Locking the DNA gate of DNA gyrase: investigating the effects on DNA cleavage and ATP hydrolysis.
Supercoiling by DNA gyrase involves the passage of one segment of double-stranded DNA through another. This requires a DNA duplex to be cleaved and the broken ends separated by at least 20 A. This is accomplished by the opening of a dimer interface, termed the DNA gate, which is covalently attached to the broken ends of the DNA. After strand passage, the DNA gate closes allowing the reunion of the broken ends. We have cross-linked the DNA gate of gyrase using cysteine cross-linking to block gate opening. We show that this locked gate mutant can bind quinolone drugs and perform DNA cleavage. However, locking the DNA gate prevents strand passage and the ability of DNA to stimulate ATP hydrolysis. We discuss the mechanistic implications of these results.
Williams
N L
NL
Department of Biochemistry, University of Leicester, UK.
Maxwell
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000378:metabolism / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D015363:Quinolones / Q000378:metabolism / Q000494:pharmacology; D059005:Topoisomerase II Inhibitors
D003432:Cross-Linking Reagents; D004278:DNA, Superhelical; D004220:Disulfides; D015363:Quinolones; D059005:Topoisomerase II Inhibitors; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II; D003545:Cysteine; D002118:Calcium