Role of Individual Monomers of a Dimeric Initiator Protein in the Initiation and Termination of Plasmid Rolling Circle Replication

Abstract

Plasmids of the pT181 family encode initiator proteins that act as dimers during plasmid rolling circle (RC) replication. These initiator proteins bind to the origin of replication through a sequence-specific interaction and generate a nick at the origin that acts as the primer for RC replication. Previous studies have demonstrated that the initiator proteins contain separate DNA binding and nicking-closing domains, both of which are required for plasmid replication. The tyrosine residue at position 191 of the initiator RepC protein of pT181 is known to be involved in nicking at the origin. We have generated heterodimers of RepC that consist of different combinations of wild type, DNA binding, and nicking mutant monomers to identify the role of each of the two monomers in RC replication. One monomer with DNA binding activity was sufficient for the targeting of the initiator to the origin, and the presence of Tyr-191 in one monomer was sufficient for the initiation of replication. On the other hand, a dimer consisting of one monomer defective in DNA binding and the other defective in origin nicking failed to initiate replication. Our results demonstrate that the monomer that promotes sequence-specific binding to the origin must also nick the DNA to initiate replication. Interestingly, whereas Tyr-191 of the initiator was required for nicking at the origin to initiate replication, it was dispensable for termination, suggesting that alternate amino acids in the initiator may promote termination but not initiation.A large number of small, multicopy plasmids in bacteria replicate by a rolling circle (RC) 1 mechanism (for reviews see Refs. 1-3). Initiation of replication of RC plasmids involves sequence-specific binding of the plasmid-encoded initiator (Rep) protein to the origin of replication (4). This is followed by nicking of one strand of the DNA within the origin by the Rep protein (5). Replication subsequently initiates by unwinding of the DNA by a helicase and extension synthesis by DNA polymerase III.Plasmids of the pT181 family in Staphylococcus aureus encode a Rep protein that functions as a dimer (6 -9). The RepC protein encoded by pT181 consists of 314 amino acids. Previous studies have identified a carboxyl-terminal domain within RepC that is involved in sequence-specific recognition of the origin. Within this region, amino acids 267-270 of RepC were found to be critical for this activity (10, 11). A more centrally located region of the Rep proteins contains its active tyrosine residue (Tyr-191 in the case of RepC), which is involved in nicking at the origin (12, 13). Mutational analysis has also shown that the DNA binding and nicking activities of RepC can be uncoupled, and both of these activities are required for replication (13). One monomer of the Rep protein becomes covalently linked to the 5Ј end of the initiator-generated nick through a phosphotyrosine bond (5, 12). After the completion of leading strand synthesis, the second free monomer of Rep is expected to cleave the displaced ssDNA and initi...

Bacillus anthracisandBacillus cereusPcrA Helicases Can Support DNA Unwinding and In Vitro Rolling-Circle Replication of Plasmid pT181 ofStaphylococcus aureus

“…Therefore, we tested whether the heterologous helicases can promote replication of plasmid pT181 DNA in vitro. For these studies, cell extracts were prepared from the pcrA3 mutant of S. aureus as described earlier (4,5). The mutant PcrA3 helicase is inactive in plasmid pT181 replication but retains functions required for cell growth and viability (5,12,29).…”

Isolation of a Minireplicon of the Virulence Plasmid pXO2 of Bacillus anthracis and Characterization of the Plasmid-Encoded RepS Replication Protein

“…as described earlier (8). The MBP-RepS protein was purified by chromatography on an amylase affinity column, and the protein was eluted using the above buffer in the presence of 10 mM maltose (8). The purity of the protein was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue.…”

“…Cells were grown in Luria-Bertani broth supplemented with 10 mM glucose at 37°C to the mid-exponential phase, and the MBP-RepS protein was overexpressed by induction with 1 mM isopropyl-␤-Dthiogalactopyranoside (IPTG) at 30°C for 2 h. The cells were lysed by several freeze-thaw cycles in a buffer containing 20 mM Tris-HCl (pH 8), 0.1 mM EDTA, 1 M NaCl, 10% glycerol, and Complete protease inhibitor cocktail tablets (Roche Molecular Biochemicals, Indianapolis, Ind.) as described earlier (8). The MBP-RepS protein was purified by chromatography on an amylase affinity column, and the protein was eluted using the above buffer in the presence of 10 mM maltose (8).…”

Plasmid Rolling-Circle Replication

“…This assumption could be valid for plasmids of the pT181 family, as the sequence-specific DNA binding and DNA relaxation activities of RepC, the initiator of pT181, are mutationally separable and lie on distant protein regions (86). Taking advantage of this property, the identification of the role of individual monomers of pT181-RepC in RCR was addressed by generating heterodimers of the initiator containing a combination of wild-type, DNA binding, and nicking mutants (89). The results demonstrated that a single monomer of RepC is sufficient for origin-specific binding and nicking.…”

“…The results demonstrated that a single monomer of RepC is sufficient for origin-specific binding and nicking. In addition, the monomer involved in sequence-specific binding to the dso must also nick the DNA to initiate replication (89).…”