The replication initiator protein of plasmid pT181 has sequence-specific endonuclease and topoisomerase-like activities.

Abstract

Initiation of pT181 DNA replication specifically requires the plasmid-encoded RepC protein. Here we demonstrate that highly purified RepC protein has sequencespecific endonuclease and topoisomerase-like activities. A maximum sequence of 127 base pairs containing the pT181 origin of replication is required for nicking-closing by RepC protein.RepC introduces a single strand break within the pT181 origin. The nick site has been shown by DNA sequencing to lie between nucleotides 70 and 71 in the bottom strand of the DNA within the origin sequence. This nick site probably corresponds to the start site of pT11 replication. The results presented here suggest that, unlike most other plasmids, pT181 replicates by a rolling circle mechanism.

Sequence requirements for the termination of rolling‐circle replication of plasmid pT181

“…This has also been observed previously in our laboratory (Zhao and Khan, 1996). It has been shown that RepC nicks at the origin between nt 70 and 71, and a 70 bp region (pT181 nt 31-101) is required for optimal origin function, including its ability to compete with the wildtype origin in vivo (Koepsel et al, 1985b;Gennaro et al, 1989). In the in vitro and in vivo experiments, the presence of a large SS DNA or a miniplasmid represents the product of termination whereas the unit-length SS DNA or plasmid represents read-through replication past the mutant origin.…”

“…The initiator proteins encoded by the RC plasmids and bacteriophages have an origin-specific nicking-closing activity (Koepsel et al, 1985b;Baas and Jansz, 1988). The free 3Ј OH end generated at the nick is used as a primer for replication of the leading strand of the DNA.…”

“…The active amino acid required for nicking at the origin corresponds to the tyrosine residue at position 191 of RepC (Dempsey et al, 1992b;Thomas et al, 1990). After nicking, Tyr-191 becomes covalently linked to the 5Ј phosphate of the DNA at the nick site (Koepsel et al, 1985b;Thomas et al, 1988). The origin of replication of pT181 contains three sets of inverted repeat (IR) elements, namely IRI, IRII and IRIII (Fig.…”

“…It is also known that binding of RepC at the origin bends the DNA and extrudes the IRII cruciform in supercoiled DNA Noirot et al, 1990). The RepC-cleavage site is located within the loop of IRII between pT181 nt 70 and 71 (Koepsel et al, 1985b). Previous studies have shown that both IRII and IRIII regions are required for the initiation of pT181 replication (Gennaro et al, 1989;Zhao and Khan, 1996).…”

Geminivirus replication proteins are related to prokaryotic plasmid rolling circle DNA replication initiator proteins

“…In all well explored systems these proteins possess a DNA nicking~losing and a topoisomerase-like activity (e.g. Koepsel et al, 1985 ;de la Campa et al, 1990). In phage ~bX174, by far the best studied RCR system, phage A protein nicks the ori site in the viral strand of the double-stranded replicative form and remains covalently linked to the 5' side of the nick.…”