Anatomy of the Replication Origin of Plasmid ColE2-P9

Abstract

The plasmid ColE2-P9 origin is a 32-bp region which is specifically recognized by the plasmid-specified Rep protein to initiate DNA replication. We analyzed the structural and functional organization of the ColE2 origin by using various derivatives carrying deletions and single-base-pair substitutions. The origin may be divided into three subregions: subregion I, which is important for stable binding of the Rep protein; subregion II, which is important for binding of the Rep protein and for initiation of DNA replication; and subregion III, which is important for DNA replication but apparently not for binding of the Rep protein. The Rep protein might recognize three specific DNA elements in subregions I and II. The relative transformation frequency of the autonomously replicating plasmids carrying deletions in subregion I is lower, and nevertheless the copy numbers of these plasmids in host bacteria are higher than those of the wild-type plasmid. Efficient and stable binding of the Rep protein to the origin might be important for the replication efficiency to be at the normal (low) level. Subregion II might be essential for interaction with the catalytic domain of the Rep protein for primer RNA synthesis. The 8-bp sequence across the border of subregions II and III, including the primer sequence, is conserved in the (putative) origins of many plasmids, the putative Rep proteins of which are related to the ColE2-P9 Rep protein. Subregion III might be required for a step that is necessary after Rep protein binding has taken place.DNA replication initiates at a specific chromosomal region called the replication origin. The DNA sequence elements required for replication origin activity have been extensively defined and characterized (30). Typically, they consist of a recognition element for the initiator protein and an AT-rich region. In the process of initiation of replication, the initiator protein binds to the origin and induces local unwinding of the duplex DNA. Subsequent events are the loading of a DNA helicase onto the unwound region followed by recruitment of additional factors to initiate DNA synthesis. In the Escherichia coli replication system, the ATP-bound DnaA proteins bind to the 9-mer DnaA boxes in oriC, and then the DnaB helicase is loaded onto unwound 13-mer AT-rich sequences, followed by loading of proteins required for initiation of DNA synthesis, including the DnaG primase (30).The plasmid ColE2-P9 (ColE2) is a circular duplex DNA molecule of about 7 kb (18) and is present at about 10 to 15 copies per host chromosome (4, 21). The plasmid specifies an initiator protein (Rep protein, 35 kDa), which is the only plasmid-specified trans-acting factor essential for its replication (21,26,60). In addition, plasmid replication requires the host DNA polymerase I (28, 50) along with other host factors. The minimal cis-acting region (origin) (Fig. 1) required for plasmid replication consists of only 32 bp, where replication initiates (23, 60). The ColE2 Rep protein specifically binds to the ColE2 origin as ...

Specific Interaction between the Initiator Protein (Rep) and Origin of Plasmid ColE2-P9

“…The E. coli strains used were AG1 recA1 (11) and BL21(DE3) (Novagen). The plasmids used have been described elsewhere (3,12,24,29,35,37), except for those described below.…”

“…The 252-bp EcoRI-EcoRV fragment of pTI51dr8 (35) and the 258-bp EcoRI-EcoRV fragment of pTI51dr4 (35) were cloned between the EcoRI and EcoRV sites of pBluescript KS(ϩ), followed by digestion with NheI and XhoI and by self-ligation to construct pHMdr8 and pHMdr4, respectively. The 419-bp EcoRI-HindIII fragment of pTI51dr56 (35) was cloned between the EcoRI-HindIII sites of pBluescript KS(ϩ) and then digested with NheI and XhoI, followed by self-ligation, to construct pHM dr56.…”

“…Mutational analyses (35) have revealed that Ori may be divided into three subregions (I, II, and III). Subregion I is important for stable binding of Rep and contains site ␤.…”

“…1A), required for ColE2 DNA replication consists of 31 bp (35). The ColE2 Rep protein binds specifically to Ori DNA, as revealed by a filter binding assay (17) and by an electrophoresis mobility shift assay (EMSA) (35). In an in vitro ColE2 DNA replication system with crude extracts of Escherichia coli, ColE2 DNA replication starts at a unique site in Ori and proceeds unidirectionally (13,32).…”

“…The minimal cis-acting region, or origin (Ori) (Fig. 1A), required for ColE2 DNA replication consists of 31 bp (35). The ColE2 Rep protein binds specifically to Ori DNA, as revealed by a filter binding assay (17) and by an electrophoresis mobility shift assay (EMSA) (35).…”