1. THE WHY AND HOW 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
topisomerasesare responsiblefor“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,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).

4. Throughthe discussionabove,one mayclearlysee how 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.