MMB vol.095 Osheroff, N. Bjornsti M.-A. (eds.) DNA Topoisomerase Vol.II Enzymology and Drugs (Humana Press, 2001)(ISBN 0896035123)(329s)

MMB vol.095 Osheroff, N. Bjornsti M.-A. (eds.) DNA Topoisomerase Vol.II Enzymology and Drugs (Humana Press, 2001)(ISBN 0896035123)(329s)

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页数:329页

时间:2019-07-20

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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,

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