Replication of single-stranded plasmid pT181 DNA in vitro.

Abstract

Plasmid pT181 is a 4437-base-pair, multicopy plasmid of Staphylococcus aureus that encodes tetracycline resistance. The replication of the leading strand of pT181 DNA initiates by covalent extension of a site-specific nick generated by the initiator protein at the origin of replication and proceeds by an asymmetric rolling circle mechanism. The origin of the leading strand synthesis also serves as the site for termination ofreplication. Replication ofpT181 DNA in vivo and in vitro has been shown to generate a single-stranded intermediate that corresponds to the leading strand of the DNA. In vivo results have suggested that a palindromic sequence, palA, located near the leading strand termination site acts as the lagging strand origin. In this paper we report the development and characterization of an in vitro system for the replication of singlestranded pT181 DNA. Synthesis of the lagging strand of pT181 proceeded in the absence ofthe leading strand synthesis and did not require the pT181-encoded initiator protein, RepC. The replication of the lagging strand required RNA polymerasedependent synthesis of an RNA primer. Replication of singlestranded pT181 DNA was found to be greatly stimulated in the presence of the paL4 sequence. We also show that paL4 acts as the lagging strand origin and that DNA synthesis initiates within this region.A number of small, multicopy plasmids from Staphylococcus aureus such as pT181, pC221, pE194, pC194, and pUB110 share similar replication properties. Many of these plasmids have also been shown to replicate in Bacillus subtilis (1, 2). These plasmids encode replication initiator proteins, which have origin-specific nicking-closing activities (3, 4). These proteins are directly involved in the generation ofa primer for the replication of the leading strand via an asymmetric rolling circle mechanism. A number of in vivo and in vitro studies have shown that single-stranded DNA (ssDNA) is an intermediate in the replication of these plasmids. The mechanism of replication of these plasmids is different from that of the more extensively studied plasmids of Escherichia coli and is in many ways similar to the replication of ssDNA bacteriophages of E. coli (5-7). These plasmids contain a palindromic sequence (palA) whose deletion results in plasmid instability, a decrease in the plasmid copy number, and the accumulation of large quantities of ssDNA in vivo (8,9). On the basis of these observations, the palA sequence has been postulated to serve as the origin of lagging strand synthesis. The palA sequences of many plasmids have considerable homology (4, 9). This sequence has been shown to be orientation dependent, can function at different locations, and is interchangeable among many small S. aureus plasmids (4,8,9).The replication properties of the pT181 plasmid of S. aureus have been extensively studied both in vivo and in vitro (4). An in vitro system for the replication of double-stranded, supercoiled pT181 DNA has been established and used to characterize the replication propert...

Sequence requirements for the termination of rolling‐circle replication of plasmid pT181

“…After one round of replication, the nick site is regenerated, and the initiators make a second cleavage and ligate the displaced SS leading strand of the DNA. The SS DNA is then converted to the double-stranded (DS) form utilizing the single-strand origin (SSO) (Baas and Jansz, 1988;Birch and Khan, 1992;Dempsey et al, 1995;Gruss et al, 1987). Previous studies have shown that the origin of replication contains sequences necessary for both the initiation and termination of RC replication (Dotto et al, 1984;Gros et al, 1987;Murray et al, 1989;Sozhamannan et al, 1990;Iordanescu and Projan, 1988;Zhao and Khan, 1996).…”

Lagging-strand origins of the promiscuous plasmid pMV158: physical and functional characterization

“…The mechanisms involved in ss + dsDNA conversion are not well understood, although some features of the process have been defined. First, it seems that only host proteins are involved in the lagging-strand synthesis, most likely RNA polymerase, as shown in vivo for the ssoU of PUB110 (Boe et al, 1989), and in vitro for the ssoA of pT181 (Birch & Khan, 1992). However, in the case of the lactococcal plasmid pWVOl (an ssoAcontaining plasmid) this conversion has been proposed to be mediated by the host DNA primase (Leenhouts e t al., 1991).…”

“…lb). Re&' r i m s similar in size have been defined for the ssoA4 of plasmid pT181 (Birch & Khan, 1992), and for the SJ-OCT o f PUB1 10 (Boe ef a/., 1989). In addition to this, u7e may also conclude that either the DNA sequence or its structure within the RS, plays an important role in the ss -+ d s D N h conversion.…”

“…RCreplicating plasmids are constructed like cassettes of genetic information (Projan & Novick, 1988), one of the modules being sso (del Solar e t al., 1993a). Based on sequence homologies, various types of sso have been described (Novick, 1989), including ssoA, which is present in plasmids pT181, pLSl and pC194 (Novick, 1989), and $sou, which is characteristic of plasmid PUB110 (Boe et a/., 1989;Birch & Khan, 1992). A more complex situation is found in the streptococcal plasmid pMV158 (from which plasmid pLSl is derived; Lacks e t al., 1986;Priebe & Lacks, 1989), because it bears both types of sso (van der Lelie e t al., 1989).…”

“…The host machinery recognizes the sso in an orientationdependent manner (Birch & Khan, 1992 ;del Solar et al, 199315). These findings suggest that unpaired sequences, within the secondary structures that constitute the sso, are important for its recognition by host proteins involved in …”