Distinct Functions of the Two Specificity Determinants in Replication Initiation of Plasmids ColE2-P9 and ColE3-CA38

Abstract

The plasmid ColE2-P9 Rep protein specifically binds to the cognate replication origin to initiate DNA replication. The replicons of the plasmids ColE2-P9 and ColE3-CA38 are closely related, although the actions of the Rep proteins on the origins are specific to the plasmids. The previous chimera analysis identified two regions, regions A and B, in the Rep proteins and two sites, ␣ and ␤, in the origins as specificity determinants and showed that when each component of the region A-site ␣ pair and the region B-site ␤ pair is derived from the same plasmid, plasmid DNA replication is efficient. It is also indicated that the replication specificity is mainly determined by region A and site ␣. By using an electrophoretic mobility shift assay, we demonstrated that region B and site ␤ play a critical role for stable Rep protein-origin binding and, furthermore, that 284-Thr in this region of the ColE2 Rep protein and the corresponding 293-Trp of the ColE3 Rep protein mainly determine the Rep-origin binding specificity. On the other hand, region A and site ␣ were involved in the efficient unwinding of several nucleotide residues around site ␣, although they were not involved in the stable binding of the Rep protein to the origin. Finally, we discussed how the action of the Rep protein on the origin involving these specificity determinants leads to the plasmid-specific replication initiation.In all organisms, DNA replication is a key event for inheritance of genetic information. Initiation of DNA replication requires interaction of the initiator protein with the specific DNA region called the replication origin and the consequent localized melting of duplex DNA, which provides a singlestranded template for establishment of replication machinery. In initiation of chromosomal DNA replication in Escherichia coli, several molecules of the initiator protein (DnaA) tightly bind to the 9-mer repeated sequences called the DnaA boxes in the chromosomal replication origin (oriC), and then the DnaB helicase is loaded onto the unwound 13-mer AT-rich region located to one side of oriC, followed by establishment of the replication machinery containing DnaG and DNA polymerase III holoenzyme (17).The plasmid ColE2-P9 (ColE2) is a circular duplex DNA molecule of about 7 kb (10) and is kept at 10 to 15 copies per chromosome (2, 12). Initiation of the plasmid replication requires host DNA polymerase I (16, 26) and a plasmid-encoded replication initiator (Rep) protein that uniquely possesses an origin-specific primase activity among bacterial plasmids (15,35), and replication proceeds in a unidirectional manner (13, 28). The Rep protein specifically binds to the replication origin (15, 33) and synthesizes a short RNA molecule of 5Ј-ppApG pA-3Ј at a specific position in the origin as a primer for initiation of DNA synthesis by DNA polymerase I (28, 29). The 32-bp minimal ColE2 origin may be divided into three functional subregions, as proposed by in vivo analyses using an exhaustive mutant set of single-base-pair substitutions (33). One of th...

Structural Basis for Replication Origin Unwinding by an Initiator Primase of Plasmid ColE2-P9

“…5A). These observations implied the involvement of these basic residues in specificity determination, and in fact, studies using the mutated Rep proteins showed some contribution of the conserved basic residues for the specificity determination between ColE2-P9 and ColE3-CA38 plasmids (21). The present crystal structure showed that the side chain of the lysine is likely to contact only the DNA phosphate backbone, and the arginine side chain is too distant to interact with the paired bases.…”

“…Previous studies showed that a critical specificity determinant for stable binding of Rep to Ori between the ColE2-P9 and ColE3-CA38 plasmids is located in the region B and site ␤ (Fig. 5, A and B), and Thr 284 in helix H7 of ColE2 Rep and its corresponding residue Trp 293 of ColE3 Rep are the key residues in binding (21,22). The crystal structure showed that ColE2 Rep Thr 284 contacts the phosphate backbone of 6Gua in the non-template strand, whereas Arg 287 in the same helix makes specific contacts with the 6Gua base (Figs.…”

“…5B). Regions A of Rep and the sites ␣ of Ori are also the determinants of plasmid specificity between ColE2-P9 and ColE3-CA38 plasmids and involved in site-specific unwinding of the origin regions (21,22). By modulating the structures of the H4 modules of the Rep proteins, the spacings between the H4 modules (especially the conserved glutamines) and the HTH module (region A and site ␣) and PriCT modules (region C and site ␥) could be adjusted to place the three modules at their proper positions on the cognate Ori regions, which is important for specific binding, duplex DNA unwinding, and subsequent primer RNA synthesis.…”

“…1B). The C-terminal region of E2Rep-DBD determines its binding specificity to Ori (17,21,22). The N-terminal region of E2Rep-DBD contains a functionally unknown region called the PriCT (primase C-terminal) domain that is commonly found near the C termini of some primases belonging to the superfamily of archaeo-eukaryotic primases (23).…”

“…The N-terminal region of E2Rep-DBD contains a functionally unknown region called the PriCT (primase C-terminal) domain that is commonly found near the C termini of some primases belonging to the superfamily of archaeo-eukaryotic primases (23). The PriCT domain of E2Rep-DBD has been shown to be involved in origin unwinding (17,21). Furthermore, Rep binds to and unwinds Ori as a monomer (24).…”