The Escherichia coli terB sequence affects maintenance of a plasmid with the M13 phage replication origin

Abstract

Replication initiated at the bacteriophage M13 origin can be affected by interaction of a properly oriented termination signal terB and the Tus protein. The effect can be alleviated by overproduction of the M13 replication gene protein II.The replication terminus of the Escherichia coli chromosome is a large region (350 kb) flanked on both sides by terminator sites that arrest replication in a polar fashion (4, 10). These sequences impede replication initiated at the E. coli chromosomal origin or at the replication origins of phages or plasmids integrated into the chromosome (4, 5, 10). Homologous terminator sites were also identified on E. coli plasmid R6K (1,15). The introduction of a ter site on ColEl-derived plasmids blocks their replication (9,12,22). This block is dependent on the orientation of the site relative to the direction of replication and requires the presence of a host-encoded protein named Tus (11). In vitro replication systems were used to characterize the mechanism of replication arrest: Tus protein binds to the ter sequence (13,18), and the Tus-ter complex inhibits the progression of DNA helicases (17,20). Strand displacement by DnaB helicase is inhibited as a result of the binding of Tus to either the R6K terR or the E. coli terB sequence. In addition, the Tus-terB complex also inhibits the action of two other E. coli helicases, UvrD (helicase II) and Rep.Most replicons used so far to study Tus-terB-dependent termination replicate by a theta mechanism and require the DnaB helicase. In contrast, the filamentous bacteriophage M13 (fl or fd) replicates by a rolling circle mechanism and requires the Rep helicase (24). In this study, we show that in vivo, Tus-terB interaction interferes with M13 replication. However, an excess of gene protein II (gpII) alleviates this effect.Plasmid pHV960 carries the ampicillin resistance gene of plasmid pBR322 and the M13 plus and minus replication origins (Fig. 1) is less stable in the absence of selective pressure in tus cells than in tus+ cells. This plasmid does not carry a ter-like signal in the region deriving from pSC101 or in gpII, but the sequence of the spectinomycin resistance gene is not known. The reasons for the effect of the tus mutation on pSCgpII are not understood. It was reported that the stability of pSC101 derivatives lacking the par site is affected by plasmid supercoiling (21), which could possibly differ in tus+ and tus cells.The three plasmids pHV960, pHV960T+, and pHV96OT-were used to transform strain JJC40 (tus+ rep' [2]) harboring plasmid pSCgpII (Table 1). In the presence of 0.02 to 0.03transformed much less efficiently than did pHV960 and pHV96OT-. In contrast, the three plasmids transformed a Atus::Kanr isogenic strain with the same efficiency (tus cells were poorly transformed by these plasmids, possibly because of the low copy number of pSCgpII; Fig. 2). These experiments show that the interaction of Tus and ter oriented to arrest replication initiated at the M13 origin interferes with plasmid transformation. This effect is not...

Interaction of the Escherichia coli Replication Terminator Protein (Tus) with DNA: A Model Derived from DNA-Binding Studies of Mutant Proteins by Surface Plasmon Resonance

“…Removing the protein requires the ordered reversal of some, at least, of the events that took place during binding. While the Tus-Ter complex appears to be capable of blocking the action of various helicases in in vitro strand separation assays (7,(10)(11)(12)(13)(14)(15)(16)(17) and RNA polymerase in transcription (32), the block is often inefficient in such assays, and the polarity is often weak. For example, in contrast to the polar block on the progress of the replisome, entrance of RNA polymerase into a Tus-Ter site promotes the dissociation of Tus from the DNA, regardless of the direction from which the polymerase approaches.…”

“…There is some controversy over whether Tus blocks the movement of the replication fork merely by acting as a clamp that prevents strand separation by the replicative helicase DnaB or whether specific interactions between Tus and some component of the replisome, probably DnaB, are also involved (8). Certainly, the Tus-Ter complex is capable of blocking the action of the DnaB, Rep, and SV40 helicases in a polar manner in assays where the action of the helicase is coupled to DNA replication (7,(10)(11)(12) and of a range of other helicases in other in vitro reactions (7,(13)(14)(15)(16)(17). With a clamp mechanism, simple thermodynamic arguments alone cannot explain the polarity of termination, so kinetic (or dynamic) arguments must be invoked.…”

Transcription‐induced deletions in Escherichia coli plasmids

“…As pTL1 A 2 kb fragment from the plasmid pSO1010 (Oehler et aL, 1990) cloned in a pACYC184 This work derivative lacking the Tc R gene pSCgpll m pSCgpll (Uzest et aL, 1991 ) is a pSC101 derivative carrying a spectinomycin resistance This work gene (cloned from plasmid pHP45-omega; Prentki and Kirsh, 1984) and gene //of M13mp2 under the control of the lacUV5 promoter (cloned from plasmid pLII; Fulford and Model, 1988). In pSCgpll m, gene I/from M13 was replaced by gene // from M13mp2 pHV960 M13 replication origin linked to the Ap R gene from pBR322 Uzest et aL (1991) Nucleotide co-ordinates used for pBR322 are from Sutcliffe (1978) and Backman and Boyer (1983); those for M13 from Van Wezenbeek et aL (1980); those for pE194 from Horinouchi and Weisblum (1982); and those for lacZ from Genbank (Kalnins et al, 1983, with nucleotide 1 of lacZat position 1286 of the lac operon). nt, nucleotide; Tc s and Tc R, tetracycline sensitive and resistant; Em, erythromycin; Ap, ampicillin.…”

“…(pACmLacl, Table 1). The inducibility of gpll was assessed by the ability of strain JJC40 (an hsdR derivative of ABl157) containing both pSCgpll rn and pACmLacl to maintain the M13-derived plasmid pHV960 (Uzest et aL, 1991) in the presence of 1 mM IPTG.…”