Abstract
The replication of pT181 and related plasmids of Staphylococcus aureus proceeds by a rolling circle mechanisms. The initiator proteins encoded by the plasmids of the pT181 family have sequence-specific DNA binding and topoisomerase activities. These proteins nick one strand of the DNA at the origin of replication. The free 3'-hydroxyl end at the nick is then used as a primer for the replication of the leading strand of the DNA. Although these initiator proteins are highly homologous, they show specificity in DNA binding and replication for their cognate DNAs. In this study, we have generated hybrid initiator proteins and studied their various biochemical activities in vitro. Our results show that 6 amino acids are sufficient to determine the DNA binding and replication specificities of such initiator proteins.
Highlights
The replication of p T l 8 l and related plasmids of staphy~ococcu8aureus proceeds bya rollingcircle mechanism
The plasmids of the pT181 family of Staphylococcus aureus include pT181, pC221,pC223,pS194,pCW7, and pUB112. They range in size from 4.2 to 4.6 kilobases, have extensive nucleotide sequence homology in their replication regions, and have similar functional organization (Projan and Novick, 1988; Novick,1989).Most of the replication initiator proteins encoded by these plasmids consist of 314 amino acids
The initiators encoded by the plasmids of the pT181 family are capable of nicking/closing all such plasmids due to the presence of a conserved tyrosine residue that is involved in this activity and an identical nucleotide sequence at thenick site within the origin (Projan and Novick, 1988, Thomas et al, 1990; Zock et al, 1990). these initiator proteins have -76% overall amino acid sequence identity, they show specificity in DNA binding and replication for their cognate DNAs (Projan et al, 1985; Thomas et al, 1990; Zock et al, 1990)
Summary
The proteins were overexpressed in E. coli and purified to region of significant amino acid divergencein these initiators is located in their COOH-terminal region (residues 238-282 of RepC) Within this region, amino acids 267-270 are very different for five of the six initiators sequenced (Fig. lA). The arginine residue at position 266 is conserved among the pT181, pC221, and pS194 initiators, butthe histidine residue at position 265 in RepC is replaced by a glutamic acid in RepD and RepE to the pT181, pC221, and pS194 origins was studied by gel mobility (Fig. lA). This glutamic acid residue was included in the shift assays (Varshavsky, 1987).
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