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![passage bytopoisomerase II.Nevertheless,itstill needstobe confirmedbyfurtherexperiments,due to
the difficultiesthatlie inthe interpretationof currentexperimentalandcomputational results.
Asthe article explains,the individual modelsabove are useful inthattheycanrationalize “certain
aspectsof type-2topoisomerase actions”,butnone of themare able to comprehensivelyexplainall the
data that scientistshave gatheredthusfaraboutthe enzyme (Liu,Diebler,Chan,&Zechiedrich,2009).
The Juxtaposition-Centric Approach
The article proposesthatthe reasonmanyof the aforementionedmodelsfail tobe comprehensive
isthat theyignore an importantfactor(Buck& Zechiedrich,2004):the role of local information
embeddedinthe substrate of type-2topoisomerase indiscriminatingwhetherDNA strandsare linkedor
unlinkedglobally,giventhe factthattopoisomerase IIissmallerthana DNA molecule inordersof
magnitudes.
To understandthis,atheorycalledjuxtaposition-centricapproachhasbeenputforward.A DNA-
DNA juxtapositionisgeneratedwhentwosegmentsof DNA molecules,be itonthe same strand or on
the differentones,come close enoughsothatthe difference betweenthemisaboutsmallerthanthe
diameterof a type-2topoisomerase molecule[37] (Buck& Zechiedrich,2004). Onlyinthissmall distance
can a topoisomerase IIfunctiontograspbothstrandsto decatenate ordeknot(Bruce,Johnson,Lewis,
Raff,Roberts& Walter,2008). One may come up withtwoassumptionsonjuxtaposition:one isthatall
existinghelix-helix juxtapositionsare the same,regardlesswhetherthe twosegmentsare unlinkedorin
a catenationor a knot;anotherone is that differentjuxtapositionisformedwhenachainissupercoiled,
linkedorknotted,comparedtowhen itislinear,relaxedorunknotted(Buck&Zechiedrich,2004).Of
the laterassumption,basically,there are three typesof juxtapositionproposed:hooked,half-hooked
and free (Liu,Zechiedrich,&Chan,2006a).
Everytime whenasegmentpassage isopenedbytopoisomeraseIIona specificjuxtapositioncan
eitherresultinanunlinkedstatusof DNA standsora linkedone.Lattice conformationalenumeration
demonstratedbyZhirongLiuandetal. has suggestthatthere is90% of chance that globally unlinked
DNA strands become linkedwhenasegmentpassage isopenedonafree juxtaposition,whereas85%of
probabilitythatthe strandsbecome unlinkedonahookedjuxtaposition(Liu,Zechiedrich,&Chan,
2006b). Additionally,the enumerationwasalsousedtosimulate the realisticinteractionsoccurring
whena segmentpassage isopenedoneachjuxtapositionmediatedbytopoisomerase II.
Inreality,atopological equilibriumisestablishedbetweenthe populationof catenanes orknotsand
unlinkedDNA segments.However,the equilibriumwill shiftinpresence of topoisomerase II.The result
isillustratedbyincorporatingtwonewfactors,whichare linkreductionfactor(RL) andknotreduction
factor(RK) (Liu,Zechiedrich,&Chan,2006a). Justas theirname implies,the higherthe value of RLand
RK is,the more probable itisthat the equilibriumwillbe shiftedtowardsthe decatenatingordeknotting
side,andvice versa.Furthermore,RLandRK are bothchain length(n) dependent,withahigh
probabilityof unlinkingforhookedandhalf-hookedjuxtaposition(linkingforfree juxtaposition) atlow
value of n, andlowprobabilityathighvalue of n.A relatedstudyhasalsoshownthat the unknotting
potential (indicatedbyRK) isalsoinfluencedbythe stiffnessof the chain(Liu,Diebler,Chan,&
Zechiedrich,2009),withthe effectenhancingasthe stiffnessincreases(exceptforfree juxtaposition).
AnotherresearchalsoshowsthatDNA supercoilingmayimprovethe efficiencyindecatenating,butnot
indeknotting(Buck&Zechiedrich,2004).](https://image.slidesharecdn.com/37240257-3d12-4e79-aa2f-0360e67eb421-160902192020/75/THE-WHY-AND-HOW-OF-DNA-UNLINKING-3-2048.jpg)
THE WHY AND HOW OF DNA UNLINKING
- 1. THE WHY ANDHOW OF DNA UNLINKING Liangkai Hu & Priyanka Soundranayagam Summary The momentWatsonandCrick revealedtheirhypothesisforthe structure of the DNA,itwas immediatelyevidenthowtheirparticularstructure wouldlenditself towardsa“functional elegance” (Liu,Diebler,Chan,&Zechiedrich,2009).The single strandsof DNA that create the anti-paralleldouble helix eachserve asa template thatcan be replicatedtocreate anidentical double helix.Scientists followingthisdiscoveryhave largelyfocusedonthe base sequencingof the strandsthemselvesasthe mainfactors that affectthe biological processeswithinthe cell.Asexplainedbythe article putforthby Liu,Deibler,ChanandZechiedrich,thisisanerroneousassumption,inthat“itis now clearthat the DNA itself playsanactive role”inthose biological processesaswell.Specifically,the topologyof the DNA,and the misregulationthereincanplayan active role in“regulatingthe abilityof the cell toextractits information”(Liu,Diebler,Chan,&Zechiedrich,2009). The article firstexplainshowthe three commontopological misregulations(supercoiling,catenation and knotting) affectthe functionalityof cells.Itthenputsforthseveral modelsthatattempttoexplain howthe cellsthemselvespreventthese misregulationsfrombeingtooharmful.The article concludes witha discussionof howthe DNA’stopological problemsthemselvessignal type-2topoisomerasesinto resolvingthose verysame problems. Topological Conformations of DNA Supercoiling Supercoilingisthe processbywhich DNA moleculestwisttorelieve the stresscausedbytheir helical shape.Negativesupercoiling,specifically,allowsforthe maintenance of the DNA ina “homeostaticallyunderwoundstate”byeithertwistingorwrithing(Liu,Diebler,Chan,&Zechiedrich, 2009). This particulartype of supercoiling,asexplainedinthe article,isessentialtothe functionalityof the cell. Firstly,negative supercoilingisresponsible forthe “controlledmeltingof the DNA duplex”,whichis the onlymethodbywhich DNA polymerases,RNA polymerasesandotherenzymescanaccessthe nucleotide sequence of eachstrand.Supercoilingalsoallowsforthe “synapsisof distance sites…witha definedspatialarrangementandorientation”.Thirdly,the chromosomal metabolismdependson negative supercoilingtoallowfora“precise meansof regulatingDNA metabolism”.The article also claimsthat “the control of DNA supercoilingcanhave importantconsequencesintermsof evolutionary fitness”,whichwouldthensuggestevolution favoursincreasednegative supercoilingincells.Finally,the mostnoteworthyaspectof negative supercoiling(assuggestedbythe article),isthatevenatrace amountof increase innegative supercoilingwill enhancethe expressionof specificgenes(suchasthe λ Int protein) significantly(Liu,Diebler,Chan,&Zechiedrich,2009). There are alsodisadvantagestothisparticulartopological conformation,asexcessive negative supercoilingcanleadto “an inhibitionof bacterial growthandRNA degradation”.The article emphasizes however,thatwhetherthe resultsare positiveornegative,itisneverthelessimportantforthe cell that supercoilingdoesinfactoccur.
- 2. Catenation Catenatesare newlyreplicated,intertwinedDNAs,whichare right-handedparallelinshape.Type-2 topisomerasesareresponsiblefor“cleaving”the DNA strandsof these catenates,“passinganother duplex throughthe transientgate”andthenresealingthe cleavage the gaptheycreated.Thisprocess, called“decatenation”,isresponsible forunliking“catenatedintermediatesof DNA replication”(Liu, Diebler,Chan,&Zechiedrich,2009).WhenexperimentingonE.coli,itwas foundthatabsence of this type-2topoisomeraseimpairedthe separationof replicatedgenetic loci.Inyeast,itleadsto“impaired chromosome segregationandcell deathatcytokinesis”.These resultswouldsuggestthatlike supercoiling,the presence of catenates(andthe subsequentdecatenationcarriedoutbytype-2 topoisomerases) canaffectthe functionalityof DNA,genome stabilityanddevelopment(Liu,Diebler, Chan,& Zechiedrich,2009). Knotting KnotsinDNA that resultfroma DNA molecule tangledwithitself canbe evenmore detrimental for cell functionalitythancatenates.Fortunately,the presence of type-2topoisomerases,the organization of DNA intonucleosomesandDNA supercoilingensurethatthe DNA staysunknottedina cell.If these factors were notinplace,the resultingincrease inknotting“blockedDNA replicationand transcription, increasedmutation,andledtolossof the replicon”.The article proposesalsothatknots“preventsthe facile returnof differentiatednuclei tothe cell cycle”.Ascanbe seen,thislastconformationof DNA has a noteworthyeffectonthe abilityof the cell tocarry out itsbiological processes(Liu,Diebler,Chan,& Zechiedrich,2009). All three of the potential topological conformations(supercoiling,catenationandknotting),ashas beenprovenbythe article thusfar,are as importantto the abilityof cellstocarry out DNA replicationas are the base sequencesof singlestrands. Models Depicting the Disentanglement Action Carried Out by Type-2 Topoisomerases A viable theoryforhowthe type-2topoisomerasefunctionsclaimsthatthe enzyme isable to recognize thathelix-helix juxtapositionsoccurmore frequentlyinDNA thathaseitherbeensupercoiled, catenatedor knotted.Severalmodelshave beencreatedtodeterminethe accuracyof this theory. The firstmodel suggestedthatDNA juxtapositionswere “phantomchains” –inother words,the helix-helix juxtapositionsweresuchthattheyallowedforone chaintofreelypassthroughitself orother chains.Thismodel,however,wasdisprovenwhenitwasshownthat type-2topoisomerases“donot turn DNA intophantomchains”.A secondmodel suggestedthatthe type-2topoisomerasesmight “activelyslide alongthe DNA totrapthe catenane orknot nodesandreduce the effectivesize of the DNA”.Again,thismodel wasabandoned(Liu,Diebler,Chan,&Zechiedrich,2009). The thirdhypothesizedmodelwasthe kineticproof-readingmodel,inwhichcollisionsbetween sequentialtype-2topoisomerase-DNA rationalizedsome of the evidenceof unknottingandunlinking scientistshave had.However,the model wasinconsistentwithexperimentaldatainitsproposed suppressionfactor.The nextmodel,the “active bendingmodel”,claimedthatthe type-2 topoisomerases“activelyuntangle DNA bybendingthe DNA gate segment”which wouldthenincrease the chance of “capturinga second‘transfer’segmentinknottedorlinkedmolecules”.Thismodel is supportedbythe simulationmentionedbelow,because itinvolvesthe selective openof segment
- 3. passage bytopoisomerase II.Nevertheless,itstillneedstobe confirmedbyfurtherexperiments,due to the difficultiesthatlie inthe interpretationof currentexperimentalandcomputational results. Asthe article explains,the individual modelsabove are useful inthattheycanrationalize “certain aspectsof type-2topoisomerase actions”,butnone of themare able to comprehensivelyexplainall the data that scientistshave gatheredthusfaraboutthe enzyme (Liu,Diebler,Chan,&Zechiedrich,2009). The Juxtaposition-Centric Approach The article proposesthatthe reasonmanyof the aforementionedmodelsfail tobe comprehensive isthat theyignore an importantfactor(Buck& Zechiedrich,2004):the role of local information embeddedinthe substrate of type-2topoisomerase indiscriminatingwhetherDNA strandsare linkedor unlinkedglobally,giventhe factthattopoisomerase IIissmallerthana DNA molecule inordersof magnitudes. To understandthis,atheorycalledjuxtaposition-centricapproachhasbeenputforward.A DNA- DNA juxtapositionisgeneratedwhentwosegmentsof DNA molecules,be itonthe same strand or on the differentones,come close enoughsothatthe difference betweenthemisaboutsmallerthanthe diameterof a type-2topoisomerase molecule[37] (Buck& Zechiedrich,2004). Onlyinthissmall distance can a topoisomerase IIfunctiontograspbothstrandsto decatenate ordeknot(Bruce,Johnson,Lewis, Raff,Roberts& Walter,2008). One may come up withtwoassumptionsonjuxtaposition:one isthatall existinghelix-helix juxtapositionsare the same,regardlesswhetherthe twosegmentsare unlinkedorin a catenationor a knot;anotherone is that differentjuxtapositionisformedwhenachainissupercoiled, linkedorknotted,comparedtowhen itislinear,relaxedorunknotted(Buck&Zechiedrich,2004).Of the laterassumption,basically,there are three typesof juxtapositionproposed:hooked,half-hooked and free (Liu,Zechiedrich,&Chan,2006a). Everytime whenasegmentpassage isopenedbytopoisomeraseIIona specificjuxtapositioncan eitherresultinanunlinkedstatusof DNA standsora linkedone.Lattice conformationalenumeration demonstratedbyZhirongLiuandetal. has suggestthatthere is90% of chance that globally unlinked DNA strands become linkedwhenasegmentpassage isopenedonafree juxtaposition,whereas85%of probabilitythatthe strandsbecome unlinkedonahookedjuxtaposition(Liu,Zechiedrich,&Chan, 2006b). Additionally,the enumerationwasalsousedtosimulate the realisticinteractionsoccurring whena segmentpassage isopenedoneachjuxtapositionmediatedbytopoisomerase II. Inreality,atopological equilibriumisestablishedbetweenthe populationof catenanes orknotsand unlinkedDNA segments.However,the equilibriumwill shiftinpresence of topoisomerase II.The result isillustratedbyincorporatingtwonewfactors,whichare linkreductionfactor(RL) andknotreduction factor(RK) (Liu,Zechiedrich,&Chan,2006a). Justas theirname implies,the higherthe value of RLand RK is,the more probable itisthat the equilibriumwillbe shiftedtowardsthe decatenatingordeknotting side,andvice versa.Furthermore,RLandRK are bothchain length(n) dependent,withahigh probabilityof unlinkingforhookedandhalf-hookedjuxtaposition(linkingforfree juxtaposition) atlow value of n, andlowprobabilityathighvalue of n.A relatedstudyhasalsoshownthat the unknotting potential (indicatedbyRK) isalsoinfluencedbythe stiffnessof the chain(Liu,Diebler,Chan,& Zechiedrich,2009),withthe effectenhancingasthe stiffnessincreases(exceptforfree juxtaposition). AnotherresearchalsoshowsthatDNA supercoilingmayimprovethe efficiencyindecatenating,butnot indeknotting(Buck&Zechiedrich,2004).
- 4. Throughthe discussionabove,one mayclearlyseehow topoisomeraseIImakesuse of the global informationembeddedinlocal juxtapositiongeometriestoselectivelydisentangle DNA strands,rather than entangle them,forbothcatenanesandknots. The Impact on DNA Topology by Biological Processes As discussedinthe secondpart,some DNA global topology,suchasknotsandcatenanes,mayhave a great impacton biological processes, suchascreatinganimpasse toDNA andRNA polymerasesbya tightknot.These effectscanbe detrimental orevenlethaltoa cell andmustbe amelioratedby topoisomeraseIIintime.Buthowcan a cell deal withthembefore the reparationtakesplace? As indicatedinthe article,manybiological activitiescanindeedrelieve thistopological stressby themselves.Asshowninthe experimentdone byVologodskii ,ithas beenproventhatmanyforcesexist invivo,suchas the slidingof polymerasesalongthe DNA,tostretchthe two endsof DNA strands and therefore localize the knotsonit(Vologodakii,2006).Thislocalizationof knotsmaybe deemedasa cell’sdefence againstDNA knotting,inthatthe knotscouldbe localizedtothe non-codingsequenceson DNA and consequentlyminimize the negative effectsof it. Asfor the DNA supercoiling,itsconformationisgreatlyaffectedbybiological activities.Itseffecton enhancedgene expressioncouldbe impairedbythe bindingof nucleoid-associationproteinsin prokaryotesorhistone proteinsineukaryotes,asexplainedinthe article.Anotherstudyreviewedinthis article alsosuggestthatthere isa type of general topological barrierincellsthatcanimpede the translocationof DNA supercoil andmaybe knotsaswell. Ultimately,the discussionof experimental resultsinthe article hasprovidedevidence of atleast some reciprocal interactions.Thisconsequentlysuggestsanintimate relationshipbetweenDNA global topologyandbiological activitiesassociatedwithDNA. Citation Buck G. R., & ZechiedrichE.L.,(2004). DNA DisentanglingbyType-2Topoisomerases.JMol Biol,340, 933-939. Liu Z.R.,ZechiedrichE.L.,&Chan H.S.,(2006a). Topological informationembodiedinlocal juxtaposition geometryprovidesastatistical mechanical basisforunknottingbytype IIDNA topoisomerases.JMol Biol,361, 268-285. Liu Z.R.,ZechiedrichE.L.,&Chan H.S.,(2006b). Inferringglobal topologyfromlocal juxtaposition geometry:interlinkingpolymerringsandramificationsfortopoisomerase action.BiophysJ,90,2344- 2355. Vologodskii A.(2006).BrownianDynamicsSimulationof KnotDiffusionalongaStretchedDNA Molecule. BiophysJ,90, 1594-1597. Zhirong,L.,Deibler,R.W.,Hue,S. C., & Zechiedrich,L.(2009). SURVEY ANDSUMMARY: The whyand howof DNA unlinking.NucleicAcidsResearch,37,661-671.