Why is the initiation nick site of an AT-rich rolling circle plasmid at the tip of a GC-rich cruciform?

Abstract

curious accident of sequence organization but has a Medical School, New York, NY 10016, USA substantive biological rationale that is derived from the 1 Corresponding author plasmid's need to prevent recycling of the initiator because recycling would obviate control of plasmid copy number. pT181 and other closely related rolling circle plasmidsRecycling is prevented by a modification of the initiator, have the nicking site for initiation of replication consisting of the attachment to one subunit of a short between the arms of a GC-rich inverted repeat oligonucleotide representing sequences 3Ј to the nick site sequence adjacent to the binding site for the dimeric in the double strand origin (DSO) (Rasooly and Novick, initiator protein. Replication is initiated by the initi-1993). The resulting heterodimer, RepC/C*, is present in ator-induced extrusion of this sequence as a cruciform, great excess, is metabolically stable, can bind to the pT181 creating a single-stranded region for nicking by the DSO and can inhibit but not initiate replication (Jin et al., protein. Nicking is followed by assembly of the repli-1997). We have proposed that this oligonucleotide is some without relaxation of the secondary structure. generated by a short 3Ј extension of the leading strand at Following termination, the initiator protein is released the end of the replication cycle and becomes attached to with a short oligonucleotide attached to one subunit, the protein via the normal termination mechanism (Rasooly which prevents it from being recycled, a necessary and Novick, 1993). feature of the plasmid's replication control system. TheSince RepC/C* is isolated from pT181-containing cells, modified initiator can cleave single-stranded substrates there must always be at least a small fraction of RepC/C, and can nick and relax supercoiled plasmid DNA corresponding to material that has not yet been used for weakly. Although it can bind to its recognition sequence replication, and accounting for the in vitro replication in the leading strand origin, the modified protein activity originally observed in extracts of pT181-concannot induce cruciform extrusion, and it is proposed taining staphylococci by Khan et al. (1981). In our initial that this inability is the key to understanding the studies of RepC/C* (Rasooly and Novick, 1993; Rasooly biological rationale for having the nicking site at the et al., 1994a,b), we attributed the weak nicking, relaxing tip of a cruciform: the need to provide the functional and replication activity seen in these preparations to initiator with a catalytic advantage over the modified this small fraction of RepC/C, which was estimated on one sufficient to offset the numerical advantage and theoretical grounds to be 5-10%. The more recent results metabolic stability of the latter.reported here and elsewhere (Jin et al., 1997) suggest that Keywords: cruciform/initiation nicking site/recycling/ the level of RepC/C present in these preparations is RepC/rolling circle considerably lower than 5% and although it can a...

A Novel Conformation of the Herpes Simplex Virus Origin of DNA Replication Recognized by the Origin Binding Protein

“…In this instance the dimeric RepC protein facilitates the extrusion of a cruciform in negatively supercoiled DNA followed by site-specific nicking by a topoisomerase-like mechanism in the newly formed loop (28). Structural information on alternative conformations occurring in other replication origins is scarce.…”

Inhibition of a naturally occurring rolling‐circle replicon in derivatives of the theta‐replicating plasmid pIP501

“…Indeed, it was found that the Rep protein is covalently linked to a small oligonucleotide after a round of replication. This modification prevents it to initiate DNA synthesis and thus ensures that it cannot be reused to start a new round of replication (Rasooly and Novick, 1993;Jin et al, 1997).…”

Purification and Characterization of the PcrA Helicase ofBacillus anthracis

“…For this, we utilized oligonucleotide probes containing a 25-bp duplex region and a 28-or 29-nucleotide (nt) ss region at their 5Ј or 3Ј ends (oligonucleotides c and d) ( Table 1). These probes represent the inverted repeat II region from the plasmid pT181 origin in which the sequences present in the 5Ј or 3Ј ss regions can assume a hairpin structure (16,17). The underlined regions in oligonucleotides c and d (Table 1) contain 5ЈCCGG3Ј sequences which (upon intrastrand base pairing) generate a cleavage site for the restriction endonuclease HpaII.…”