Previous genetic studies have suggested that a putative chromosome-encoded helicase, PcrA, is required for the rolling circle replication of plasmid pT181 in Staphylococcus aureus. We have overexpressed and purified the staphylococcal PcrA protein and studied its biochemical properties in vitro. Purified PcrA helicase supported the in vitro replication of plasmid pT181. It had ATPase activity that was stimulated in the presence of single-stranded DNA. Unlike many replicative helicases, PcrA was highly active as a 5 3 3 helicase and had a weaker 3 3 5 helicase activity. The RepC initiator protein encoded by pT181 nicks at the origin of replication and becomes covalently attached to the 5 end of the DNA. The 3 OH end at the nick then serves as a primer for displacement synthesis. PcrA helicase showed an origin-specific unwinding activity with supercoiled plasmid pT181 DNA that had been nicked at the origin by RepC. We also provide direct evidence for a proteinprotein interaction between PcrA and RepC proteins. Our results are consistent with a model in which the PcrA helicase is targeted to the pT181 origin through a protein-protein interaction with RepC and facilitates the movement of the replisome by initiating unwinding from the RepC-generated nick.

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...

Oenothera plants homozygous for a recessive allele at the plastome mutator (pm) locus show non-Mendelian mutation frequencies that are 1000-fold higher than spontaneous levels. Characterization of RFLP sites in a collection of mutants indicates that insertion-deletion hot spots in the pm lines are defined by tandem direct repeats, implicating replication slippage or misalignment during recombination. Several sites known to contain very short direct repeats were examined, and all were found to have been targeted in one or more plants of the mutant collection. To determine if replication slippage was occurring, two oligo-A stretches in non-coding DNA were examined, and 3 of 12 plants were found to have an additional adenine in a 13-base track. To search for other mutations that would not be visible as restriction fragment length polymorphisms, PCR-amplification products of the psbB gene were digested with a restriction endonuclease, denatured, and examined for single-strand conformational polymorphisms. Among 21 mutants, one 4-bp insertion and one point mutation were identified in psbB. The discovery that the plastome mutator can cause base substitutions as well as repeat-mediated insertions and deletions points to a likely defect in a component of the cpDNA replication machinery.