Analysis of DNA cleavage by reverse gyrase from Sulfolobus shibatae B12.
Reverse gyrase is a type I-5' topoisomerase, which catalyzes a positive DNA supercoiling reaction in vitro. To ascertain how this reaction takes places, we looked at the DNA sequences recognized by reverse gyrase. We used linear DNA fragments of its preferred substrate, the viral SSV1 DNA, which has been shown to be positively supercoiled in vivo. The Sulfolobus shibatae B12 strain, an SSV1 virus host, was chosen for production of reverse gyrase. This naturally occurring system (SSV1 DNA-S. shibatae reverse gyrase) allowed us to determine which SSV1 DNA sequences are bound and cleaved by the enzyme with particularly high selectivity. We show that the presence of ATP decreases the number of cleaved complexes obtained whereas the non-hydrolyzable ATP analog adenosine 5'-[beta, gamma-imido]triphosphate increases it without changing the sequence specificity.
Jaxel
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C
Laboratoire d'Enzymologie des Acides Nucléiques, Université Paris Sud, France. jaxel@dsvidf.cea.fr
Duguet
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Nadal
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004278:DNA, Superhelical / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*
D004278:DNA, Superhelical; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D019286:Endopeptidase K; C041813:DNA reverse gyrase; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II