Ligase enzyme

Ligase enzyme

1. DNA ligase
Dr.Manikandan Kathirvel M.Sc., Ph.D., (NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
(Reaccredited with "A" Grade by NAAC)
Affiliated to BengaluruNorthUniversity,
K. Narayanapura, Kothanur(PO)
Bengaluru 560077

2. The main enzymes involved in genetic engineering.
The enzymes are:
1.Restriction Endonuclease
2.DNA Ligase
3.Alkaline Phosphatase
4.Polynucleotide kinase
5.DNA Polymerase and the KlenowFragment
6.Reverse Transcriptase
7.DNase
8.Rnase
9.DNA polymerase
Enzymes involved in genetic engineering

3. DNA ligase

4. WhatisDNAligase?
•TheDNAligaseisaclassoftheenzymethathelpsintheformationofphosphodiesterbondbetweenthe5’end
ofonesidetothe3’endofanothersideusinganenergymolecule(ATPorNAD
+
).
•FirstDNAligaseenzymewaspurifiedandcharacterizedbyWeissandRichardsonin1967.
•DNAligasecatalysestheformationofphosphodiesterbondbetweentwodeoxynucleotideresiduesoftwoDNA
strands
•DNAligaseenzymerequiresafreehydroxylgroupatthe3´-endofoneDNAchainandaphosphategroupatthe
5´-endoftheotherandrequiresenergyintheprocess.
••E.coliandotherbacterialDNAligaseutilizesNAD+asenergydonor,whereasinT4bacteriophage,T4DNA
ligaseusesATPascofactor.
ThemostwidelyusedDNAligaseisisolatedfromT4bacteriophage.T4DNAligaseneedsATPasacofactor.
•TheenzymefromE.coliusescofactorNAD.

5. Alltheeukaryoticenzymeworksefficientlyatbodytemperature(37°C).However,someoftheDNAligasesliketheT4
DNAligaseworkefficientlyat16°Ctemperature,invitro.Even,theligationcanalsobeachievedat4°CduringinVitro
reaction.
ComponentsofDNAligaseenzyme:
TheT4DNAligasecontainsthreepartsmadeupof
thesinglepolypeptidechain.
•Nucleotide-bindingsite
•DNAbindingcleftinthecentre
•Anactivesitecontainingthelysineresidue

6. MechanismofDNAligation:
1.The5’endoftheDNAbreaksiscalled“donor”whilethe3’endsare
called“acceptor”.
2.Thecatalyticreactionofligationisstartedwiththerecognitionofthe
ligationsiteasnick.
3.Thereactionstartswiththenucleophilicattack.
Inthefirststep,oneATPorNAD
+
(incaseofbacterialligase)energy
moleculesreactwithligaseenzymeandhelpsinformingtheenzyme-
AMPcomplexlinkedtoaminogroupofthelysine.TheAMPattached
withthelysineaminoacidpresentintheactivesiteoftheenzyme
byreleasingthePPi.Thus,theactivecentreoftheenzyme
becomesadenylatedbyadditionofAMPtoitslysineandstarts
theenzymaticreaction.Thebondformedbetweenthelysineof
enzymeandtheAMPiscalledthephospho-amidebond.
Inthesecondstep,theactivesiteoftheenzymewiththeAMPmoiety
activatesthephosphategroupatthe5´-endoftheDNAmoleculetobe
joined[andreleasestheAMPfromthelysineandattacheditwiththe
phosphateofthedonor].ThustheligaseenzymetransferstheAMPto
the5’phosphateend.
Thefinalstepisanucleophilicattackbythe3´-hydroxylgrouponthis
activatedphosphorusatom.Nowtheenzymeattachesthe5’tothe3’
bycreatingthephosphodiesterbondandreleasestheAMPfromthe
activesite.

7. DifferenttypesofDNAligase:
EukaryoticDNAligase:
DNAligaseI:ligatestheDNAonthelaggingstrand,especially,thegapsbetweentheOkazakifragments.
DNAligaseII:TheDNAligaseIIisnotconsideredasatrueligasebecauseitdoesnothaveitsowngene,the
eukaryoticDNAligaseIIsynthesizedfromthegenethatencodestheDNAligaseIII.
ItismajorlyinvolvedintheDNArepairpathway.
DNAligaseIII:itisrequiredintheDNArepair,especially,inthenucleotideexcisionrepair.Itistheonlykindof
ligasethatpresentinmitochondrialDNAtoo.
DNAligaseIV:TheDNAligaseIVisveryspecialbecauseitjointsthedouble-strandedDNA.Itisinvolvedinthe
double-strandbreakrepairpathway,particularly,inthenon-homologousend-joining.
Furthermore,itisalsorequiredfortheV(D)Jfragmentjoining.
Note:theeukaryoticDNAligaseutilizedATPasaco-factorinsteadofNAD+.

8. ProkaryoticDNAligases:
E.coliDNAligase:
TheE.coliDNAligaseusesNAD
+
,nicotinamideadeninedinucleotideasacofactororenergysourceforformingthe
phosphodiesterbond.
ItcanjoinonlyDNA-DNAmoleculeandunabletojoinDNA-RNA.Inadditiontothis,onlystickyendscanbesealedby
E.coliDNAligase.
TheE.coliDNAligaseisencodedbythe“lig”geneofE.coli.
T4DNAligase:
In1083,ArmstrongandcoworkersdiscoveredthestructureandgeneticorganizationofT4DNAligase,however,thefirst
T4DNAligasewasclonedbyWilsonandMurrayin1979.
TheT4DNAligaseisextractedfromthebacteriophageT4,anditisoneofthemostcommonlyusedDNAligasesin
recombinantDNAtechnology.
ItisoneofthebestenzymesforresearchlabsbecauseofitstremendousadvantagesoverE.coliDNAligase.
Itcanjoindouble-strandedDNA
LigateDNA-DNA
DNA-RNA
RNA-RNAmolecule
Also,itcancapableofligatingstickyendsaswellasbluntends.
Theoptimumactivityofitcanbeachievedatalowertemperature.
InsteadofNAD
+
,itutilizesATPasanenergymoleculeforperformingtheenzymaticreaction.

9. Protein Accession
number
P00970
Gene Gp30(gene30)ofT4bacteriophage
pH 7.8-8.0
Molecularweight
77kDa(Sedimentation,Lehman1974)
74±3kDa(SDSPage,Lehman1974)
55.3kDa(Theoretical)
Isoelectricpoint 6.14
Activators ATP,Mg2+,sulfhydrylreagents
Optimumtemperature 16°C
Inhibitors AhigherconcentrationofNaCl
Information related to the T4 DNA ligase
ThetemperaturevstheactivitygraphoftheT4DNA
ligaseisshownhere,

10. ThefunctionofDNAligase:
ThemainfunctionofDNAligaseistoligatetwoDNAstrandseithersinglestrand
ordoublestrand.Although,differentligaseisusedfordifferentfunctioninvivo
andinvitroprocesses.
1.ThefunctionofDNAligaseinReplication:
Replicationisaprocessinwhichthefourdifferentdaughtersingle-strandedDNA
moleculesgeneratedfromasingleDNAduplex(dsDNA).Differentenzymes
workindifferentstepsforcompletionreplication.
TheprocessofDNAreplicationstartswiththeadditionofRNAprimerbythe
primaseenzyme.The3′endoftheprimerisusedasastartingpointforthe
additionofnucleotidesbytheDNApolymeraseattheleadingstrand.
ThereplicationendsatthelaggingstrandbythesynthesisofOkazakifragments.
Inthefinalsteps,theprimerisremovedandthenucleotidesarefilledinthegaps
betweentheOkazakifragmentsbyDNApolymerasebutthenewlysynthesised
strandsarestillnotjoined.
DNALigaseperformsthefunctionoffillingsgapsbycreatingphosphodiester
betweenthegaps,createdaftertheremovalofprimerandbetweentheOkazaki
fragments.
Itperformsligationbyusingthe5′endoftheonestrandand3′endofanother
endandjoinsitbyremovingthepyrophosphatefromthetriphosphate.
However,theligaseusedintheDNAreplicationcannotligatethedouble-
strandedDNAorblunt-endeddsDNA.

11. ThefunctionofDNAligaseinrecombinantDNAtechnology:
EukaryoticDNAligasealikeDNAligaseI,DNAligaseIIorDNAligaseIVcannotbeusedinthecloningexperiments,
instead,thephageT4DNAligaseisusedforperformingdifferentligationmethods.
RestrictiondigestionofDNAgeneratestwotypesofDNAendsvizstickyendsorbluntends.
Differentendsaregeneratedfordifferentmolecularbiologytechniques.
Processofblunt-endligation:
Bluntendsaregeneratedduetotherestrictiondigestionatthesamebasepaironboththesestrands.
Theendsaresimpleand,directandnoncohesive.SpecialtypesofDNAligaseareusedtoligatethesetypesofDNAends.
Theprocessofblunt-endligationasshownintothefigurebelow,

12. Processofstickyendligation:
RestrictiondigestionwhichgeneratesfewnucleotideoverhangsonboththeDNAstrandsiscalledstickyends.
Theseendsarecohesiveandhavefewbasepairspalindromicsequencesonboththestrands(mostcases).
Theprocessofstickyendligationisshowninthefigurebelow:
Thestickyendsaregeneratedforinsertingageneofinterestintheplasmid.Itworksbetterincomparisonwith
thebluntendligation.

13. Preparationofligationreaction:
InsertDNA:100ng
PlasmidDNA:~30ng
T4DNAligase:0.5to1μl
Ligationbuffer:10X
D/wtotalvolumeof10μl.
10XligationbufferComposition:
Tris-HCl:500mM
MgCl2:100mM
Dithiothreitol:100mM
ATP:10mM
AdjustpH7.5storeatroomtemperature.
Incubation:
Thetimeandtemperatureforincubationofligation
reactionsareverycrucial.AlltheDNAligasesaregenerally
activeatroomtemperature,however,theoptimumactivity
ofDNAligaseobtainedat16°Ctemperature.
Youcanincubatethesampleatroomtemperaturefor2
hoursorat16°Covernight.
Interestingly,
OneofthecommerciallyavailableDNAligasecalled
“Ampligase”DNAligasecanworkefficientlyevenata
highertemperatureofmorethan95°C.
The“ampligase”isisolatedfromthethermophilicbacteria
havingahalf-lifeof48hoursat65°Cand1hourat95°C.

14. ApplicationofDNAligase:
Ligatesthecohesivebluntendsaswellasstickyends.
Ligatesingle-strandedaswellasdouble-strandedDNA.
Usedinaligasechainreaction
UsedduringtheDNArepairmechanism
UseduringDNAreplication
Usedtoinsertageneintheplasmid

15. Ligationtechniques
1.LigationdependentCloning(LC)-usingT4DNAligase/E.coliDNAligase
2.LigationIndependentCloning(LIC)
•LICisacloningmethodthatmakesuseofannealingofsingle-strandedcomplementaryoverhangsonthe
targetvectorandaPCR-generatedinsertofatleast12basesoverlapregion.
•Single-strandedoverhangscanbegeneratedbyusingT4DNApolymeraseandonlyonedNTPinthe
reactionmix,leadingtoanequilibriumof3'->5'-exonucleaseand5'->3'-polymeraseactivityatthesiteofthe
firstoccurrenceofthisnucleotide.
•TheincubationisdonewithT4DNApol.for30min.andstoppedbyaddingdCTPtothereactionmix.After
annealingofvectorandinsert,themixtureisusedtotransformE.coli.
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