MCB 502A-2014. Lecture # 9.Topoisomerases. The replisome.— Topoisomerase I— DNA gyrase— Type I and type II topoisomerases— Bacterial NucleoidReplication factory— The composition of DNA polymerase III— The structure and function of the replication fork in E. coli.— The replisome that moves in opposite directions at the same timeTopoisomerase I-1— James Wang has isolated an enzyme, asingle polypeptide of 110 kD, from E. colithat, when reacted with negativelysupercoiled plasmids, relaxed them.— The first suspicion about relaxation of asupercoiled DNA is, of course, that this isa just a nicking by an endonuclease.— In contrast, a real swivelase should notonly nick the supercoiled DNA, but shouldalso covalently close the molecule whendone with the relaxation.— And this is what Wang has observed ifthe reaction was stopped in the middle.The enzyme relaxed the cccDNA, butincompletely, producing a distribution ofcovalently closed molecules.R-SC-Enzyme – +Run tocompletionJames WangTopoisomerase I-2— This looked like magic — the enzymechanged nothing about the DNA moleculebesides increasing its linking number!— The different bands in this distributionrepresent ccc molecules of the same size,but with different topologocal structures:no supercoils, one supercoil, twosupercoils, three supercoils, etc....— These are called topoisomers, and theenzymes that catalyze transition betweenvarious topoisomers of the same moleculeare called topoisomerases.— Some topoisomerases require energy tocatalyze their reactions, some other do not.In particular, Wang’s enzyme required noenergy.TopoisomersR-SC- – + Enzyme (Topoisomerase)Stopped in the middleTopoisomerase I-3— To learn how the enzyme did its trick Wangadded a powerful peptidase (an enzyme thatdegrades other proteins) to the reaction.— He observed a fast and complete relaxationof the circles, because one of the strands nowcontained a single-strand break.— A-ha, this meant that the protein wasbreaking and re-closing DNA bonds, and Wangcaught it in the middle of this act!— Further studies revealed that the enzymewas reducing negative supercoiling in steps ofone, which was consistent with breaking asingle strand.R-SC-– + Topoisomerase+ + PeptidaseJust startedΔL# = 1Topoisomerase I-4— It was postulated that the enzyme breaks aphosphodiester bond, turns one of the ends of thebroken DNA strand around the other strand, andreseals the break.— Recall that the chemically analogous reactionof DNA ligation requires input of energy.— Since no energy of ATP is spent in thereaction catalyzed by Wang’s enzyme, theenzyme needs to somehow preserve the energy ofthe broken bond.Topoisomerase I-5— Wang has found out how the enzyme did itwhen he added alkali to the ongoing reaction todenature the relaxed DNA.— Not only did he observed denaturation of thetwo strands and their complete separation(because one of the strands was now linearized),but he also found that the denatured protein wasnow dangling from the 5’-end of the linearizedstrand!— Subsequent chemical analysis had shown thatthe enzyme formed a covalent intermediate viaone of its tyrosines with the 5’-phosphate end ofthe break, thus preserving the energy of the bond.Should besupercoiled!5'3'Topoisomerase+ alkaliTopoisomerase I-6— One last reaction that the enzyme was foundto catalyze was knotting of a single DNA strand,suggesting that the enzyme can also operate onssDNA.— However, Wang's enzyme also catalyzedconversion of two individual but complementarycircular strands into a complete circular duplex, areaction that could start only when the twostrands form a short duplex region.— When Wang tried his enzyme with a single-strand DNA in conditions promoting pairing, hefound that knotting was greatly stimulated.— Therefore, the enzyme was binding to duplexregions to promote knotting, and there was nossDNA binding.Topoisomerase I-7— Although originally Wang called hisenzyme a swivelase, to reflect the initialgeneral belief that this activity can relievesupercoiling in front of the replicationfork, eventually he had to abandon thissuggestive terminology.— The act of DNA unwinding by areplication fork should generate positivesupercoils in the duplex region in front ofthe fork, so an enzyme relieving torsionalstress in front of the fork should be able torelax positively-supercoiled DNA.— However, Wang’s swivelase showedabsolutely no activity on DNA containingpositive supercoils and so could not be aswivel at the replication fork.Relaxed DNA(+)sc-DNATopoisomerase I-8— The only supercoiled substrate forWang’s enzyme was negatively-supercoiled DNA, — a somewhatcounterintuitive finding, because thenegative supercoiling of DNA is believedto be actively maintained by the cell.— So, Wang’s enzyme was eventuallysimply called "topoisomerase I", becausemore topoisomerases were found in E.coli.Relaxed DNANegatively-supercolied DNAPositively-supercolied DNATopo ITopo IDNA gyrase-1— The second topoisomerase, discovered in E.coli by Martin Gellert (the co-discoverer ofligase), proved to be very different fromtopoisomerase I.— First of all, it catalyzed the oppositereaction, converting a relaxed or positively-supercoiled cccDNA into a highly negatively-supercoiled one.— Second, it required ATP for this activity(without ATP, the enzyme would relaxnegatively-supercoiled DNA, but notpositively-supercoiled DNA).— Third, the enzyme changed the linkingnumber of DNA in steps of two, rather than insteps of one, as Topo I did.ATP ATPPositivescNegativescNo ATPΔL# = 2Marty GellertGyrBGyrBDNA gyrase-2— Fourth, the enzyme was a heterotetramerof two subunits: two GyrA and two GyrB.— The enzyme was called "Topo II", but ismore frequently called DNA gyrase, whichmeans "revolvase" or "spiralase" in regularEnglish (from "gyrate" — to revolve, tomove in a circle or spiral).— The elucidation of the mechanism ofDNA gyrase action was greatly aided by thethree characteristics of the enzyme:1) the two subunits can be isolated separatelyand then put together in vitro to form a fullyfunctional enzyme;GyrAGyrBGyrBGyrAGyrAGyrAGyrBGyrBDNA gyrase-32) either subunit can be inhibited by aspecific antibiotic (4-quinolone derivativesfor GyrA (NAL, for nalidixic acid),novobiocin derivatives for GyrB (COU, forcoumermycin));3) mutations in gyr genes, conferringresistance to either antibiotic, are readilyisolated, because both
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