The interaction of the F plasmid killer protein, CcdB, with DNA gyrase: induction of DNA cleavage and blocking of transcription.
We have studied the interaction of the F plasmid killer protein CcdB with its intracellular target DNA gyrase. We confirm that CcdB can induce DNA cleavage by gyrase and show that this cleavage reaction requires ATP hydrolysis when the substrate is linear DNA, but is independent of hydrolysis when negatively supercoiled DNA is used. The 64 kDa domain of the gyrase A protein, which can catalyse DNA cleavage in the presence of the B protein and quinolone drugs, is unable to cleave DNA in the presence of CcdB unless the C-terminal 33 kDa domain of the gyrase A protein is also present. CcdB-induced DNA cleavage by gyrase requires a minimum length of DNA (> approximately 160 bp), whereas in the presence of quinolone drugs gyrase can cleave much shorter DNA molecules. We show that CcdB, like quinolones, can form a complex with gyrase which can block transcription by RNA polymerase. A model for the interaction of CcdB with gyrase involving the trapping of a post-strand-passage intermediate is suggested. We conclude that CcdB can stabilise a cleavage complex between DNA gyrase and DNA in a manner distinct from quinolones but, like the quinolone-induced cleavage complex, the CcdB-stabilised complex can also form a barrier to the passage of polymerases.
Critchlow
S E
SE
Department of Biochemistry, University of Leicester, Leicester, LE1 7RH, UK.
O'Dea
M H
MH
Howells
A J
AJ
Couturier
M
M
Gellert
M
M
Maxwell
A
A
D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D003603:Cytotoxins / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D014158:Transcription, Genetic; D014764:Viral Proteins
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068355:CcdA protein, Bacteria; C068356:CcdB protein, Plasmid F; D003603:Cytotoxins; D004279:DNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004250:DNA Topoisomerases, Type II