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1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMVOLUME95DNADNATOPOISOMERASETOPOISOMERASEPROTOCOLSPROTOCOLSEnzymologyandDrugsEnzymologyandDrugsEditedbyEditedbyNeilOsheroffNeilOsheroffandandMary-AnnBjornstiMary-AnnBjornstiHUMANAPRESSRelaxationActivity11AssayingDNATopoisomeraseIR
2、elaxationActivityLanceStewartandJamesJ.Champoux1.IntroductionTypeItopoisomerasescatalyzetopologicalchangesinduplexDNAbyreversiblynickingonestrand,whereastypeIIenzymescatalyzethetransientbreakageofbothstrandssimultaneously.ThetypeIenzymesalterthelinkingnumberofcovalentlyclosedcircu
3、larDNAinstepsofone,presumablybyallowinganunbrokensegmentofonestrandoftheDNAtomovethroughthetransientsingle-strandbreakintheotherstrand.ThetypeIIenzymesalterthelinkingnumberinstepsoftwobyallowinganunbrokensegmentofduplexDNAtopassthroughthetransientdouble-strandbreak.Alltopoisomeras
4、esconservephosphodiesterbondenergyduringcatalysisbytransientlyformingaphosphotyrosinebondbetweentheactive-sitetyrosineresidueandthephosphateatoneendofthebrokenstrand(s)(1).ThetypeItopoisomerasesfallintotwocategoriesdependingonthepolarityoftheircovalentattachment.Thecellularandvira
5、leukaryotictopoisomeraseIenzymes(2)andthearchebacterialtopoisomeraseV(3)areclassifiedastypeI-3',sincetheybecomelinkedtothe3'-endofthebrokenstrand(1).Theseenzymesalsohavethedistinctivecharacteristicthattheycanrelaxbothpositivelyandnega-tivelysupercoiledDNAintheabsenceofanenergycofa
6、ctorordivalentmetalcation(2).TheothertypeIenzymes—topoisomerasesIandIIIofprokaryotes(1,4,5),reversegyraseandtopoisomeraseIIIofarchebacteria(6–9),andeukaryotictopoisomeraseIII(10)—becomelinkedtothe5'-endofthebrokenstrandandareclassifiedastypeI-5'.TheseenzymesrequireMg2+foractivitya
7、ndcanonlyrelaxnegativelysupercoiledDNA.ThetypeIItopoisomerases(seeChapters2and3)—whethervirallyencodedorisolatedfromarchebacteria,eubacteria,oreukaryotes—invariablyrequirebothATPandMg2+foractivityandbecomelinkedtothe5'-endofthebrokenstrands(1,11,12).From:MethodsinMolecularBiology,
8、Vol.95:DNATopoisomeraseProtocols,