Competition of bacteriophage polypeptides with native replicase proteins for binding to the DNA sliding clamp reveals a novel mechanism for DNA replication arrest in Staphylococcus aureus.
Bacteriophages have evolved specific mechanisms that redirect bacterial metabolic pathways to the bacteriophage reproduction cycle. In this study, we characterized the bactericidal mechanism of two polypeptides from bacteriophages Twort and G1 that target the DNA sliding clamp of Staphylococcus aureus. The DNA sliding clamp, which tethers DNA polymerase to its template and thereby confers processivity upon the enzyme, was found to be essential for the viability of S. aureus. Expression of polypeptides TwortORF168 and G1ORF240 in S. aureus selectively inhibited DNA replication which in turn resulted in cell death. Both polypeptides specifically inhibited the S. aureus DNA replicase that was reconstituted in vitro but not the corresponding replicase of Streptococcus pyogenes. We demonstrated that inhibition of DNA synthesis is multifaceted and occurs via binding the DNA sliding clamp: TwortORF168 and G1ORF240 bound tightly to the DNA sliding clamp and prevented both its loading onto DNA and its interaction with DNA polymerase C. These results elucidate the impact of bacteriophage polypeptide expression upon DNA replication in the growing cell.
Belley
Adam
A
Targanta Therapeutics, St-Laurent, QC, Canada.
Callejo
Mario
M
Arhin
Francis
F
Dehbi
Mohammed
M
Fadhil
Ibtihal
I
Liu
Jing
J
McKay
Geoffrey
G
Srikumar
Ramakrishnan
R
Bauda
Pascale
P
Bergeron
Dominique
D
Ha
Nhuan
N
Dubow
Michael
M
Gros
Philippe
P
Pelletier
Jerry
J
Moeck
Greg
G
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001667:Binding, Competitive; D004258:DNA Polymerase III / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D008969:Molecular Sequence Data; D013211:Staphylococcus aureus / Q000235:genetics / Q000821:virology*; D017108:Streptococcus Phages / Q000502:physiology*; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D014764:Viral Proteins; C023140:DNA replicase; C446929:PolC protein, bacteria; C101139:beta subunit, DNA polymerase III; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase