Functional Domains of an ATP-

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Introduction

D lularis re portainclu (LeEngler & Richardson, 1982), and tdamaged DNA, as evidenced by thviruses which have genes encodingDNA ligases. The enzymes appear togroups, those requiring NAD

(Lehman, 1974) and those requiring ARichardson, 1982; Lindahl & Barneseukaryotic enzymes all require ATPvira owiety in,103 ymfor t T7enzy cotheir ed(Tom onsdiffelarglian

T7 enzyme represents one of the smallest knownDNA ligases, and sequence similarity with otherligases shows that it corresponds to the highly con-

ii EpA pDNA !AppDNA E

PrBiocLond

Aand

E-wigl

Article No. jmbi.1998.2301 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 285, 6371

0022rences are due to N-terminal extensions in theer DNA ligases, with the exception of mamma-DNA ligases III and IV (Wei et al., 1995). The

iiiDNA-OHAppDNA !DNA-p-DNAAMPA conserved motif (KxDG) includes a lysine resi-

due which is known to be at the active site of anumber of enzymes that catalyse nucleotidyl trans-fers including all DNA (Lehman, 1974; Engler &

esent address: A. J. Doherty, Department ofhemistry and Molecular Biology, University Collegeb

-lly encoded enzymes. They shof molecular masses (Kletz

kDa for the human type I enzhe enzyme from bacteriophagemes show striking amino acidC-terminal region when alignkinson et al., 1991) and the con, Gower Street, London WC1E 6BTbreviations used: ssDNA, dsDNA, sin

double-stranded DNA, respectively.mail address of the corresponding autey@eric.path.ox.ac.uk

2836/99/01006309 $30.00/0enzyme thatnt processes

hman, 1974;he repair ofe number of

their ownfall into twofor activityTP (Engler &, 1992). The, as do thea wide var-1992) frome to 41 kDa

. DNA ligasenservation inby active siteiderable size

served C-terminal region, with only a 33 aminoacid N-terminal extension beyond the active sitelysine (Kletzin, 1992).

The catalytic mechanism of ligases proceeds viaa number of steps beginning with the hydrolysis ofATP or NAD to yield AMP covalently attached tothe active site lysine residue (i), with the release ofpyrophosphate or nicotinamide mononucleotide.The adenylated enzyme then transfers the AMPmoiety from this lysine residue to the free 50-phos-phoryl group at the end of a DNA strand (ii) eitherat a nick site or at the end of a DNA duplex. Thefinal step is phosphodiester bond formation withconcomitant release of AMP from the adenylatedDNA intermediate (iii):

i E pppA !EpA PPiNA ligase is an essential celquired for a number of imding replication of DNAan J. Doherty and Dale B. Wigley*

illiam Dunn School oflogy, University ofd, South Parks Roadd OX1 3RE, UK

The crystal structure of anphage T7 revealed that theorder to investigate the biooverexpressed them separalarger N-terminal domainthough both at a reduceddomain is stimulated by ththat a conformational changprotein. The DNA bindingbeen studied. The larger ddouble-stranded DNA, whidouble-stranded DNA. Theligases for nick sites in Ddifferent DNA binding activ

Keywords: bacteriophage T7sponding authorRicha(Heapet al.,ping

, UK.gle-stranded

hor:dependent DNA Ligase

ATP-dependent DNA ligase from bacterio-rotein comprised two structural domains. In

hemical activities of these domains, we haveely and purified them to homogeneity. The

retains adenylation and ligase activities,level. The adenylation activity of the largepresence of the smaller domain, suggestingis required for adenylation in the full length

properties of the two fragments have alsomain is able to band shift both single andthe smaller fragment is only able to bind to

e data suggest that the specificity of DNAA is produced by a combination of these

ities in the intact enzyme.# 1999 Academic Press

DNA ligase; ligation; DNA bindingrdson, 1982; Lindahl & Barnes, 1992), RNAhy et al., 1987) and tRNA ligases (Baymiller1994) as well as the eukaryotic mRNA cap-

enzymes (Fresco & Buratowski, 1994;

# 1999 Academic Press

Shumenzymvia athe Glysinelysinemotifalso ctRNAsee Ssuggeenzymthat tture (suppomutaenzymBurat1997)T7 DPBCV1997)regioligase& Schtheseshowsupposuper& Ru

WetallisaThe sture oing oenzymlogenthe Aknowspecifienzymdoma16 kDsituatbetweshowthe ativityet al.,produfragmC-termity to

MomRNdeter(HakacrystaferedmolecWhenrequi(Ho e

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expression and purification of domainsd 2 of T7 ligase

order to dissect the activity of T7 ligase, thedomains (see Figure 1) were cloned and pro-d separately as described in Materials andods. In both cases there was a large pro-on of recombinant protein with the expectedcular mass and, unlike the recombinant T7e proteolysis fragments (Doherty et al., 1996b),f the expressed protein was soluble. The twoains were purified using the procedures out-

above, both giving final yields of 50-60 mg/lcterial culture. The purity of the proteins waser than 99 % as determined by Coomassie-ed SDS/polyacrylamide gels.

uie

64 Functional Domains of an ATP-dependent DNA Ligasean et al., 1994). Studies of mRNA cappinges have revealed that the reaction proceeds

covalent GMP-enzyme intermediate in whichMP moiety is attached to the protein via a

residue (Shuman & Hurwitz, 1981). Thisresidue is part of the conserved active site

. Furthermore, there are other motifs that areonserved between DNA ligases, RNA ligases,ligases and capping enzymes (for a review,

human & Schwer, 1995). These similaritiesst that nucleotidyl transfer by all of thesees must share a common mechanism and

he enzymes are likely to have a similar struc-Shuman & Schwer, 1995). This proposal wasrted initially by biochemical experiments on

nt ligase (Shuman & Ru, 1995) and cappinges (Cong & Shuman, 1993, 1995; Fresco &

owski, 1994; Shuman et al., 1994; Wang et al.,and more recently by the crystal structures ofNA ligase (Subramanya et al., 1996) and-1 mRNA capping enzyme (Hakansson et al.,

. Sequence alignments indicate clearly severalns of amino acid conservation between DNAs and the mRNA capping enzymes (Shumanwer, 1995). Invariant residues within some ofmotifs have been mutated and have been

n to be essential for both classes of enzyme,rting the concept of a nucleotidyltransferase

family (Cong & Shuman, 1993, 1995; Shuman, 1995; Wang et al., 1997).

have reported the characterisation and crys-tion of T7 DNA ligase (Doherty et al., 1996a).ubsequent determination of the crystal struc-f this enzyme has increased our understand-f the enzymatic mechanism of DNA ligasees (Subramanya et al., 1996). Five of the phy-

etically conserved motifs are located aroundTP bound at the active site and key residuesn to be essential for catalytic activity makec contacts with the ATP molecule. The T7e comprises two domains, an N-terminal

in 1 of 26 kDa and a C-terminal domain 2 ofa (Figure 1). The nucleotide binding pocket ised in the larger domain, at the base of a cleften the two domains. The structure also

ed an exposed surface loop in the T7 ligase,ccessibility of which explains the hyper-sensi-of the enzyme to protease digestion (Doherty1996b). Limited proteolysis of T7 DNA ligaseces two fragments, an N-terminal 16 kDaent containing the active site lysine and ainal 26 kDa fragment which retains the abil-

bind to DNA (Doherty et al., 1996b).re recently we have crystallised the PBCV-1A capping enzyme (Doherty et al., 1997), andmined the structure at 2.5 A resolutionnsson et al., 1997). The structures of the twollographically independent molecules dif-substantially in conformation, despite bothules containing a bound GTP molecule.challenged with manganese ions, which are

red as cofactor in the guanylation reactiont al., 1996), only one of these two capping

enzytionThissubstionposebe re

Inhypoby wtionsmighexpreligaspropThesmonsupechanficityof sithe edupl

Res

Over1 an

IntwoduceMethductimoleligasall odomlinedof bagreatstain

FigDomaresidue molecules was able to undergo guanyla-ithin the crystals to give the GMP-adduct.bservation provided direct evidence for antial conformational change during guanyla-

the capping enzymes. By analogy, we pro-that a similar conformational change might

uired for catalysis by ligases.rder to provide biochemical evidence for thishesis, and to analyse further the mechanismich DNA ligases perform their catalytic reac-

and how the domains and conserved motifscontribute to these processes, we have

sed separately the two domains of T7 DNAThe catalytic activities and DNA binding

rties of these domains are presented below.data provide conclusive evidence for a com-mechanism for this nucleotidyltransferaseamily that involves a large conformationale during catalysis. Furthermore, the speci-of these domains, with regard to the bindinggle or double-stranded DNA, explains howzyme is able to recognise nick sites on DNAxes.

re 1. Domain structure of T7 DNA ligase.n 1 consists of residues 1 to 240 and domain 2 ofs 241 to 359.

Domais stim

Basintereactivitthis aInitialbecomwith esencetion amagn(dataishedthe inexpectnylatisite mto disadenyof domtrationnylatiobservsuggeand ththe rawe obligasevage oSincesensitiunlike

Figudomai[a-32P]5 mM15 % praphy. ure 3. Adenylation of domain 1 is enhanced by

in 2. (a) Adenylation assays of domain 1 per-d in the presence and absence of domain 2 (see

rials and Methods). The proteins were separated onpolyacrylamide gel and stained with Coomassie

Lane 1, 5 mg of domain 1 and 2, and 5 mM PPi;2, 5 mg of domain 1 and 2, and 5 mM EDTA; laneg domain 1; lanes 4 to 7, 5 mg of domain 1 and aasing concentration of domain 2 (7, 5, 3 and 1 mg

Functional Domains of an ATP-dependent DNA Ligase 65in 1 has intrinsic adenylation activity thatulated by domain 2

ed on the structural information it was ofst to discover if domain 1 had adenylation

re 2. Adenylation of T7 DNA ligase. T7 ligasen 1 was incubated with either [a-32P]dATP orATP in 50 mM Tris-HCl (pH 7.5), 5 mM DTT,MgCl2. The samples were separated on an SDS/olyacrylamide gel and exposed to autoradiog-

FigdomaformeMatea 15 %blue.lane3, 5 mdecrey or whether it was possible to reconstitutectivity by mixing the two domains in vitro.

experiments revealed that domain 1es adenylated after incubating the proteinither [a-32P]ATP or [a-32P]dATP in the pre-of Mg2 (Figure 2), although the adenyla-ctivity of this domain is over an order of

itude lower than that of the intact enzymenot shown). Addition of EDTA or PPi abol-this activity completely, in common withtact enzyme (Doherty et al., 1996a). Ased, domain 2 alone had no detectable ade-

on activity since it does not retain the activeotif (KxDG). However, we were surprisedcover that it was possible to stimulate thelation activity of domain 1 by the addition

ain 2 (Figure 3). In fact, when the concen-s of the two domains were equal, the ade-

on activity of domain 1 was restored to thated for full-length ligase. This result

sts that domains 1 and 2 come into contactat this association of the domains enhances

te of adenylation significantly. Interestingly,served previously that preincubation of T7with ATP prior to proteolysis reduced clea-f the protein greatly (Doherty et al., 1996b).

the ATP-binding site and the proteolyticallyve site are distant from each other, it isly that the altered proteolytic sensitivity is

due tothat thformatmoredencebetweecrucialof ATP

Anastratedomaidomaiand abelutionexperimof domabsenc

respectigraph olationautoradshown)enhancreactionbinding of ATP per se. It is more likelye binding of the nucleotide induces a con-ional change which makes the proteinresistant to proteolysis. This indirect evi-

suggests that there is an interactionn the N and C-terminal domains which isfor the binding and subsequent hydrolysis.

lytical gel filtration was employed to demon-any such interactions between the two

ns in vitro. Stoichiometric amounts ofns 1 and 2 were preincubated in the presencesence of Mg2 and ATP. Figure 4 shows theprofile results from the gel filtration. Theseents confirmed that there is an associationains 1 and 2, which is stable even in the

e of ATP or Mg2.

vely); lane 8, 5mg of domain 2. (b) Autoradio-f the same gel (see above). A low level of adeny-

can be observed for domain 1 (lane 3) if theiograph is exposed for a longer time (data not. The level of adenylation of domain 1 ised dramatically by titration of domain 2 into the

(lanes 4 to 7).

Domaligas

Thestratecohesined.sive-eactiviparedthe aactividomaenzymendedunclestabil

Figuelutio30 mlwas cprotei(1 mgbrated5 mMlectedas indsamplively.imentabsendatafollowlengthof pea

u

66 Functional Domains of an ATP-dependent DNA LigaseFigfor 15in 1 of T7 ligase possesses DNAe activity

ability of domain 1 to ligate the known sub-s of intact T7 DNA ligase such as nicked,ive-ended and blunt-ended DNA was exam-Domain 1 was able to ligate nicked and cohe-nded DNA, but with a much reducedty (approximately 20 to 50-fold lower) com-

to the full-length enzyme (Figure 5). Unlikedenylation activity of domain 1, the ligationty was not stimulated by the addition ofin 2 (data not shown). In contrast to the intacte, domain 1 was unable to ligate blunt-DNA fragments. The reason for this is

ar, though it is possibly due to a reducedity of the ligase/DNA complex. Although

doma(see bdetectssDNA

DNA b

We1 anddsDNthat ddsDNcharacbindinin othstaphyDNA-proteissDNAwhichdomafor ssDconcendsDN2 is abSinceappeabetwebothfrompreviosurfacsite obetwe1996).

re 4. Analytical gel filtration of T7 ligase. Then profile of a mixture of domains 1 and 2 from aSuperdex S-200 gel filtration column. The columnalibrated by monitoring the elution of standardns. Samples (100 ml) of domain 1 and domain 2/ml) were loaded onto the column (pre-equili-

in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl,DTT and 2 mM EDTA). Fractions (1 ml) were col-and peak fractions were analysed by SDS-PAGEicated. Lanes 1 and 2 are markers containing

es of purified domain 2 and domain 1, respect-Although the data shown above are for an exper-in which domains 1 and 2 were incubated in the

ce of ATP or Mg2 prior to gel filtration, thesewere indistinguishable from similar experimentsing preincubation with ATP and Mg2. Full-DNA ligase elutes at a position equivalent to that

k 5.

(see Mradiolaadditioducts oacrylam3, 5 mPPi; laDNA lin 1 has significant ssDNA binding activityelow), no ssDNA ligation activity could beed. The intact enzyme is also unable to ligate

substrates (Doherty et al., 1996a).

inding affinity of domains 1 and 2

re 5. DNA ligase assay. Reactions were incubatedminutes at room temperature in ligation bufferaterials and Methods) and contained 8 pmol ofbelled DNA and 25 mg of domain 1, with otherns as indicated. Autoradiograph of ligation pro-f domain 1 separated on a denaturing 15% poly-ide gel. Lane 1, no ATP; lane 2, 5 mM ATP; lane

M AMPPNP; lane 4, 10 mM EDTA; lane 5, 5 mMne 6, single-stranded DNA control; lane 7, noigase.examined the substrate specificity of domaindomain 2 of T7 ligase using ssDNA and

A 40mer oligonucleotides. Figure 6 showsomain 1 has a very high affinity for bothA and ssDNA. Interestingly, domain 2 has ateristic OB (oligopeptide/oligonucleotide-g) fold (Murzin, 1993) that has been founder nucleic acid binding proteins includinglococcal nuclease (Hynes & Fox, 1991), the

binding domain of a bacterial cold shockn (Schindelin et al., 1993) and the gene V

-binding protein (Skinner et al., 1994), all ofbind to single-stranded DNA. However,

in 2 does not have any measurable affinityNA, even under conditions in which similartrations of domain 1 can shift all of the

A and ssDNA (Figure 6). In contrast, domainle to bind dsDNA quite effectively (Figure 6).both domain 1 and domain 2 bind dsDNA, itrs likely that the DNA binding site is situateden the domains in the intact protein withcontributing to the recognition, presumablyeither side of the duplex. We have shownusly that the electrostatic potential of thee of the protein is also consistent with thef DNA binding being located in the cleften the two domains (Subramanya et al.,

Disc

WprodC-terThelysinthe awasenzyprotelocatandthedigeshowdomdomdommenalsoingdomdom

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Functional Domains of an ATP-dependent DNA Ligase 67an Ato avidu

Thligaspectdomthisactivdomactivthattionlevepresactiv2. Tby droleconcT7woutheussion

e reported that limited proteolysis of T7 ligaseuces an N-terminal 16 kDa fragment and aminal 26 kDa fragment (Doherty et al., 1996b).16 kDa fragment contained the active sitee residue while the 26 kDa fragment retainedbility to bind to ligation sites on DNA but

inactive at ligation. The crystal structure of theme (Subramanya et al., 1996) revealed that theolytically sensitive region was actuallyed in an exposed surface loop of the protein,the accessibility of this loop region explainshyper-sensitivity of this region to proteasestion. Consequently, although the structureed that the enzyme did comprise two distinct

ains, domain 1 (residues 1 to 240; 26 kDa) andain 2 (residues 241 to 359; 16 kDa), theseains did not correspond to the proteolytic frag-ts. The structure of a complex with ATP wassolved and showed that the nucleotide bind-pocket is situated on the larger N-terminalain, at the base of the cleft between the twoains. The availability of the first structure of

indicdomabetwmoretratiodomature.explaconfotactschangmotifcontaimpliRu, 1protecondadenalthohydrobounstrucwhic(Subrlar care lchangnucleligasedays,nylatprolocause

Th1 mRhas cdepeprovimatioenzym

ure 6. DNA gel shift assays. Autoradiograph of aolyacrylamide gel showing the shifting of a 32P-

ed double-stranded (ds) and single-stranded (ss)r DNA fragment by T7 ligase domains 1 and 2.ions contained 8 pmol of radiolabelled DNA.s 1 and 2, 5 and 10 mg of domain 1, respectively,s 40-mer DNA; lanes 3 to 6, 2, 5, 10 and 15 mg ofin 1, respectively, and ds 40-mer DNA; lane 7, ss

er DNA only; lanes 8 and 9, 10 and 20 mg ofin 2, respectively, and ss 40-mer DNA; lanes 10

11 contained 10 and 20 mg of domain 2, respect-, and ds 40-mer DNA; lane 12, ds 40-mer only.TP-dependent ligase has allowed us to beginsign functions to the two domains and indi-l conserved residues of the enzyme.

e observation that domain 1 is an active, albeit with reduced activity, was unex-d and raised the question of the role ofin 2 in the enzyme activity. The answer touestion became clearer when the adenylationty of domain 1 was investigated. Althoughin 1 is capable of adenylation by ATP, thisty was reduced considerably compared toof the intact enzyme. However, the adenyla-activity of domain 1 could be restored to asimilar to that of the intact enzyme, in thence of domain 2. In contrast, the ligasety of domain 1 was not stimulated by domaine stimulation of the adenylation of domain 1,omain 2, indicates that both domains play an the adenylation reaction. At first sight, thisusion is at odds with the crystal structure ofgase complexed with ATP. This interactiond not be predicted from the conformation ofrotein seen in the crystal structure, which

ates that there are few contacts between theins. In particular, there are no contacts

een domain 2 and the bound ATP. Further-, the association of the two domains on gel fil-n suggests greater contact between theins than is indicated from the crystal struc-However, these apparent anomalies are

ined if the protein were able to undergo armational change that could increase the con-between the domains. Such a conformationale in the protein might also bring regions of

s V and VI, that are in domain 2, into closerct with the active site, since these have beencated in the adenylation activity (Shuman &995). The structure of the ATP complex of thein was obtained by soaking the crystals underitions in which the enzyme would becomeylated rapidly in solution. Surprisingly,ugh ATP was bound with high occupancy,lysis did not take place in the crystals and no

d magnesium ion was evident. In the crystalture, the protein is in an open conformation inh the domains are not in close proximityamanya et al., 1996). Inspection of the molecu-

ontacts within the crystal suggest that theseikely to prevent any large conformationale in the enzyme that might be required for

otide hydrolysis. This could explain whycrystals soaked in ATP, even after several

are unable to catalyse formation of the ade-ed intermediate. It is probably significant thatnged soaking in ATP and magnesium ionss the crystals to eventually shatter.e recent structure determination of the PBCV-NA capping enzyme (Hakansson et al., 1997)onfirmed the structural similarity with ATP-ndent DNA ligases. Furthermore, the structureded conclusive evidence for a large confor-nal change during guanylation of thee. Within the crystals there are two different

confopebounwitheculeGMPthewithligasdatabothchana prbothalsothewhoof th

Wassatheabilibut1996mensiteretaiveryligasteinposewhicas ahasnia DsyntthenucldatadomWhito ddsDNbindintacduplcoultheirdiffethatrecoferreDNAthisduplto bdomopporecotherespecin th

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68 Functional Domains of an ATP-dependent DNA Ligaseormations of the enzyme, referred to asn and closed, although both contain ad GTP molecule. However, when challengedmanganese ions, only one of the two mol-

s is able to undergo catalysis to yield the-adduct. This GMP-adduct is equivalent to

AMP-adduct formed upon reaction of ligaseATP. An equivalent conformational change in

e would be consistent with the biochemicalpresented here. We therefore propose thatenzymes undergo similar conformational

ges during the activation step of the reaction,ocess that requires participation of residues in

domains of the proteins. These observationsexplain the roles of a number of residues in

conserved motifs (notably those in motif VI)se roles were unclear from the crystal structuree T7 DNA ligase.e have shown, using native gel mobility shiftys, that removal of 130 amino acid residues atN terminus of T7 ligase does not affect thety of the truncated protein to bind to DNA,destroys its catalytic activity (Doherty et al.,b). The loss of catalytic activity of the frag-t is presumably due to removal of the activelysine residue (Lys34). However, the fragmentns the ability to bind to dsDNA, and is also aeffective competitive inhibitor of intact DNA

e (Doherty et al., 1996b). The truncated pro-retains the cleft between the domains, the pro-d DNA-binding site (Subramanya et al., 1996),h probably explains why the fragment can actcompetitive inhibitor of the intact enzyme. It

been demonstrated (Shuman, 1996) that vacci-NA ligase can form a stable complex with a

hetic nicked dsDNA substrate. Formation ofcomplex is reduced significantly if a oneeotide gap is present in the DNA. With thesein mind, the DNA binding properties of the

ains of T7 ligase proved to be interesting.le domain 1 is able to bind to either ssDNA oruplex DNA, domain 2 can only bind toA despite having a fold similar to ssDNA

ing proteins (Subramanya et al., 1996). Thet enzyme only binds dsDNA or nickedexes (Doherty et al., 1996b). This observationd explain how ligases are able to recognise

DNA substrates. The DNA at a nick site hasrent properties on either side of the helix atpoint, and the enzyme requires an ability to

gnise these features. The AMP has to be trans-d from the enzyme to the 50 free end of the

at the nick and domain 1 needs to recognisefree end but only as a part (or an end) of aex. Consequently, domain 1 needs to be ableind both ssDNA and dsDNA. However, sinceain 2 binds to the DNA duplex on the sidesite to the nick, it only needs to be able to

gnise dsDNA. The selectivity for nick sites isfore achieved by a combination of these two

ificites, in an appropriate spatial organisation,e intact enzyme.

ThevidedomDomreprebothfullWhation?the scompobvioise th

Mat

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AllenzymLtdresins(Uppfrom[a-35SLtd. Tkin-Eobtain

Strain

Thhosts(supEproABfor thsingle(hsdSgeneoveregrowagar

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Allenzymtocolsmaniprevi1989)Applcleoticia) a

Clon

Totities,T7 gewerethe dGTGGGGGAA(50-GACTGdata presented here provide biochemicalnce for a dual role for both domain 1 andin 2 in catalysis and DNA recognition.in 1, the N-terminal catalytic core, appears toent the smallest domain which can perform

ATP hydrolysis and DNA ligation, althoughigase activity requires the intact enzyme.is the role of domain 2 in the ligation reac-A complete answer to this question awaitsructure of a complex of the intact enzymelexed with a DNA substrate, though oneus possibility is that domain 2 helps to stabil-

binding of ligase at ligation sites on DNA.

rials and Methods

als

restriction endonucleases and modificationes were obtained from Boehringer Mannheim UKewes, UK) or Gibco-BRL. Protein purificationand columns were obtained from Pharmacia

ala, Sweden). All other chemicals were obtainedigma (Poole, Dorset, UK) unless stated otherwise.

dATP was obtained from Amersham Internationalaq polymerase (AmpliTaq) was obtained from Per-

er Cetus, Emoryville, CA. Sequenase II wased from United States Biochemicals, Ohio, USA.

s and cloning vectors

following strains of Escherichia coli were used asor pET21 (Novagen) derived constructs. XL1-Blue4, hsdR17, recA1, endA1, gyrA96, thi-, reclA, [F0, lac Iq Z?M15, Tn10 (tetR)] ) (Bullock et al., 1987)e propagation of clones and the preparation ofstranded DNA for sequencing, and B834(DE3)gal, (lcIts857, ind1, met-, Sam 7, nin 5, lacUV5-T7) (Wood, 1966; Studier & Moffatt, 1986) for thepression of T7 DNA ligase. E. coli strains wereaerobically in Luria broth (LB) or on Luria broth

lates containing the appropriate antibiotics.

al techniques

restriction endonucleases and modificationes were used according to the manufacturers pro-

All the transformations, DNA isolations andulations were performed essentially as describedusly unless stated otherwise (Sambrook et al.,

Oligonucleotides were synthesised using and Biosystems 381A DNA synthesiser. The oligonu-es were desalted on NAP G200 columns (Pharma-cording to the manufacturers instructions.

g of T7 ligase domain 1 and domain 2

enable production of the domains in large quan-we amplified the respective gene fragments fromomic DNA using PCR. Oligonucleotide primersesigned to be based on the 50 and 30 sequences ofmain regions: a 50 primer (50-GA TAT ACC ATGAC ATT AAG ACT AAC-30) and a 30 primer (50-

TTT GAA TTC CTA ACG GAT TGG TTC AACTAA-30) for the PCR of domain 1 and a 50 primer:

TAT ACC ATG GAT AAA GTT CCC TTT AAGAC-30) and 30 primer (50-GGG TTT TAA GCT

TACdomaprimethe Pbased

PCandAmpPCR(Gibcand pThefurthdephtransagarmethnucleSinglXL1-Baccor

Over

Twampilateding t[pT70.6-0.0.5 mthreeat 50requi(w/v(pHPMSF20,00to pu

Thdomavolumthe presushepar90 %2 M sfurthwas40 %tainedThe cthe 2NaCllowequiliwithbuffewereS-75conta

ThammtivityroseA shwas rNaCl

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Functional Domains of an ATP-dependent DNA Ligase 69ATT TTC TCT TGA GGG-30) for the PCR ofin 2. The 50 primers contained an NcoI site, the 30rs had a HindIII site after the stop codon allowingCR products to be cloned into the T7 promoterexpression vector pET21d (Novagen).

R experiments were performed with these primersbacteriophage T7 genomic DNA (Sigma) usingliTaq cycled as described (Saiki et al., 1988). Theproduct was electrophoresed in 1 % (w/v) agaroseo-BRL) and the DNA band excised from the gelurified using a Qiaex DNA extraction kit (Qiagen).

DNA was digested with NcoI and HindIII, ander gel purified. The fragments were ligated toosphorylated, NcoI and HindIII cleaved pET21d,formed into E. coli XL1-Blue and plated onto LBplates containing 100mg/ml ampicillin. The dideoxyod (Sanger et al., 1977) was used to confirm theotide sequence of the cloned T7lig fragments.e-stranded template DNA was produced in E. colilue by using helper phage M13KO7 (Pharmacia)

ding to the manufacturers instructions.

expression and purification of ligase domains

o litre cultures of Luria broth containing 100 mg/mlcillin and 50 mg/ml chloramphenicol were inocu-with a 5 ml culture of B834(DE3)[pLysS] contain-

he appropriate domain construct ([pT7 Dom 1] orDom 2]) and grown at 37 C until the A600 reached7. The cultures were induced by the addition ofM IPTG, and growth was continued for a furtherhours before harvesting the cells by centrifugation

00 g. The cell pellets were stored at 20 C untilred. The pellets were lysed by sonication of a 10 %) cell suspension in buffer A (50 mM Tris-HCl7.5), 2 mM EDTA, 5 mM DTT) containing 100 mM. The cell debris was pelleted by centrifugation at

0 g. Different purification strategies were employedrify the different domains.e cell lysates containing either T7 domain 1 orin 2 were precipitated by addition of an equale of a saturated ammonium sulphate solution andrecipitate pelleted at 20,000 g. The pellets were

pended in buffer A. Domain 1 was applied to ain-Sepharose column (20 ml) equilibrated withbuffer A and 10 % buffer B (buffer A containingodium chloride). After washing the column with a

er two column volumes of this mixture the proteinstep eluted by washing with buffer A containingbuffer B. SDS-PAGE confirmed that this peak con-

the semi-purified enzyme at high concentrations.onductivity of the pooled peak fractions containing6 kDa fragment was reduced to that of 200 mMby dilution with buffer A. This was loaded onto a

substitution Blue-Sepharose column (40 ml) pre-brated in buffer A. The column was then washed80 ml of buffer A before eluting the protein withr A containing 40 % buffer B. The peak fractionspooled, concentrated and applied to a Superdex

column which was pre-equilibrated with buffer Aining 10 % buffer B.e domain 2 protein was purified by loading theonium sulphate cut fraction (diluted to a conduc-

less than that of 100 mM NaCl) onto a Q-Sepha-column which was pre-equilibrated in buffer A.allow gradient of 0.2 M to 1 M NaCl in buffer Aun, with domain 2 eluting at approximately 0.7 M. The peak fractions were pooled, concentrated and

applibrate

Pr(w/vGelsdestameth

DNA

LigDNA10 m20 mstoppfor fiphorbefor

DNA

Th& Crby inmmo45 mThe uthroureact(pHwererun o(w/v

DNA

DNby i100 m50 un37 CratedS-200assayChanoligoM13mallowincub10 mthe pcatedThe rcinging aseparurea

Ack

Ththankfor syd to a Superdex S-75 column which was pre-equili-with buffer A containing 10 % buffer B.

tein samples were analysed by SDS-PAGE in 15 %gels with 4 % (w/v) stacking gels (Laemmli, 1970).ere stained with Coomassie brilliant blue and

ned in 10 % (v/v) acetic acid and 25 % (v/v)nol.

igase labelling

ase-AMP adducts were produced by incubating T7ligase (1 mg/ml) in 50 mM Tris-HCl (pH 7.5),

MgCl2, 2 mM DTT and 1 mCi of [a-32P]ATP in

for 15 minutes at 21 C. The reactions wered by boiling in SDS sample buffer (Laemmli, 1970)e minutes and products were analysed by electro-sis on 15 % PAGE-SDS gels. The gels were driedbeing autoradiographed on Fuji RX X-ray film.

obility shift assays

se were performed essentially as described (Friedthers, 1981). Oligonucleotide (20 mg) was labelledubation with 100 mCi of [g-32P]ATP (3000 Ci/

) and 50 units of T4 polynucleotide kinase forutes at 37 C followed by ten minutes at 70 C.

nincorporated label was removed by centrifugationh a S-200 micro spin column (Pharmacia). Bindingns were carried out in 10 ml of 50 mM Tris-HCl.5), 5 mM DTT, 5 mM MgCl2. Reaction mixturesncubated at room temperature for 20 minutes andn 6-8 % polyacrylamide gels which contained 5 %glycerol.

igase assay

A ligase assay substrate (22mer) was radio-labelledcubating 2.5 nmol of the oligonucleotide withi of [g-32P]ATP (3000 Ci/mmole, Amersham) and

ts of T4 polynucleotide kinase for 45 minutes atfollowed by ten minutes at 70 C. The unincorpo-label was removed by centrifugation through amicro spin column (Pharmacia). The DNA ligasewas performed essentially as described (Yang &1992). The complementary 18mer and 22mer

ucleotides were annealed to single-strandedp19 by incubation at 70 C for two minutes andd to cool for one hour. The annealed DNA wasted with ligase buffer (50 mM Tris-HCl (pH 7.5),

MgCl2, 5 mM DTT) unless stated otherwise, inesence of enzyme and nucleotide cofactors as indi-in a total volume of 10 ml for 15 minutes at 25 C.actions were terminated by the addition of sequen-top buffer (Sequenase kit, USB) followed by heat-95 C for five minutes. The ligation products wereted by electrophoresis on a 15 % polyacrylamide-el and autoradiographed with Fuji RX X-ray film.

owledgements

s work was supported by the Wellcome Trust. WeHu Pan for technical assistance and Val Cooperthesising oligonucleotides.

Refe

BaymKseo

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70 Functional Domains of an ATP-dependent DNA Ligaserences

iller, J., Jennings, S., Kienzle, B., Gorman, J. A.,elly, R. & McCullough, J. E. (1994). Isolation andquence of the transfer-RNA ligase-encoding gene

f Candida albicans. Gene, 142, 129-134.k, W. O., Fernandez, J. M. & Short, J. M. (1987).L1-blue a high efficiency plasmid transformingcA Escherichia coli strain with beta-galactosidaselection. Biotechniques, 5, 376.P. J. & Shuman, S. (1993). Covalent catalysis in

ucleotidyl transfer - a KDG motif essential forzyme-GMP complex formation by messenger

NA capping enzyme is conserved at the active-tes of RNA and DNA ligases. J. Biol. Chem. 268,256-7260.

P. J. & Shuman, S. (1995). Mutational analysis ofessenger RNA capping enzyme identifies amino-

cids involved in GTP binding, enzyme-guanylatermation, and GMP transfer to RNA. Mol. Cell.

iol. 15, 6222-6231.ty, A. J., Ashford, S. R., Subramanya, H. S.

Wigley, D. B. (1996a). Bacteriophage T7NA ligase: cloning, over-expression, crystallisation

nd characterisation. J. Biol. Chem. 271, 11083-11089.ty, A. J., Ashford, S. R. & Wigley, D. B. (1996b).haracterisation of proteolytic fragments of bac-riophage T7 DNA ligase. Nucl. Acids Res. 24,

281-2287.ty, A. J., Hakansson, K., Ho, C. K., Shuman, S. &igley, D. B. (1997). Crystallisation of the RNA

uanyltransferase of Chlorella virus PBCV-1. Actarystallog. sect. D, 53, 482-484., M. J. & Richardson, C. C. (1982). DNA ligases.The Enzymes (Boyer, P. D., ed.), vol. 15, pp. 3-29,

cademic Press, New York., L. D. & Buratowski, S. (1992). Active-site of theessenger RNA capping enzyme guanylyltransfer-

se from Saccharomyces cerevisiae - similarity toe nucleotidyl attachment motif of DNA andNA ligases. Proc. Natl Acad. Sci. USA, 91, 6624-628.M. & Crothers, D. M. (1981). Equilibria and kin-ics of lac repressor-operator interactions by poly-

crylamide-gel electrophoresis. Nucl. Acids Res. 9,505-6525.sson, K., Doherty, A. J., Shuman, S. & Wigley,. B. (1997). X-ray crystallography reveals a largenformational change during guanyl transfer byRNA capping enzymes. Cell, 89, 545-553.y, S., Sing, M. & Gait, M. J. (1987). Effect of single

mino-acid changes in the region of the adenylyla-on site of T4 RNA ligase. Biochemistry, 26, 1688-696.

. K., Vanetten, J. L. & Shuman, S. (1996).xpression and characterization of an RNA cappingzyme encoded by Chlorella virus PBCV-1. J. Virol.

0, 6658-6664., T. R. & Fox, F. O. (1991). The crystal-structure ofaphylococcal nuclease refined at 1.7 A resolution.roteins: Struct. Funct. Genet. 10, 92-105.n, A. (1992). Molecular characterization of a DNAgase gene of the extremely thermophilic archaeonesulfurolobus ambivalens shows close phylogeneticlationship to eukaryotic ligases. Nucl. Acids Res.

0, 5389-5396.li, U. K. (1970). Cleavage of structural proteins

uring the assembly of the head of bacteriophage4. Nature, 227, 680-685.

Lehm

Lind

Mur

Saiki

Samb

Sang

Schin

Shum

Shum

Shum

Shum

Shum

Skin

Stud

Subr

Tom

Wanan, I. R. (1974). DNA ligase: structure, mechanism,nd function. Science, 186, 790.hl, T. & Barnes, D. E. (1992). Mammalian DNAgases. Annu. Rev. Biochem. 61, 251-281.in, A. G. (1993). OB (oligonucleotide oligosacchar-e binding)-fold - common structural and func-

onal solution for nonhomologous sequences.MBO J. 12, 861-867.R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J.,iguchi, R., Horn, G. T., Mullis, K. B. & Erlich,. A. (1988). Primer-directed enzymatic amplifica-on of DNA with a thermostable DNA polymerase.cience, 239, 487-491.rook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecu-r Cloning: A Laboratory Manual, 2nd edit., Coldpring Harbor Laboratory Press, Cold Springarbor, NY.r, F., Nicklen, S. & Coulson, A. R. (1977). DNAequencing with chain-terminating inhibitors. Proc.atl Acad. Sci., USA, 74, 5463-5467.

delin, H., Marahiel, M. A. & Heinemann, U. (1993).niversal nucleic acid-binding domain revealed by

rystal structure of the Bacillus subtilis major cold-hock protein. Nature, 364, 164-168.an, S. (1996). Vaccinia virus DNA ligase - speci-city, fidelity, and inhibition. Biochemistry, 34,6138-16147.an, S. & Hurwitz, J. (1981). Mechanism ofessenger RNA capping by vaccinia virus guany-ltransferase - characterization of an enzyme gua-

ylate intermediate. Proc. Natl Acad. Sci. USA, 78,87-191.an, S. & Ru, X. M. (1995). Mutational analysis ofaccinia DNA ligase defines residues essential forovalent catalysis. Virology, 211, 73-83.an, S. & Schwer, B. (1995). RNA cappingnzyme and DNA ligase - a superfamily ofovalent nucleotidyl transferases. Mol. Microbiol.7, 405-410.an, S., Liu, Y. Z. & Schwer, B. (1994). Covalent cat-lysis in nucleotidyl transfer reactions - essentialotifs in Saccharomyces cerevisiae RNA capping

nzyme are conserved in Schizosaccharomyces pombend viral capping enzymes and among polynucleo-de ligases. Proc. Natl Acad. Sci. USA, 91, 12046-2050.er, M. M., Zhang, H., Leschnitzer, D. H., Guan, Y.,ellamy, H., Sweet, R. M., Gray, C. W., Konings,. N. H., Wang, A. H. J. & Terwilliger, T. C. (1994).tructure of the gene V protein of bacteriophage F1etermined by multiwavelength X-ray-diffractionn the selenomethionyl protein. Proc. Natl Acad. Sci.SA, 91, 2071-2075.r, F. W. & Moffatt, B. A. (1986). Use of bacterio-hage T7 RNA polymerase to direct selective high-vel expression of cloned genes. J. Mol. Biol. 189,13-130.manya, H. S., Doherty, A. J., Ashford, S. R. &igley, D. B. (1996). Crystal structure of an ATP-

ependent ligase from bacteriophage T7. Cell, 85,07-615.inson, A. E., Totty, N. F., Ginsburg, M. & Lindahl,. (1991). Location of the active-site for enzyme-denylate formation in DNA ligases. Proc. Natlcad. Sci. USA, 88, 400-404., S. P., Deng, L., Ho, C. K. & Shuman, S. (1997).hylogeny of mRNA capping enzymes. Proc. Natlcad. Sci. USA, 94, 9573-9578.

Wei, Y.-F., Robins, P., Carter, K., Caldecott, K., Pappin,D. J. C., Yu, G. L., Wang, R. P., Shell, B. K., Nash,R. A., Schar, P., Barnes, D. E., Haseltine, W. A. &Lindahl, T. (1995). Molecular cloning andexpression of human cDNAs encoding a novelDNA ligase IV and DNA ligase III, an enzymeactive in DNA repair and recombination. Mol. Cell.Biol. 15, 3206-3216.

Wood, W. B. (1966). Host specificity of DNA producedby Escherichia coli: bacterial mutations affecting therestriction and modification of DNA. J. Mol. Biol.16, 118-133.

Yang, S. W. & Chan, J. Y. H. (1992). Analysis of the for-mation of AMP-DNA intermediate and the succes-sive reaction by human DNA ligase I and ligase II.J. Biol. Chem. 267, 8117-8122.

Edited by A. R. Fersht

(Received 26 May 1998; received in revised form 24 September 1998; accepted 7 October 1998)

Functional Domains of an ATP-dependent DNA Ligase 71

Functional Domains of an ATP-dependent DNA LigaseIntroductionResultsFigure 1Figure 2Figure 3Figure 4Figure 5Overexpression and purification of domains 1 and 2 of T7 ligaseDomain 1 has intrinsic adenylation activity that is stimulated by domain 2Domain 1 of T7 ligase possesses DNA ligase activityDNA binding affinity of domains 1 and 2

DiscussionFigure 6

Materials and MethodsMaterialsStrains and cloning vectorsGeneral techniquesCloning of T7 ligase domain 1 and domain 2Overexpression and purification of ligase domainsDNA ligase labellingDNA mobility shift assaysDNA ligase assay

AcknowledgementsReferences