Uncoupling of the DNA topoisomerase and replication activities of an initiator protein.

Abstract

The replication initiator proteins encoded by the pT181 and related plasmids have sequence-specific DNA binding and topoisomerase activities. These proteins create a site-specific nick in one strand of the DNA at the origin of replication that serves as a primer for the initiation of replication. To define the regions of the pT181-encoded initiator protein, RepC, that are involved in its DNA binding, topoisomerase, and replication activities, we have carried out site-directed mutagenesis of the repC gene. Analysis of mutant RepC proteins in vitro and in vivo has identified the amino acids that are critical for its various biochemical activities. The DNA binding domain of RepC was found to be located near its C-terminal region and was different from the domain involved in its sequence-specific topoisomerase activity. These studies also showed that the DNA topoisomerase activity ofthe initiator protein can be uncoupled from its tight noncovalent DNA binding and replication activities.Plasmid pT181 is a prototype of a family of small, multicopy staphylococcal plasmids that replicate by a rolling-circle mechanism (1, 2). The plasmids of this family include pT181, pC221, pC223, pS194, pCW7, and pUB112, range in size from 4.2 to 4.6 kilobase pairs (kb), and have extensive sequence homology in their replication regions. These plasmids encode replication initiator proteins with 76% overall amino acid sequence identity (1). The initiators encoded by pT181 and pC221 (RepC and RepD, respectively) have been purified and shown to have origin-specific DNA nicking-closing activities (3, 4). These activities resemble those of CisA protein of 4X174, gene 2 proteins of the filamentous phages of Escherichia coli, and type I DNA topoisomerases (5-7). These initiator proteins nick one strand ofthe DNA at a specific site, and the free 3' OH end serves as a primer replication by an asymmetric rolling-circle mechanism (2, 8).The RepC protein consists of 314 amino acids. It binds to a 32-base-pair (bp) sequence located between positions 37 and 68 and nicks the bottom strand of the DNA between nucleotides 70 and 71 within the pT181 origin of replication (3,(9)(10)(11). Genetic studies have suggested that a divergent amino acid sequence located near the C-terminal region ofthe initiator proteins of the pT181 family is involved in their replication activities (1,(12)(13)(14)

An 18-base-pair sequence is sufficient for termination of rolling-circle replication of plasmid pT181

“…The binding of the RepC initiator protein to the pT181 origin was studied by gel mobility shift assays as described previously (5). The wild-type pT181 origin and the 18-bp subregion of IRII were cloned into the multiple cloning site of pUC19.…”

“…1). Subsequent studies showed that initiation of pT181 replication requires stable, sequence-specific binding of RepC to the origin (4,5). We performed gel mobility shift assays to test the requirement for RepC-origin interaction during the termination of replication.…”

“…The DNA binding domain of the initiators of the pT181 family has been identified, and amino acids corresponding to 265 to 270 in RepC were found to be critical in determining their DNA binding specificity (4). The active tyrosine residue involved in the nicking activity of the initiators of pT181-type plasmids corresponds to position 191 in RepC (5,30).…”

Plasmid Rolling-Circle Replication

“…A modular structure based on the presence of at least two domains involved in origin nicking and specific recognition of the dso sequence has been assumed to be a general feature of the initiator proteins of RCR plasmids (85)(86)(87)(88). 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).…”

“…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).…”