Biochemical characterization of the Staphylococcus aureus PcrA helicase and its role in plasmid rolling circle replication.
Previous genetic studies have suggested that a putative chromosome-encoded helicase, PcrA, is required for the rolling circle replication of plasmid pT181 in Staphylococcus aureus. We have overexpressed and purified the staphylococcal PcrA protein and studied its biochemical properties in vitro. Purified PcrA helicase supported the in vitro replication of plasmid pT181. It had ATPase activity that was stimulated in the presence of single-stranded DNA. Unlike many replicative helicases, PcrA was highly active as a 5' --> 3' helicase and had a weaker 3' --> 5' helicase activity. The RepC initiator protein encoded by pT181 nicks at the origin of replication and becomes covalently attached to the 5' end of the DNA. The 3' OH end at the nick then serves as a primer for displacement synthesis. PcrA helicase showed an origin-specific unwinding activity with supercoiled plasmid pT181 DNA that had been nicked at the origin by RepC. We also provide direct evidence for a protein-protein interaction between PcrA and RepC proteins. Our results are consistent with a model in which the PcrA helicase is targeted to the pT181 origin through a protein-protein interaction with RepC and facilitates the movement of the replisome by initiating unwinding from the RepC-generated nick.
Chang
Tseh-Ling
TL
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Naqvi
Asma
A
Anand
Syam P
SP
Kramer
M Gabriela
MG
Munshi
Rajan
R
Khan
Saleem A
SA
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D001483:Base Sequence; D004265:DNA Helicases; D017931:DNA Primers; D004261:DNA Replication / Q000502:physiology*; D004269:DNA, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D013211:Staphylococcus aureus / Q000201:enzymology*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; C043650:RepC protein, Staphylococcus aureus; C084217:pcrA protein, Bacteria; D004265:DNA Helicases