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

Abstract

The replication initiator protein (Rep) of plasmid ColE2-P9 (ColE2) is multifunctional. We are interested in how Rep binds to the origin (Ori) to perform various functions. We used the wild type and variants of Rep to study the Rep-Ori interaction by both in vitro and in vivo approaches, including biochemical analyses of protein-DNA interactions and an in vivo replication assay. We identified three regions (I, II, and III) of Rep, located in the C-terminal half, and three corresponding binding sites (I, II, and III) in Ori which are important for Rep-Ori interaction. We showed that region I, containing a putative helix-turn-helix motif, is necessary and sufficient for specific Ori recognition, interacting with site I of the origin DNA from the major groove. Region II interacts with site II of the origin DNA, from the adjacent minor groove in the left half of Ori, and region III interacts with site III, next to the template sequence for primer synthesis, which is one and one-half turn apart from site I on the opposite surface of the origin DNA. A putative linker region located between the two DNA binding domains (regions II and III) was identified, which might provide Rep an extended conformation suitable for binding to the two separate sites in Ori. Based on the results presented in this paper, we propose a model for Rep-Ori interaction in which Rep binds to Ori as a monomer.

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

“…In addition, region II ( Fig. 1) across region C of the ColE2 Rep protein binds to site b in the origin (9). Therefore, the amino acid residue(s) in region C might specifically recognize the nucleotide residue(s) of site ␥ in the cognate origin.…”

“…EMSA was performed essentially as described previously (33) except that the origin-containing 32 P-labeled DNA segments were prepared by treatment of the 0.14-kb BamHI fragments of plasmids pKA2/2a, pKA3/3a, pKA2/3a, and pKA3/2a, which carry the minimal ColE2, ColE3, ColE2/ColE3 (2/3), and ColE3/ColE2 (3/2) origins, respectively, with [␣-32 P]dCTP and Klenow fragment of DNA polymerase I as described previously (9).…”

“…The SELEX (systematic evolution of ligands by exponential enrichment) experiment and electrophoretic mobility shift assay (EMSA) using the mutant ColE2 origins showed that those sites, especially sites a and b, are required for high-affinity binding of the ColE2 Rep protein (32). The analyses using the truncated ColE2 Rep proteins suggested that sites a, b, and c of the origin are recognized by regions I, II, and III of the Rep protein, respectively (9) (Fig. 1).…”

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

“…This Rep specifically binds to the replication origin of the plasmid (Ori; 31 bp) (12)(13)(14)(15), and the Ori-bound Rep locally unwinds duplex DNA in the Ori and uniquely exhibits origin-specific primase activity, synthesizing the primer RNA, ppApGpA (16,17). The primer is used by the host DNA polymerase I to initiate DNA synthesis (16,18,19).…”

“…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).…”