Abstract
A large number of plasmids have been shown to replicate by a rolling circle (RC) mechanism. The initiators encoded by these plasmids have origin-specific, nicking-closing activity that is required for the initiation and termination of RC replication. Since the initiators of many RC plasmids are rate-limiting for replication, these proteins are usually inactivated after supporting one round of replication. In the case of the pT181 plasmid, inactivation of the initiator RepC protein occurs by the attachment of an oligonucleotide to its active tyrosine residue. We have generated the inactivated form of RepC, termed RepC*, in vitro and investigated the effects of attachment of the oligonucleotide on its various biochemical activities. Our results demonstrate that while RepC* is inactive in nicking-closing and replication activities due to the blockage of its active tyrosine residue, it is competent in origin DNA binding and DNA religation activities. We have investigated the oligomeric state of RepC and RepC* and found that RepC exists as a dimer in solution and can oligomerize on the DNA. We have generated heterodimers in vitro between the wild-type and epitope-tagged RepC proteins. In electrophoretic mobility shift experiments, the initiator heterodimers generated a novel DNA-protein complex, demonstrating that it binds to DNA as a dimer. We have shown that a DNA binding mutant of RepC can be targeted to the origin in the presence of the wild-type protein primarily through a protein-protein interaction. Interestingly, RepC* is defective in its ability to oligomerize on the DNA. RepC* inhibited the DNA binding and replication activity of wild-type RepC to only a very limited extent, suggesting that it may play only a minor regulatory role in replication in vivo. Based on these and earlier results, we propose a model for the role of RepC during the initiation and termination of pT181 RC replication.
Highlights
Bacterial plasmids provide useful model systems to study the mechanism and regulation of DNA replication
To prevent the reutilization of an initiator molecule that has been used for a round of replication, RepC is inactivated by the attachment of an approximately 11-nt-long oligonucleotide corresponding to pT181 positions 70 – 60 located immediately downstream of the initiator nick site [26, 27]
Preparations of the initiator protein from cells containing either wild-type pT181 or its copy mutants contain an approximately 1:1 mixture of the RepC and RepC* forms [27, 28]. Based on this and other findings, it has been proposed that RepC exists as a dimer in solution and that attachment of the oligonucleotide to one subunit results in the inactivation of the other, wild-type subunit by an as yet unknown mechanism (25, polyacrylamide gel electrophoresis; bp, base pair(s); electrophoretic mobility shift assays (EMSAs), electrophoretic mobility shift assay
Summary
Purification of RepC and MBP-RepC Proteins—Wild-type RepC protein was purified using an overexpression system as described earlier [16]. One microgram of supercoiled pT181cop608 DNA was incubated with various amounts of the RepC or RepC* proteins at 32 °C for 30 min in TEKEM buffer, and the reaction mixtures were subjected to electrophoresis on 0.7% agarose gels using TBE (Tris borate-EDTA) buffer containing 1 g/ml ethidium bromide. 5Ј-end-labeled oligonucleotides (pT181 nt 83– 60 containing the IRII and nt 75– 60 representing the right arm and loop of IRII (Fig. 1) were incubated with 200 ng of the RepC or RepC* proteins for 30 min at 32 °C in TEKEM buffer in a reaction volume of 10 l. Reaction mixtures (20 l) containing the 32P-labeled oligonucleotides (2–5 pmol), 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 1 mM EDTA, 5 mM dithiothreitol, 10% ethylene glycol, 100 ng of poly(dI-dC), and the indicated amounts of the initiator proteins were incubated at room temperature for 10 min and electrophoresed on native polyacrylamide gels using TBE buffer. Fractions were assayed for the topoisomerase activity of RepC as described above
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