In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein

Abstract

Rolling-circle replication of plasmid pLS1 is initiated by the plasmid-encoded RepB protein, which has nicking-closing (site-specific DNA strand transferase) enzymatic activity. The leading-strand origin of pLS1 contains two regions, (i) the RepB-binding site, constituted by three directly repeated sequences (iterons or the bind region), and (ii) the sequence where RepB introduces the nick to initiate replication (the nic region). A series of plasmids, belonging to the pLS1 family, show features similar to those of pLS1 and have DNA sequences homologous to the pLS1 nic region. In addition, they all share homologies at the level of their Rep proteins. However, the bind regions of these plasmids are, in general, not conserved. We tested the substrate specificity of purified RepB of pLS1. The RepB protein has a temperature-dependent nicking-closing action on supercoiled pLS1, as well as on recombinant plasmid DNAs harboring the pLS1 nic region. The DNA strand transferase activity of pLS1-encoded RepB was also assayed on two plasmids of the pLS1 family, namely, pE194 and pFX2. DNAs from both plasmids were relaxed by RepB, provided they had a proper degree of supercoiling; i.e., it was necessary to modulate the supercoiling of pE194 DNA to achieve RepB-mediated DNA relaxation. Single-stranded oligonucleotides containing the nic regions of various plasmids belonging to the pLS1 family, including those of pE194 and pFX2, were substrates for RepB. In vitro, the RepB protein does not need to bind to the iterons for its nicking-closing activity.Many multicopy plasmids isolated from gram-positive and -negative bacteria replicate by an asymmetric rolling-circle (RC) mechanism (reviewed in references 10, 13, and 23). Characterization of various staphylococcal replicons has led to the proposal that plasmids replicating by the RC mode are constructed as interchangeable genetic modules (26). Only one of these modules is essential for plasmid replication, the leading-strand initiation and control region (10). On the basis of homologies in the DNA sequence and in the genetic organization of the leading-strand initiation and control region, four families have been defined, and their prototypes are pT181, pSN2, pC194/pUB110, and pMV158 (10, 13, 23). The latter plasmid has been studied in some detail for its derivative pLS1, which lacks the mob gene involved in conjugative mobilization (19,25). The leading-strand initiation and control region includes the plasmid double-strand origin (dso) and the gene encoding the initiator of replication (Rep) protein. The dso can be physically and functionally divided into two regions, (i) bind, where the Rep protein binds, and (ii) nic, which includes the Rep-mediated nick site used to initiate replication (11). The two regions can be contiguous (as in pT181) or separated by dozens of bases (as in pLS1). The nic region contains one or two stem-loop structures which have been mapped by determining the sensitivity of plasmids pLS1 and pT181 to nuclease S1 in vitro and in vivo (21, 27). The DNA seq...

Nicking activity of the pMV158 MobM relaxase on cognate and heterologous origins of transfer

“…RepB from pMV158 was able to nick DNA from pE194 (with which there is homology 439 at the dso) only when the supercoiling of pE194 was altered (Moscoso et al, 1995). 440…”

Precise Determination, Cross-Recognition, and Functional Analysis of the Double-Strand Origins of the Rolling-Circle Replication Plasmids in Haloarchaea

“…In five other plasmids, including pZMX201, the nick site is in a stem region but near an unpaired position. As in bacterial plasmids, the nick site is always located in the loop region of a hairpin structure to facilitate the nicking by the Rep protein (30). It is possible that the hairpin structure in pZMX201 might serve as a target for recognition by the Rep protein and might melt from the unpaired position to expose the nick site upon the Rep/DSO interaction.…”

“…Among plasmids from the same family, the nick site is highly conserved but the binding site is not, suggesting that the origin's specificity is provided by the sequence differences in the binding site (5). The DNA sequence of the binding site can be either an inverted repeat or a set of direct repeats, and the nick site is always located in the loop region of a hairpin structure (7,31,33), which is regarded as necessary for the Rep protein to introduce the nick, especially when the plasmid is in a tightly packed super-coiled form (4,30,31).In the domain Archaea, two distinct RCR plasmid families have been identified according to the homologies of Rep proteins and DSO sequences. One family includes plasmids pGT5 (8), pRT1 (40), and pTN1 (36), isolated from hyperthermophilic archaea.…”

Plasmid Rolling-Circle Replication

“…The Rep nick sequence is generally located on an unpaired region within these hairpins, as exemplified by IRII of pT181 and IR-I of pMV158, which accounts for the requirement of plasmid DNA supercoiling to render the cleavage sequence a suitable ssDNA substrate for replication (66)(67)(68). The presence of secondary structures is likely to be involved in efficient recruitment and utilization of the initiator protein.…”