kDa with 62% overall amino acid sequence identity. The initiation of replication in vivo by each of the Rep proteins is plasmid specific, acting in trans only at the cognate replication origin (ori) of the encoding plasmid. Previous studies in vitro of the RepC protein of pT181 demonstrated replication initiator, topoisomerase-like, and DNA binding activities, which appeared to be specific for the origin (oriC) of pT181 when compared with unrelated staphylococcal plasmids. Although RepD, specified by the inc4 chloramphenicol resistance plasmid pC221, has a range of activities similar to those noted previously for RepC, manipulation of in vitro conditions has revealed discrete steps in the overall reaction of RepD with oriD. In addition, factors have been identified which are necessary not only for sequence-dependent discrimination in vitro by Rep proteins for all pT181-like plasmids but also for the absolute specificity of RepD for its cognate pC221 replication origin (oriD), the latter occurring in vivo and a function of the topological state of the ori-containing target DNA. Here we also demonstrate the presence of a covalent phosphoryl-tyrosine linkage between the RepD protein of plasmid pC221 and an oligonucleotide substrate corresponding to its replication origin (oriD). The reactive tyrosine (Tyr-188) was identified from amino acid sequences of 32P-labeled peptide-oligonucleotide fragments. Substitution of Tyr-188 with phenylalanine confirms the importance of the tyrosyl hydroxyl group since the Y188F protein retains the sequence-specific DNA-binding capabilities of wild-type RepD but is unable to attach covalently to the replication origin or participate in the nicking-closing reaction in vitro.
Highlights
Previous studies in vitro of the RepC protein of pT181 demonstrated replication initiator, topoisomerase-like, and DNA binding activities, which appeared to be specific for the origin of pTl81 when compared with unrelated staphylococcal plasmids
The manipulation of buffer compositions has allowed dissection of the overall process facilitated by RepD into apparent partial reactions and has defined factors likely to be important in the sequence specificity of RepD for its origin
As a single RepD-induced cleavage site has been identified, in the (+) strand only (Fig. 6), it follows that RepD acts as a type I topoisomerase
Summary
Preparation of RepD-Purification of RepD yielded significantly greater amounts of soluble protein when buffers of high ionic strength (300 mM KCl) were used instead of the lower concentrations (50-100 mM KCl) used in the isolation of RepC [4]. The precise location of the nick site was determined by cleavage with RepD of oriD-containing DNA fragments labeled strand-followed by size determination of the product by electrophoresis in parallel with sequencing tracks. DNA gyrase activity in the S. aureus cell extract) it was found that replication initiator specificity was confined to pC221cop903, the incorporation of labeled precursors into relaxed pT181cop608 and pCW7 being drastically reduced compared with that incorporated into its SC form (Table 3) Such specificity mirrors that observed in Go, where the Rep proteins are known to act in trans to initiate replication only at their cognate ori sequences [13, 17]. The origin sequences of other plasmids relaxed by RepD in this study are presented (sequences are identical to that of oriD except where indicated)
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