The Inactive pT181 Initiator Heterodimer, RepC/C, Binds but Fails to Induce Melting of the Plasmid Replication Origin

Abstract

Staphylococcus aureus plasmid pT181 replicates via a rolling circle mechanism. The synthesis of the pT181 initiator protein (RepC) is regulated by antisense RNAs, and RepC is inactivated after usage by the attachment of an oligonucleotide to one of its subunits. The inactivated heterodimeric RepC/C* has been shown be unable to initiate replication in vitro (Rasooly, A., and Novick, R. P. (1993) Science 262, 1048 -1050). The inactive RepC/C* has been found to be very stable and constitute about 90 -95% of the total RepC antigen inside the cell. We studied the specific interaction of the RepC/C and RepC/C* complex with the pT181 double strand origin. The results indicated that RepC/C and RepC/C* footprint supercoiled DNA differently although their footprints on linear DNA are similar; we also find that RepC/C is able to enhance cruciform extrusion while RepC/C* cannot. RepC/C* binds and bends the double strand origin much more weakly than does RepC/C. These results suggest that the attached oligonucleotide induces a conformational change in the RepC/C* molecule that is responsible for its lack of activity.All known bacterial plasmids encode a specific initiator and control their replication primarily by negatively regulating its synthesis. Often, the linkage between replication and the control of initiator synthesis is indirect in that the regulatory factors act in trans and respond to the copy number of the plasmid. This type of replication control requires that the initiator be utilized stoichiometrically rather than catalytically since the latter would obviate such control. Plasmids such as pT181 and its relatives, which replicate by the rolling circle mechanism, inactivate the initiator protein automatically as a part of the termination process (1). ColE1 and its relatives, which replicate by the mechanism, utilize a long RNA primer as initiator and automatically degrade this primer as replication proceeds. Plasmids such as R1, which also replicate by the mechanism, require new protein synthesis for each round of replication (2), suggesting that the initiator is inactivated after use.The inactivation mechanism for pT181 is unique; the initiator protein, RepC, is a dimer, referred to as RepC/C, and following the completion of a round of replication, an oligonucleotide, representing sequences immediately 3Ј to the initiation nicking site is attached to the active site tyrosine of one of its subunits (1). Our initial experiments suggested that the heterodimeric derivative, RepC/C*, lacked the nicking, relaxation, and replication activities of the native protein. This was puzzling. Since only one of the two subunits was modified, the unmodified subunit would be expected, a priori, to have nicking activity, the reversal of which would result in relaxation, and to be able to initiate but not terminate replication. Testing of these possibilities was complicated by the fact that preparations of total RepC antigen from pT181-containing cells generally consist of 90 -95%

An Oligonucleotide Inhibits Oligomerization of a Rolling Circle Initiator Protein at the pT181 Origin of Replication

“…7). The above results are consistent with the previous observation that RepC binds to both the IRIII and IRII regions in supercoiled DNA, whereas RepC/RepC* binds only to the IRIII region (29). The inability of RepC/RepC* to bind to IRII and oligomerize on the DNA may result in a lack of melting of the pT181 origin involving IRII, which is critical for nicking by the initiator protein.…”

“…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 (25,27). Recently, the RepC/RepC* heterodimer isolated from pT181-containing cells was found to retain its DNA binding activity but was defective in its ability to induce cruciform extrusion at the origin (25,28,29). Since preparations of RepC* from pT181-containing cells always contain a 1:1 mixture of RepC and RepC* forms, we have generated RepC* in vitro.…”

In vitro inhibitory activity of RepC/C*, the inactivated form of the pT181 plasmid initiation protein, RepC

“…As can be seen, the retarded RepC/C* band in lane 3 is much weaker than the RepC/C-DNA band in lane 1, indicating that the affinity of RepC/C* for DNA is less than 50% of that of RepC/C. We have calculated that the binding constants of the two forms actually differ by a factor of about 4 (10).…”

Plasmid Rolling-Circle Replication

“…Thus, the second transesterification allows completion of the termination process by avoiding recycling, i.e., continuous synthesis of the leading strand driven by the same initiator molecule (117). Analysis of the different interaction patterns of RepC/C and RepC/C* with the pT181-dso DNA by in vitro footprinting and binding-bending assays revealed that, although RepC/C* retains the ability to bind to DNA, it is unable to promote cruciform extrusion to expose the nick sequence in ssDNA form (118). This may explain why the formation of a RepC/ RepC* heterodimer inactivates the protein in addition to uncoupling termination of leading-strand replication and initiation of a new replicative round.…”