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J. Biol. Chem., Vol. 277, Issue 48, 45880-45886, November 29, 2002
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,
From the Department of Molecular Genetics and Biochemistry,
University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 15261
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.
Present address: Universidad de Navarra, Dept. Medicina Interna,
Pamplona, Navarra, Spain.
§
To whom correspondence should be addressed: Dept. of Molecular
Genetics and Biochemistry, University of Pittsburgh School of Medicine,
East 1240 Biomedical Science Tower, Pittsburgh, PA 15261. Tel.:
412-648-9025; Fax: 412-624-1401; E-mail: Khan@pitt.edu.
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