ligation i

Ligation: Theory andPracticeKarthikeyan Kandavelou, Johns Hopkins University, Maryland, USA

Mala Mani, Johns Hopkins University, Maryland, USA

Sekhar PM Reddy, Johns Hopkins University, Baltimore, Maryland, USA

Srinivasan Chandrasegaran, Johns Hopkins University, Baltimore, Maryland, USA

Ligation is theprocess bywhichNADDNA ligases catalyse the formationof phosphodiester

bonds between juxtaposed 3-hydroxyl group and 5-phosphate termini in duplexdeoxyribonucleic acid (DNA). Ligases use adenosine triphosphate or nicotinamide

adenine dinucleotide (NAD) as cofactors for this covalent joining of DNA.

Introduction

One of the key steps in recombinant deoxyribonucleic acid(DNA) technology is the joining of two separate DNAfragments covalently to form a single DNA molecule thatis capable of autonomous replication in cells. The cons-truction of recombinant DNAmolecules involves the join-ing of insert sequences into plasmids or bacteriophagecloning vectors. This critical step is performed by DNAligases. Like type II restriction enzymes, DNA ligases areessential tools in recombinant DNA technology for ana-lysing and manipulating DNA. DNA ligases are essentialenzymes in the synthesis of new DNA during cell divisionand for maintaining the integrity of the genome.

DNA Ligase Reaction Properties andIntermediates

DNA ligases catalyse the formation of phosphodiesterbonds between juxtaposed 5-phosphate groups and a 3-hydroxyl termini in duplex DNA. They can repair single-stranded nicks in duplex DNA as well as covalently joinrestriction fragments with compatible cohesive ends orblunt ends (Figure 1). The commonly used DNA ligases innucleic acid research are T4 DNA ligase and Escherichiacoli DNA ligase. T4 DNA ligase was originally puriedfrom T4 phage-infected cells of E. coli; it is the product ofgene 30 of phage T4. E. coli DNA ligase is the product ofthe lig gene. Both genes have been cloned and the enzymesare obtained from overproducing strains. T4 DNA ligaseand E. coli ligase differ from each other in two importantproperties: (1) T4DNA ligase uses adenosine triphosphate(ATP) as a cofactor, whereas E. coli ligase uses NAD as acofactor; (2) under normal reaction conditions, only T4DNA ligase joins blunt ends efciently.WhileE. coliDNAligase repairs single-stranded nicks in duplex DNA and

joins restriction fragments with compatible cohesive orsticky ends, it does not ligate restriction fragments withblunt ends under normal reaction conditions. See also:BacteriophageT4;DNAligases;DNArepair by reversal ofdamage; Nucleic acids: general propertiesDNA ligases catalyse the DNA joining reaction in three

distinct steps: (1) the formation of a covalent enzymeadenosine monophosphate (AMP) complex; (2) the trans-fer of the AMP moiety to the 5-phosphate at a nick in theduplex DNA; and (3) the formation of the phosphodiesterbond with concomitant release of AMP. See also: DNA-binding enzymes: structural themesThe rst step of the reaction is the best characterized.

Studies with ATP-dependent T4 DNA ligase and NAD-dependentE. coliDNA ligase have shown the formation ofa covalent enzymenucleoside monophosphate reactionintermediate. It appears that the AMPmoiety is linked viaa phosphoramidite bond to a lysine residue in an active sitemotif, KXDGXR, that is diagnostic for DNA ligases(Figure2).A further ve conservedmotifs have been denedby protein sequence alignments of DNA ligases. See also:Protein motifs for DNA bindingThe crystal structure of bacteriophage T7DNA ligase has

revealed that this enzyme comprises two distinct domainslinked together to form a cleft. The active site motif,KXDGXR, is at thebaseof the cleft, and theother conservedmotifs are located on the surface of the two domains close tothe site of adenylation. It appears that the nicked duplexDNA binds the cleft between the two protein domains.

Manipulating and RecombiningRestriction Fragments with DNA Ligase

Figure 3a shows the cloning of a HindIII restriction frag-ment into a vector or plasmid containing a single HindIII

Article Contents

Introductory article

. Introduction

. DNA Ligase Reaction Properties and Intermediates

. Manipulating and Recombining Restriction Fragmentswith DNA Ligase

. Optimizing the Formation of Recombinant Molecules

. Biochemical Properties of DNA Ligases

. In vivo Function of DNA Ligases

. Ligase Chain Reaction

. Summary

. Acknowledgements

doi: 10.1038/npg.els.0003838

1ENCYCLOPEDIA OF LIFE SCIENCES & 2005, John Wiley & Sons, Ltd. www.els.net

site. The rst step is to cleave the vector at the uniqueHindIII site by using theHindIII restriction endonucleaseto produce the vector with homologous cohesive ends. Thecleaved vector is then treated with calf intestinal phospha-tase to remove the 5-phosphates from the termini. Thisprocess inhibits self-ligation of the vector. Gel electro-phoresis is used to purify the cleaved vector away from theuncleaved vector. Uncleaved and self-ligated vector DNAare the major source of background in the cloning of therestriction fragments. Because all the HindIII cohesiveends are equivalent, the restriction fragment will be clonedin either of the two possible orientations with respect to thevector sequences. These two possible recombinant prod-ucts can be distinguished by restriction mapping by usingan enzyme that cleaves asymmetrically within the insertDNA. See also: Restriction enzymesAlternatively, directional cloning of the restriction frag-

ments with heterologous ends may be used to force theinsert in a particular orientation with respect to the vectorsequences (Figure 3b). The restriction fragments that are tobe subcloned have heterologous ends. These are generatedby cutting theDNAwith twodifferent restriction enzymes,for example BamHI and HindIII, respectively. The vectoror plasmid DNA containing unique BamHI and HindIIIsites is also digested withBamHI andHindIII. The cleavedvector DNA with heterologous ends is gel-puried awayfrom the uncleaved vectorDNA to reduce background.Asthe heterologous ends of the vector DNA are not compat-ible, there is no self-ligation.Directional cloning is possiblebecause the heterologous ends of the insert are compatiblewith those of the vector DNA in only one orientation. Theother orientation results in noncompatible ends for liga-tion and, hence, this recombinant product is eliminatedfrom the reaction mixture. See also: Gel electrophoresis:one-dimensional; PlasmidsT4 DNA ligase can join DNA fragments with blunt

ends, but the ligation efciency is much lower than thatachieved by restriction fragments with cohesive ends. Theligation efciency may be increased by increasing the

concentration ofDNAand by usingmoreDNA ligase. Theblunt ends are equally ligatible, irrespective of the restric-tion enzyme used to generate the ends. The ligation of co-hesive ends requires compatible termini. The ligation ofblunt ends producedbydifferent restriction enzymes resultsin recombinants that cannot be cleaved by either enzyme.

Optimizing the Formation ofRecombinant Molecules

During the ligation reaction, several factors appear to in-uence the formation of recombinant molecules: (1) thepurity of the DNA, (2) the relative concentrations of theinsert and vector DNA and (3) increased amount of T4DNA ligase for blunt-end ligations. The probability ofobtaining the desired recombinant DNA molecules isgreatly improved with the purity of the individual DNAcomponents of the ligation reaction. Highly puried re-striction fragments and vector DNA free of minor con-taminants are required. The minor contaminants arisefrom incomplete digestion by restriction endonucleases.For example, even if the cleavage efciency of the vectorDNA by a restriction enzyme is 99%, the uncleaved 1% ofvector DNA readily transfects the competent cells duringtransformation. This results in an unacceptably high back-ground in these experiments. Gel purication of thecleaved vector DNA away from the uncleaved vectorgreatly reduces this background.Theothermajor source ofbackground is from intramolecular self-ligation of vectorDNA. This can be eliminated by using vector DNA withheterologous ends. In the case of vector or plasmid DNAwith homologous ends, intramolecular joining can be pre-vented by removing the 5-phosphate from each end withcalf intestinal phosphate. This 5 dephosphorylation can-not be applied to both the vector DNA and the DNAfragment tobe ligated.Thus, the untreatedpartner remainscapable of self-ligation. Recently, Ukai and colleagues

5 33 5

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Figure 1 T4 and E. coliDNA ligase activity at single-stranded breaks or nicks (a) and at double-stranded breaks with cohesive or sticky ends. (b) T4 DNAligase activity at blunt ends. (c) ATP, adenosine triphosphate; NAD, nicotinamideadenine dinucleotide. They catalyse the formation of phosphodiester

bonds between juxtaposed5 phosphate groups and3 hydroxyl termini in duplexDNA.While T4DNA ligase uses ATP as a cofactor, E.coli ligase usesNAD.

Ligation: Theory and Practice

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proposed a new technique to overcome this problem. Thistechnique replaces the 2 deoxyribose at the 3-end of theDNA fragment with 2,3dideoxyribose to prevent self-ligation. The 5-phosphate in this fragment remainscapable of ligation to the 3-OH of the vector DNA. Bycombining this 3 replacement technique with 5 de-phosphorylation of vector DNA, self-ligation of both thepartners in the reaction can be simultaneously prevented.The relative concentrations of the insert DNA and vectorDNA in the ligation mixture may also inuence the

frequency of specific ligation products. Cloning experi-ments using vectorDNArequire the joining of two ormoreseparateDNAmolecules followed by the circularization ofthe product. The optimal cloning efciency results inDNAconcentrations that are high enough to permit sufcientintermolecular ligation, but not so high as to reduce intra-molecular ligation. In situations where the background islow, the DNA concentration has been determined experi-mentally to be between 1 and 50mgmL21. It must bepointed out that genetic methods are available to identify

O

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Figure 2 Reactionmechanisms for T4 DNA ligases. (a) T4 DNA ligase (L) reacts with ATP to form a phosphoramide-linked AMPwith the amino group ofthe active lysine site. Pyrophosphate (PPi) is released. (b) The 5-phosphate at the nick attacks the activated phosphoryl group of the AMP to form anadenylatedDNA. (c) The enzyme catalyses joining of the 3-OH of theDNA at the nick to the activated 5-phosphate to form the phosphodiester bond andconcomitant release AMP.


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colonies containing the desired DNAmolecule from back-ground colonies. These include the use of a combination ofdrugmarkers, as well as the use of designed cloning vectorsto select or screen for recombinants on appropriate indi-cator plates. Furthermore, polymerase chain reaction(PCR) methods may be employed directly to screen col-onies for recombinants by using oligonucleotide primerscomplementary to the vector sequences anking the inser-

tion site. Finally, it is important to perform appropriatecontrol experiments simultaneously with the ligation re-action of interest. The controls reect on the relative suc-cess of the cloning experiment. This is particularly helpfulbecause the preparation and analysis of the recombinantDNAmolecules is a laborious and time-consuming part ofthe cloning process. Blunt-end ligation is facilitated by us-ing an even higher concentration ofDNA components andabout 10-fold more ligase. All ligations are carried out at168C overnight in a relatively low reaction volume, usually1020mL. See also: Bacterial restrictionmodicationsystems; DNA: methods for preparation; Polymerasechain reaction (PCR); Polymerase chain reaction (PCR):specialized reactionsIn practice, the critical parameters for the design of a

ligation reaction include the number of inserts, the type ofDNA ends, the concentration of the DNA fragment, thepreparation and purity of the restriction fragments, andthe availability of a selection or screening system that canbe employed to identify the desired recombinant DNAmolecule. Sometimes, it is important to obtain maximumnumberof recombinants especially during the constructionof genomic or complementary DNA (cDNA) libraries.This ensures a complete coverage or representation of thegenomeor cDNA. In other instances, it is better to obtain afew colonies containing the desired recombinant productthan a large number of colonies inwhich only relatively fewcontain the desired product. In each case, the experimentalconditions of ligation are adjusted to favour the desiredrecombinant product. See also: Microorganisms: applica-tions in molecular biologyFormation of the desired recombinant product during

the ligation reaction can be increased by using an excess ofthe dephosphorylated vector or plasmid DNA to that ofthe insert that is to be cloned. An alternative method is toperform the ligation of the vector to the insert with com-patible ends in the presence of the restriction enzyme thatwas used to generate the inserts. The enzyme will inhibitligation of the inserts to themselves, as these will be cleavedcontinuously by the enzyme to regenerate the monomerinserts. On the other hand, the ligation of the inserts to thevector DNA is favoured because it leads to the generationof a fusion site that is not recognized by the restrictionendonuclease, and hence not cleaved. This results in theaccumulation of the desired recombinantmolecules duringthe ligation reactions.Selection or screeningmethods are currently available to

identify the desired recombinant molecule from a ligationmixture. Cloning vectors have been designed to screen orselect for the recombinants. pUC or M13mp derivativesare the commonly used vectors. They contain the a-complementation region of the E. coli lacZ gene. Theyproduce blue colonies or plaques on appropriate indicatorplates. A white colony or plaque is seen when there is asuccessful insertion of a restriction fragment in the lacZregion of the vector. This white colony or plaque is easily

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Figure 3 Ligation of a restriction fragment into a plasmid or vector DNA.(a) Cloning of a restriction fragment into a vector with cohesive ends. Theinsert can be cloned into the vector in two possible orientations with

respect to the vector sequences. Both recombinant molecules are shownand they can be distinguished by restriction mapping. (b) Directional

cloning of a restriction fragment into a vector with heterologous ends. Therestriction fragment is digested with two different restriction enzymes to

generate heterologous ends. The plasmid or vector DNA is also cleavedwith the same enzymes to generate heterologous ends. This results in the

cloning of the insert DNA in only one particular orientation with respect tothe vector sequences. The other orientation results in noncompatible ends

for ligation, and hence this recombinant product is eliminated from theligation reaction mixture. ATP, adenosine triphosphate.


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detected or identied among a background of blue trans-formants. An alternative approach is to use plasmids orvectors that contain two selectable drug markers, such asampicillin or tetracycline resistance, as in the case ofpBR322. Insertion of a restriction fragment into one ofthese marker genes renders the colony sensitive to thatdrug. These recombinant colonies can easily be screened bymeans of replica plating. See also: Genetic engineering:reporter genes

Biochemical Properties of DNA Ligases

TheATP-dependentDNA ligases arewidespread in naturefrom bacteriophage to mammals. The NAD-dependentDNA ligases are unique to bacteria. This uniqueness ofNAD-dependent ligases to eubacteria has led to the sug-gestion that it could be a potential target for novel anti-biotics. In addition to ATP or NAD as cofactors, theseenzymes require Mg2+ for activity. The lower organismsappear to encode only a single essential DNA ligase,whereas mammalian cells carry multiple genes. DNAligases vary widely in their molecular mass, ranging from41 kDa (T7 ligase) to 100 kDa (mammalian DNA ligase I).Protein regions with additional regulatory functions ac-count for much of the variation in the size. For instance,mammalian DNA ligase I comprises two distinct and sep-arable domains: a carboxy (C)-terminal catalytic domainand an amino (N)-terminal region with regulatory func-tions. The N-terminal domain is not required for the cata-lytic activity. However, it is required both for nuclearlocalization and for recruitment of the enzyme at the rep-lication sites during the S phase.While significant sequencehomology has been observed between ATP-dependentligases, none is found between NAD-dependent DNAligases of eubacteria. See also: Adenosine triphosphate;Coenzymes and cofactors; NAD+ and NADP+ asprosthetic groups for enzymes; ProteinDNA complexes:specicBarany and co-workers have compared the biochemical

properties of seven NAD-dependent DNA ligases ofthermophilic bacteria, collected from Thermus speciesworldwide. The enzymes are highly homologous, withamino acid sequence identities ranging from 85 to 98%.The enzymes have different levels of tolerance for mis-match ligation when Mn2+ is substituted for Mg2+. Thesequence divergence and subtle structural variation amongthese DNA ligases appear to underlie the enzymes recog-nition preferences toward differentmismatched base pairs.

In vivo Function of DNA Ligases

Study of mammalian DNA ligases has shown that theyplay crucial roles in cellular functions such as DNA rep-

lication, DNA repair and genetic recombination. Fourdistinct DNA ligase activities have been shown to arisefrom three mammalian genes (I, III, IV) encoding DNAligases. DNA ligase I joins the Okazaki fragments gener-ated by lagging strand DNA synthesis during DNA rep-lication. Ligase I is also involved in base excision repair(BER). It directly interacts with DNA polymerase b, theenzyme responsible for BER. DNA ligase I-decient celllines are sensitive to DNA damage by alkylating agents,ionizing radiation and ultraviolet exposure. This suggeststhat DNA ligase I is involved in DNA repair pathways aswell. See also: Developmentally programmed DNArearrangements; DNA repair; Eukaryotic replication forkIt appears that two forms of DNA ligase III, IIIa and

IIIb, are produced by alternative splicing in a tissue- andcell type-specific manner. DNA ligase IIIa interacts withthe DNA strand-break repair protein XRCC1 to form acomplex during the repair of DNA single-stranded breaksin all tissues and cells. This interaction is mediated bydimerization of their carboxy terminal BRCT (BRCA C-terminal) modules. BRCT modules are present in variousproteins involved in cell cycle and DNA replication and ithas been proposed that these processes are controlled inpart by interaction of these BRCT domains. Ligase IIIa isalso involved in the repair and replication ofmitochondrialDNA.DNA ligase IIIb does not interact with XRCC1 andappears to be involved in the meiotic recombination.Ligase IV takes part in the nonhomologous end joining(NHEJ) repair of DNA double-strand breaks caused byradiation and chemical agents. This enzyme interacts withXRCC4, which stabilizes and directs it to DNA double-strand breaks. It has been shown to be essential for em-bryonic development and V(D)J recombination in mice.DNA ligases also appear to be involved in the repair ofDNA double-stranded breaks by homologous or non-homologous recombination pathways. See also: Alterna-tive splicing: cell-type-specic and developmental control;Meiotic recombination pathways; Recombinational DNArepair in eukaryotesThe clones carrying chimaeric nucleases can be made

more viable by increasing the levels of the DNA ligasewithin the cells. As there are no counterpart methylasesavailable for the hybrid endonucleases that are formed bythe fusion of the isolated nuclease domain of FokI to otherDNA-binding motifs, production of these engineered nu-cleases in vivo is often lethal to cells.

Ligase Chain Reaction

The availability of cloned thermostable ligases makes itpossible for the application of ligases in DNA diagnostics.This method is called the ligase chain reaction (LCR). Itcomplements the PCR that has revolutionized DNA diag-nostics. PCR is a simple and powerful in vitromethod that


5

allows enzymatic synthesis of specific DNA sequences, us-ing two oligonucleotide primers that hybridize to oppositestrands and ank the region of interest in the target DNA.A repetitive series of automated cycles, which include tem-plate denaturation, primer annealing and the extension ofthe annealed primers by a thermostable DNA polymerase,results in the exponential amplication of a specific frag-ment whose termini are dened by the 5-ends of the prim-ers. LCR, on the other hand, utilizes a thermostable ligaseboth to amplify DNA and to discriminate a single basesubstitution in target DNA (Figure 4). The thermostableligase specifically links two juxtaposed oligonucleotideswhen hybridized at 658C to a complementary target at thejunction. Oligonucleotide products are amplied expo-nentially by thermal cycling of the ligation reaction in thepresenceof a second setof adjacentoligonucleotides, comp-lementary to the rst set and the target. A single basemismatch at the junction prevents ligation, and hence am-plication. Barany has used this method to discriminatebetween normal bA and sickle bS globin genotypes of hu-man population by using 10-mL blood samples. More re-cently, Barany and co-workers have developed a universalDNA microarray method for multiplex detection of lowabundance point mutations in cancer by combining PCRwith LCR.Alternatively, pointmutations and SNPs can besensitively detected by using padlock probes. These areoligonucleotides, which include one target-complementarysequence at each end and are designed such that the twoends are placed immediately next to each other on hybridi-zation with a perfectly matched target sequence. These

perfectly matched probes can be linked covalently byligase. Hence, they can specifically discriminate point mu-tations on the target sequence. The circularized probes canbe amplied by rolling circle amplication (RCA) usingsingle primer and f 29 DNA polymerase. This reaction,unlike PCR, proceeds at 378C for 3 h and yields a tandemrepeat of the target sequence. The RCA product can bedirectly stained with Sybr-Gold and visualized under UVlight.

Summary

DNAligases play essential roles inmany cellular functions,including DNA replication, DNA repair and DNA re-combination. They catalyse the formation of phosphodi-ester bonds between adjacent 3-hydroxyl and 5-phosphate termini at single- or double-stranded breaks.DNA ligases fall into two major classes based on theircofactor requirements: (1) enzymes that use ATP and (2)enzymes that are NAD dependent. Four distinct DNAligase activities from three mammalian genes encodingDNA ligases have been identied. They have been shownto play important roles in the maintenance of genomicstability and integrity.DNA ligases have also proven to be essential tools in the

recombinant DNA technology. These enzymes are used tojoin two or more separate DNA segments to generate asingle DNA molecule that is capable of autonomous rep-lication in cells and bacteria.LCR is a powerful in vitromethod that utilizes a thermo-

stable ligase both to amplify DNA and to discriminate asingle base substitution in target DNA. LCR thus comple-ments PCR, which has revolutionized DNA diagnostics.

Acknowledgements

The work in Dr Chandrasegarans lab is funded by a grantfrom NIH (GM 53923).

Further Reading

Barany F (1991) Genetics disease detection and DNA amplication us-

ing cloned thermostable ligase.Proceedings of theNationalAcademyof

Sciences of the USA 88: 189193.

Ciarrocchi G, MacPhee DG, Deady LW and Tilley L (1999) Specific

inhibition of eubacterial DNA ligase by arylamino compounds.

Antimicrobial Agents and Chemotherapy 43: 27662772.

Doherty AJ and Wigley DB (1999) Functional domains of an ATP-

dependent DNA ligase. Journal of Molecular Biology 285: 6371.

Frank KM, Sekiguchi JM, Seidi KJ et al. (1998) Late embryonic

lethality and impaired V(D)J recombination in mice lacking DNA

ligase IV. Nature 396: 173177.

Grossman L (1997) DNA repair. Encyclopedia of Human Biology

3: 447454.

Matched target

Denature DNAand

anneal oligonucleotides

Ligation

Exponential amplification

Mismatched target

No ligation

Figure 4 Diagram depicting DNA amplification and detection bymeans of the LCR. The target DNA is heat denatured and fourcomplementary oligonucleotides are then hybridized to the target at

658C. A thermostable ligase is used to link covalently adjacentoligonucleotides that are perfectly matched to the target. Products from

one cycle of ligation become targets for the next cycle, and thus thenumber of products increases exponentially. Oligonucleotides that

contain a single base mismatch at the junction do not ligate efficiently,and therefore do not amplify the ligated product. The single base

mismatch at the junction is shown in red.


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Gumport RI and Lehman IR (1971) Structure of theDNA-ligase adeny-

late intermediate: lysine-linked adenosine monophosphoramidite.

Proceedings of the National Academy of Sciences of the USA

68: 25592563.

Kubota Y, Nash RA, Klungland A et al. (1996) Reconstitution of DNA

base extension-repair with puried human proteins: interaction be-

tween DNA polymerase b and the XRCC1 protein. EMBO Journal15: 66626670.

Struhl K and Tabor S (2000) Enzymatic manipulation of DNA and

RNA: DNA ligases. In: Ausubel FM, Brent R, Kingston RE et al.

(eds) Current Protocols in Molecular Biology, vol. I, chap. 3,

pp. 3.143.14.3. New York: John Wiley.

Timson DJ and Wigley DB (1999) Functional domains of an NAD+-

dependent DNA ligase. Journal of Molecular Biology 285: 7383.

Timson DJ, Singleton MR and Wigley DB (2000) DNA ligases in the

repair and replication of DNA.Mutation Research 460: 301318.

Tomkinson AE and Levin DS (1997) Mammalian DNA ligases. Bio-

Essays 19: 893901.

UkaiH,Ukai-TadenumaM,OgiuT andTsujiH (2002)Anew technique

to prevent self-ligation ofDNA. Journal of Biotechnology 97: 233242.

Qi X, Bakht S, Devos KM, Gale MD and Osbourn A (2001) L-RCA

(liagation-rolling circle amplication): a general method for geno-

typing of single nucleotide polymorphisms (SNPs). Nucleic Acids

Research 29: E116.


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