MOLECULAR AND CELLULAR BIOLOGY, Jan. 1994, p. 310-3170270-7306/94/$04.00+0Copyright © 1994, American Society for Microbiology

Aberrant DNA Repair and DNA Replication Due to anInherited Enzymatic Defect in Human DNA Ligase ICLAUDE PRIGENT, MASAHIKO S. SATOH, GRAHAM DALY, DEBORAH E. BARNES,

AND TOMAS LINDAHL*Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms,

Hertfordshire EN6 3LD, United KingdomReceived 27 July 1993/Returned for modification 11 October 1993/Accepted 18 October 1993

Two missense mutations in different alleles of the DNA ligase I gene have been described in a patient (46BR)with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents. One of the mutant allelesproduces an inactive protein, while the other encodes an enzyme with some residual activity. A subline ofidentical phenotype that is homozygous (or hemizygous) for the mutant allele encoding this partially activeenzyme has facilitated characterization of the enzymatic defect in 46BR. This subline retains only 3 to 5% ofnormal DNA ligase I activity. The intermediates in the ligation reaction, DNA ligase I-AMP and nickedDNA-AMP, accumulate in vitro and in vivo. The defect of the 46BR enzyme lies primarily in conversion ofnicked DNA-AMP into the final ligated DNA product. Assays of DNA repair in 46BR cell extracts and of DNAreplication in permeabilized cells have clarified functional roles of DNA ligase I. The initial rate of ligation ofOkazaki fragments during DNA replication is apparently normal in 46BR cells, but 25 to 30% of the fragmentsremain in low-molecular-weight form for prolonged times. DNA base excision repair by 46BR cell extractsshows a delay in ligation and an anomalously long repair patch size that is reduced upon addition of purifiednormal DNA ligase I.

DNA ligase I is the major DNA ligase in mammalian cellsand is induced upon cell proliferation (16). A human cDNAencoding this 102-kDa protein can complement the replica-tion defect of a DNA ligase-deficient, conditional-lethal cdc9mutant of Saccharomyces cerevisiae, and the yeast andhuman enzymes show 37% sequence homology (2). Mis-sense mutations in both alleles of the DNA ligase I gene havebeen identified in a female patient exhibiting stunted and frailgrowth, severe immunodeficiency, and sun sensitivity, whodied at the age of 19 years with lymphoma (3, 32). Fibro-blasts from the patient, 46BR, are hypersensitive to killingby several DNA-damaging agents and also by 3-aminoben-zamide, an inhibitor of poly(ADP-ribose) polymerase (27,28). Moreover, 46BR cells are anomalously sensitive toinduction of sister chromatid exchanges and show delayedstrand break rejoining after DNA damage (10). Retardedjoining of Okazaki fragments during DNA replication hasalso been reported for 46BR cells (10, 15, 17). These obser-vations suggest a role for DNA ligase I in both lagging-strandDNA synthesis and DNA repair processes.One of the mutations in 46BR fibroblasts results in an

inactivating Glu-566--Lys replacement within the highlyconserved active site of the enzyme. The mutation in theother allele, Arg-771-+Trp, also occurs in a region of theprotein showing marked evolutionary conservation (3). Thelatter mutation is inherited from the mother and is present intwo brothers of the patient. All three heterozygotes areclinically normal (32). A simian virus 40-transformed sub-line, 46BR. lGl, that shows the same physiological defectsas the primary 46BR fibroblast strain has been established(15). 46BR. lGl is homozygous or hemizygous for the allelecontaining the mutated Trp-771 residue and consequentlyhas a normal Glu residue at position 566 (3). Using the46BR. lGl cell line, we have investigated the effect of the

* Corresponding author. Fax: (44) 71-269-3803.

Arg-771--*Trp mutation on the activity of DNA ligase I, aswell as the altered function of the mutant enzyme in in vitroassays for DNA repair and replication.

MATERIALS AND METHODS

Cell lines. The simian virus 40-transformed human cell line46BR. lGl was derived from the DNA ligase I-defectiveprimary fibroblast strain 46BR (3) following transfection withpSV3gpt (15, 20). The simian virus 40-transformed lineMRC5V1 was established from normal human fibroblasts(11). The cells were propagated in Dulbecco's modifiedEagle's medium with 10% fetal bovine serum. HeLa cellswere grown in suspension culture under standard conditions.

Immunopurification of DNA ligase I from cell extracts.Frozen cell pellets were thawed and incubated for 30 min onice in lysis buffer containing several protease inhibitors (1ml/107 cells): 50 mM Tris-HCl (pH 8.5), 125 mM NaCl, 1%

Nonidet P-40, 2 mM EDTA, 100 mM Na3PO4, 170 ,ug ofphenylmethylsulfonyl fluoride per ml, 27 ,ug of aprotinin perml, and 0.5 ,ug each of leupeptin, pepstatin, chymostatin,and tosyllysine chloromethylketone (TLCK) per ml. Thelysate was centrifuged at 5,000 x g for 15 min at 4°C, and thesupernatant was recovered. Ten microliters of rabbit poly-clonal antiserum (13) raised against homogeneous bovineDNA ligase I was added per ml of extract, and then themixture was incubated at 0°C for 1 h. Immunocomplexeswere affinity purified on 2% (wtlvol) protein A-Sepharosebeads (4 Fast Flow; Pharmacia) at 4°C for 1 h with continu-ous mixing. The protein A-Sepharose beads were thenwashed three times with 1 ml of lysis buffer. ProteinA-Sepharose-bound immunocomplexes containing DNA li-gase I were used in in vitro DNA ligation assays. Forimmunoblotting analysis, DNA ligase I was released fromthe beads by heating at 90°C for 10 min in 20 ,ul of sodiumdodecyl sulfate (SDS) sample buffer, electrophoresedthrough an SDS-7.5% polyacrylamide gel, transferred onto

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nitrocellulose, and detected with DNA ligase I polyclonalantibodies and alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (Bio-Rad).DNA joining assay. The double-stranded polynucleotide

substrates [5'-32P]oligo(dT)16. poly(dA) and [5'-32P]oligo(dT)16. poly(rA) were made as described previously (22).Protein A-Sepharose-bound DNA ligase I obtained from 1 mlof cell lysate was incubated at 25 or 37°C in 50 pl of ligationbuffer containing 60 mM Tris. HCI (pH 8.0), 10mM MgCl2,50 pg of bovine serum albumin (BSA) per ml, 5 mMdithiothreitol (DTT), and 1 mM ATP with 1,ug of substrate(-100,000 cpm). Five-microliter aliquots were removed attimes indicated, and the reaction was stopped by adding 20,u of formamide (90%)-dyes, heating at 90°C for 10 min, andimmediately cooling on ice. 32P-labelled oligo(dT) multimerswere resolved by electrophoresis through 10% polyacryl-amide-8 M urea gels, detected by autoradiography, andquantitated by densitometry. DNA ligase activity is ex-pressed in femtomoles of [5'-32P]oligo(dT)16 ligated.Formation of the DNA ligase I-AMP reaction intermediate.

Where indicated, protein A-Sepharose-bound DNA ligase Iwas deadenylylated by pretreatment with 0.2 mM sodiumPPi in a reaction buffer containing 60 mM Tris. HCl (pH8.0), 10mM MgCl2, 50,ug of BSA per ml, and 5 mM DTT(adenylylation buffer) at 25°C for 10 min. The beads werethen washed three times with the buffer to remove the PPi.For the adenylylation reaction, DNA ligase I was incubatedat 25°C, for 1 min when not otherwise stated, in adenylyla-tion buffer containing [a-32P]ATP (3,000 Ci/mmol; Amer-sham) diluted with nonradioactive ATP to a specific activityof 250 Ci/mmol and added to a final concentration of 2 pMATP. The beads were then washed twice with lysis bufferand incubated for 10 min at 90°C in 20 ,ul of SDS samplebuffer, and the released protein-AMP complexes were ana-lyzed by electrophoresis through SDS-7.5% polyacrylamidegels. DNA ligase I-AMP was visualized by autoradiography.Formation of the DNA-AMP reaction intermediate. Protein

A-Sepharose-bound DNA ligase I was first deadenylylatedand then adenylylated with [a-32P]ATP as described above.The beads were washed with a reaction buffer containing 50mM morpholineethanesulfonic acid (MES)- NaOH (pH6.5), 10mM MgCl2, 50 pg of BSA per ml, and 5 mM DTT.The DNA ligase I-AMP was then incubated in the samereaction mixture with 60 ng of nonradioactive oligo(dT)16.poly(dA) at 25°C. Samples were removed at various times,and the reaction was stopped by adding formamide-dyes,heating at 90°C for 10 min, and immediately cooling on ice.Samples were analyzed by electrophoresis through 10%polyacrylamide-8 M urea gels. DNA-AMP intermediateswere visualized by autoradiography and quantitated bydensitometry.

Joining of the [32PJoligo(dT)-AMP- poly(dA) reaction inter-mediates. [5'-32P]oligo(dT)32 with a 5'-terminal AMP residuewas prepared essentially as described by Yang and Chan(35). Briefly, T4 DNA ligase was incubated with [5'-2P]oligo(dT)16. poly(dA) in the presence of ATP, and reac-tion products were subjected to electrophoresis through a15% polyacrylamide gel containing 8 M urea. [32P]oligo(dT)32-AMP was recovered as a minor band migratingslightly more slowly than [32P]oligo(dT)32, electroelutedfrom the gel, and hybridized with poly(dA) to generate thedouble-stranded substrate [32P]oligo(dT)32-AMP- poly(dA).Protein A-Sepharose-bound DNA ligase I, previously dead-enylylated, was incubated with this substrate in a reactionmixture containing 60 mM Tris. HCl (pH 8.0), 10 mMMgCl2, 50 ,ug ofBSA per ml, and 5 mM DTT but without any

ATP. Reaction products were detected by 10% polyacryl-amide-8 M urea gel electrophoresis and autoradiography andwere quantitated by densitometry.DNA repair by a cell extract of a plasmid containing a single

uracil residue. A single-strand break was introduced in thepBluescript SK plasmid (Stratagene) by digestion withBamHI in the presence of ethidium bromide (EtBr), and thenicked DNA was purified by EtBr-CsCl gradient centrifuga-tion. A single-strand DNA gap was then generated by brieflydigesting the nicked DNA with Escherichia coli exonucleaseIII. Plasmids containing a single-strand gap spanning lessthan 28 residues were eliminated after digestion with SacII;this restriction enzyme has a recognition site 28 bp upstreamof the BamHI site. Plasmids containing a single-strand gapspanning more than 28 residues remained circular after SacIItreatment. A single uracil residue was introduced by anneal-ing the synthetic oligonucleotide 5'-pGCTCTAGAACTAUTG-3' at the single-strand gap followed by repair withKlenow fragment and T4 DNA ligase to obtain a covalentlyclosed plasmid, which was purified by EtBr-CsCl gradientcentrifugation. The resulting construct contained a singleuracil residue opposite a C at position 730 of pBluescript SK,and the sequence flanking the U was 5'-TAUTGGATC-3'.Cell extracts were prepared from MRC5 and 46BR fibroblastlines as described by Manley et al. (19). DNA repair assayswith cell extracts were performed as described previously(25, 33). Substrate plasmid DNA (0.3 ,ug) was incubated in a50-p1 reaction mixture containing 45 mM N-2-hydroxyeth-ylpiperazine-N'-2-ethanesulfonic acid (HEPES) KOH (pH7.8), 70 mM KCI, 5mM MgCl2, 1 mM DTT, 0.4 mM EDTA,2 mM ATP, 20 ,uM each dCTP, dATP, and TTP, 8 ,uMdGTP, 2 pCi of [a-32P]dGTP (3,000 Ci/mmol), 40 mMphosphocreatine, 2.5 ,ug of creatine phosphokinase, 3%glycerol, 20 pg of BSA per ml, either 0 or 2 mM NAD, and10 pl of cell extract (-50 pg of protein) at 30°C for varioustimes. Where indicated, reaction mixtures were supple-mented with different amounts of bovine DNA ligase I(>97% pure [29]), equivalent to 2.5 to 10U of T4 DNA ligase(New England Biolabs). Reactions were stopped in 0.6%SDS-20 mM EDTA-240 pg of proteinase K per ml, andmixtures were incubated for 30 min at 37°C. After phenol-chloroform extraction, samples were ethanol precipitated,redissolved in 20 pl of 10 mM Tris- HCl (pH 8.0)-0.1 mMEDTA, and treated with RNase A (200 p.g/ml) for 10 min at37°C. The samples were then subjected to electrophoresisthrough 1% agarose gels, the DNA was visualized with EtBr,the gel was photographed, and the negative was used fordensitometry; the gel was dried and autoradiographed tomeasure repair replication and estimate repair patch sizes asdescribed previously (5, 25).

Analysis of newly synthesized DNA. Okazaki fragmentswere isolated and analyzed by the method of Burhans et al.(4). Cells (about 80% confluent) were synchronized at theG1/S boundary by isoleucine deprivation for 48 h followed byrelease in complete medium containing aphidicolin (10 p,g/ml) for 12 h. Cells were then washed with phosphate-buffered saline and incubated 5 min at 37°C in mediumwithout aphidicolin to allow transit to S phase. After collec-tion by low-speed centrifugation, 50 pl of packed cells wassuspended in 200 pl of replication mixture containing 30 mMHEPES KOH (pH 7.8), 0.1 mM each dGTP, dCTP, anddTTP, 0.2 mM each GTP, CTP, and UTP, 4 mM ATP, 10mM MgCl2, 0.1 mg of BSA per ml, and 1 mM DTT at 4°C.The cells were permeabilized by addition of Nonidet P-40 to0.4% and transferred to 34°C after addition of 100 ,uCi of[ct-32P]dATP (3,000 Ci/mmol). In some experiments, the

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312 PRIGENT ET AL.

replication mixture was supplemented with 10 ,uM dUTP or2 mM uracil. DNA replication was allowed to proceed for 60s, and the reaction was either chased for various times byadding 2 ,ul of 0.1 M nonradioactive dATP or directlystopped by lysing the cells with 40 mM Tris - HCl (pH 7.8)-8mM EDTA-0.3 M NaClI-.5% SDS-200 ,ug of RNase A perml for 1 h at 37°C. After addition of 200 ,ug of proteinase Kper ml, reaction mixtures were incubated for an additional 1h at 37°C. DNA solutions were then passed through a23-gauge needle to shear large DNA into fragments of about30 kb. After phenol-chloroform treatment, DNA was ethanolprecipitated and redissolved in 300 ,ul of H20 for 15 h at 4°C.Samples were analyzed by electrophoresis through 10%polyacrylamide-8 M urea gels and autoradiography, and theconversion of Okazaki fragments to high-molecular-weightDNA was quantitated by densitometry.

RESULTS

DNAjoining activity ofDNA ligase I from 46BR. lGl cells.In addition to DNA ligase I, human cells contain two otherDNA ligases (30), as well as a protein inhibitor of DNAligase I (34), which interfere with attempts to measure DNAligase I activity in cell extracts and partially purified proteinfractions. In this work, DNA ligase I was specifically recov-ered from human cell extracts by addition of nonneutralizingantibodies raised against homogeneous bovine DNA ligase I(13). Monoclonal antibody, 7A12, which recognizes anepitope in the nonessential N-terminal region of the enzyme,as well as polyclonal rabbit antibodies were used and yieldedequivalent results (data not shown). Immunocomplexeswere attached to protein A-Sepharose beads, washed toremove interfering human proteins, and assayed for DNAligase I activity. The joining of single-strand interruptions inthe double-stranded radioactive substrate, [5-32P]oligo(dT)16- poly(dA), was monitored by autoradiography anddensitometry of 2P-labelled multimers of (dT)16 separated indenaturing polyacrylamide gels.DNA ligase I from 46BR. lGl cells retained a small

amount of enzymatic activity in the DNA joining assay whenassayed at either 25 or 37°C, but this was reduced about20-fold in comparison with DNA ligase I from MRC5V1 andHeLa cells (Fig. 1). 46BR. lGl cells contain normal levelsofDNA ligase I protein (3), and immunoblotting experimentsshowed that similar amounts of DNA ligase I protein wererecovered from all three cell lines following immunoprecip-itation with DNA ligase I-specific antibodies and proteinA-Sepharose beads (Fig. 1D). The residual DNA ligaseactivity in the 46BR. lGl immunoprecipitate could not beascribed to DNA ligase II or III, because the antibodies useddo not cross-react with the latter enzymes (21a, 30). More-over, DNA ligases II and III differ from DNA ligase I inbeing able to join strand interruptions in the hybrid substrate[5 '-32P]oligo(dT) poly(rA) (30), and no detectable joining ofthis substrate by the DNA ligase I-containing immunocom-plexes from 46BR. lGl, MRC5V1, or HeLa cells wasobserved even after prolonged incubation times and autora-diographic exposures (data not shown). As an additionalcontrol, DNA ligase I was partially purified from 46BR* lGland MRC5V1 cells by ammonium sulfate fractionation andgel filtration to remove most of the DNA ligases II and IIIprior to immunoprecipitation (29), and the results remainedsimilar to those shown in Fig. 1A to C. Furthermore, DNAligase I requires phosphorylation in its N-terminal region bycasein kinase II for activation (23), but the reduced activityof the immunoprecipitated DNA ligase I from 46BR. lGl

cells did not appear to be due to underphosphorylationbecause incubation of the enzyme with purified casein kinaseII and GTP did not result in increased DNA ligase activity(data not shown).Formation of the DNA ligase I-AMP reaction intermediate.

DNA ligase I acts by the general reaction mechanism ini-tially established for microbial DNA ligases (7, 14, 16). In thefirst step of the reaction, the enzyme is activated by cleavageof the ATP cofactor to generate a covalent DNA ligase-AMPintermediate with a lysine-adenylate phosphoamide bond. Inagreement with a previous observation on DNA ligase I notassayed as an immune complex (3), the formation of aradioactively labelled DNA ligase I-AMP intermediate wasreduced 10- to 20-fold with the enzyme from 46BR. lGlcells compared with that from a control cell line (Fig. 2, lanes8 and 9). However, it was now found that if the DNA ligaseI-containing immunocomplexes attached to protein A-Seph-arose beads were pretreated with PPi to dissociate anyenzyme-AMP that might be present prior to incubation with[a-32P]ATP, both 46BR. lGl DNA ligase I and the controlMRC5V1 enzyme were effectively adenylylated with similarkinetics and to the same extent (Fig. 2, lanes 1 to 7). Thus,the mutated DNA ligase I from 46BR- lGl cells is normal inits ability to generate the enzyme-AMP reaction intermedi-ate, but in contrast to the control cell line, DNA ligase Iisolated from 46BR. lGl cells is apparently almost entirelypresent in an activated adenylylated form in vivo.Formation of the DNA-AMP reaction intermediate. The

DNA ligase I-AMP reaction intermediate interacts with aDNA single-strand break, and the AMP residue is trans-ferred to the 5' terminus of the break by the formation of aDNA-AMP PPi bond. The DNA-AMP complex is a transientreaction intermediate and does not accumulate to a signifi-cant degree during the normal DNA joining reaction, be-cause phosphodiester bond formation occurs immediatelyafter the transfer of the AMP residue to DNA (14). Inagreement with this scheme of events, only a small amountof [32P]AMP could be recovered in the form of DNA-AMPafter incubation of radiolabelled MRC5V1 DNA ligaseI-AMP with oligo(dT) poly(dA) (Fig. 3). In contrast, thereaction between 46BR lGl DNA ligase I-AMP and thesubstrate was highly unusual in that the covalent DNA-AMPintermediate accumulated during the reaction (Fig. 3). Whenthe disappearance of covalently bound [32P]AMP from theDNA ligase I-AMP intermediate in the presence of excessoligo(dT). poly(dA) substrate was monitored, it was foundto be slightly slower for the 46BR. lGl enzyme than for thecontrol enzyme (data not shown).The difference in the reaction of DNA ligase I-AMP with

the nicked DNA substrate was not due to a general decreasein DNA binding affinity of DNA ligase I from 46BR. lGlcells, because this protein and the MRC5V1 enzyme elutedat identical positions during enzyme purification by gradientchromatography on DNA-cellulose, with the peak fraction ofDNA ligase activity appearing at 92 + 5 mM NaCl in eachcase (data not shown).

Phosphodiester bond formation. In the final step of DNAjoining, nonadenylylated DNA ligase I catalyzes a reactioninvolving nucleophilic attack of the DNA 3' OH group on theactivated 5' P residue to generate a phosphodiester bond. Toinvestigate this step, [5'-32P]oligo(dT)32 with a 5'-terminalAMP residue was prepared enzymatically (35) and annealedwith poly(dA) to generate double-stranded nicked DNA-AMP. Upon incubation of this substrate with MRC5V1 DNAligase I attached to protein A-Sepharose beads in a reactionmixture without ATP, formation of phosphodiester bonds

MOL. CELL. BIOL.

DEFECTIVE HUMAN DNA LIGASE I 313

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FIG. 1. Residual DNA joining activity of DNA ligase I from 46BR * lGl cells. Protein A-Sepharose-bound DNA ligase I immunopurifiedfrom the control cell lines MRC5V1 (A) and HeLa (B) or from 46BR- lGl cells (C) was incubated with the polynucleotide substrate[5'-32P]oligo(dT)16- poly(dA) at 37°C for 0 (lane 1), 1 (lane 2), 5 (lane 3), 15 (lane 4), 30 (lane 5), or 105 (lane 6) min. Following ligation ofsingle-strand breaks in the substrate, oligo(dT)16 multimers were separated in polyacrylamide-urea gels and visualized by autoradiography.(D) Quantitation of protein A-Sepharose-bound DNA ligase I by SDS-polyacrylamide gel electrophoresis and immunoblotting. DNA ligaseI-[ 2P]AMP migrates as a 125-kDa protein that is detected with DNA ligase I-specific polyclonal antibodies and alkaline phosphatase-conjugated secondary antibody (13). The portion of the gel corresponding to approximately 65 to 200 kDa is shown, excluding theimmunoglobulin bands. (E) DNA joining activity of equivalent amounts of protein A-Sepharose-bound DNA ligase I from 46BR * lGl (0),MRC5V1 (0), and HeLa (El) cells as determined from the data in panels A to D and expressed in femtomoles of [5'-32P]oligo(dT)16 ligated.The inset shows longer reaction times.

occurred with generation of multimers of (dT)32 (Fig. 4). TheDNA ligase I from 46BR. lGl was clearly deficient in thisreaction step and could catalyze the formation of phosphod-iester bonds at only 3 to 5% of the rate of the control enzyme(Fig. 4). Thus, analysis of the individual steps in the DNAligation process indicates that the altered DNA ligase I from46BR- lGl cells can cataly2 the formation of enzyme-AMPnormally and retains its DINA-binding properties. The 20-fold-slower overall reaction rate is mainly due to a defect inthe last step, with impaired ability of the 46BR- lGl enzymeto catalyze cleavage of the PPi bond of the DNA-AMPintermediate to generate a DNA phosphodiester bond. Thismolecular defect also explains the unusual accumulation ofreaction intermediates observed with DNA ligase I from46BR. lGl cells (Fig. 5).

Complementation of defective DNA repair in 46BR- lGlcell extracts by purified DNA ligase I. Having identified thecatalytic defect of DNA ligase I in 46BR cells, we investi-gated the activity of the altered versus normal enzyme in

DNA repair. Human cell extracts prepared by the method ofManley et al. (19) can perform DNA base excision repairinvolving replacement of a dUMP residue (5). DNA incisionis rapidly catalyzed by uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease, whereas the consecutive stepsof excision, gap filling, and ligation occur more slowly. Inconsequence, a covalently closed circular DNA moleculecontaining a single damaged base, used as the substrate insuch an in vitro assay, is rapidly converted to an opencircular form. The DNA is then more slowly subject toshort-patch DNA repair and rejoined into a closed circularform (5, 25). The latter steps of the repair reaction areinhibited by the abundant cellular protein poly(ADP-ribose)polymerase, which is present in the cell extracts and bindsstrongly to DNA strand interruptions, but NAD-dependentautomodification releases this enzyme from DNA and re-lieves the inhibition (24).

Cell extracts of MRC5 and 46BR fibroblasts were com-

pared by this in vitro DNA repair assay. Both extracts

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FIG. 2. DNA ligase I-AMP formation. Equal amounts of proteinA-Sepharose-bound DNA ligase I from 46BR. lGl or the controlcell line MRC5V1 were deadenylylated by treatment with PP; (lanes1 to 7). From different immunoprecipitates, equal amounts of DNAligase I from 46BR. lGl and MRC5V1 were also used without PPipretreatment (lanes 8 and 9, duplicate samples). Aliquots wereadenylylated in the presence of [a-32P]ATP at 25°C for 0 s (lane 2),5 s (lane 3), 15 s (lane 4), 30 s (lane 5), 1 min (lane 6), 5 min (lane 7),or 1 min (lanes 8 and 9). After electrophoresis through SDS-polyacrylamide gels, adenylylated DNA ligase I was visualized byautoradiography. 14C-methylated protein size markers (Amersham)are shown in lane 1.

rapidly incised a uracil-containing plasmid. However, sub-sequent rejoining into repaired, closed circular DNA wasdelayed in 46BR compared with MRCS extracts althoughsome residual DNA ligase activity was present in the 46BRcell extract (Fig. 6). Addition ofNAD strongly promoted therepair reaction in both cell extracts (25), but the rate ofrejoining was still delayed in 46BR extracts. After longerincubation periods in the presence of 2 mM NAD, theamount of repaired, closed circular DNA in both MRC5 and46BR cell extracts apparently reached a maximal level forthe assay conditions. A similar delay in DNA rejoining by46BR cell extracts was observed when plasmids containingsingle-strand breaks introduced by y irradiation (24) wereused as the substrate (data not shown). Furthermore, when

1.0

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Time (min)FIG. 3. Formation of the DNA-AMP reaction intermediate.

Transfer of [32P]AMP from DNA ligase I-[32P]AMP tooligo(dT)16- poly(dA) was measured. Aliquots (5 fmol each) ofprotein A-Sepharose-bound DNA ligase I-[32P]AMP from46BR- lGl (-) and MRC5V1 (0) cells were incubated at 25°C andpH 6.5 for various times with nonradioactive oligo(dT)16 * poly(dA).The formation of oligo(dT)16-[32P]AMP was monitored by gel elec-trophoresis and quantitated by densitometry of autoradiograms.

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treated protein A-Sepharose-bound DNA ligase I from 46BR * lGl(i) and MRC5V1 (0) cells was incubated at 37°C for various timeswith [5'-32P]oligo(dT)-AMP poly(dA) in the absence of ATP. Thereaction products were analyzed by autoradiography following gelelectrophoresis as in Fig. 1, and DNA joining activity was deter-mined as femtomoles of oligo(dT)-AMP ligated.

the amount of DNA repair replication occurring during thereaction was measured, there was again a distinct differencebetween the two cell extracts. Control MRC5 extracts car-ried out short-patch repair, as estimated by the amount ofradioactive material incorporated into the repaired, co-valently closed circular plasmid (5). In contrast, 46BR ex-tracts produced repair patches about four times longer in aplasmid containing a single uracil residue (Fig. 7B, lanes 2and 7). The anomalously increased repair patch size gener-ated by 46BR cell extracts most likely reflects an increasedamount of DNA strand displacement, exonuclease action,and extension of the newly synthesized patch at the repairsite occurring as a consequence of slow and inefficient DNAligation. In a direct test of this model, purified calf thymusDNA ligase I was added to cell extracts in an attempt tonormalize this DNA repair defect. An incubation time of 60min in the presence of NAD was chosen, when the amountsof DNA rejoining in the two extracts were similar (Fig. 6 and7A, lanes 2 and 7). A small decrease in DNA repair synthesiswas apparent for control MRC5 extracts with added DNAligase I (Fig. 7B). However, addition of purified DNA ligaseI to 46BR extracts strongly reduced the repair patch in therejoined, closed circular DNA to a size similar to that seenwith MRC5 extracts and consequently complemented theDNA repair defect (Fig. 7).

Persistence of newly synthesized DNA fragments in perme-abilized 46BR. lGl cells. To investigate the possible role ofDNA ligase I in lagging-strand DNA replication and theeffect of the defective enzyme on this process in 46BR cells,synthesis and joining of Okazaki fragments (4) were mea-sured in permeabilized 46BR- lGl and control MRC5V1fibroblasts synchronized at the G1/S boundary and pulse-labelled with [a-32P]dATP. After 60 s, newly synthesizedradiolabelled DNA fragments were chased with an excess ofnonradioactive dATP, and samples were collected at differ-ent times to monitor the rate of conversion of the pulse-labelled fragments into high-molecular-weight DNA by elec-trophoresis in denaturing polyacrylamide gels andautoradiography (4).

314 PRIGENT ET AL.

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VOL. 14, 1994 DEFECTIVE HUMAN DNA LIGASE I 315

Ligase + ATP Ligase - AMP +PP_

Ligase - AMP + nicked DNA ([nicked DNA - AMP I + Ligase

Ligasenicked DNA - AMP --;=--+ sealed DNA + AMP

FIG. 5. Schematic representation of the reaction mechanism of the defective DNA ligase I from 46BR- lGl cells. The boxed-in reactionintermediates show anomalous accumulation due to the slow reaction rate in the final step. Since most of the enzyme is present as ligase-AMPrather than free ligase, which is required in the final step, the overall reaction is further retarded.

Similar amounts of newly synthesized DNA were recov-ered as Okazaki-size fragments, 60 to 250 nucleotides inlength (1), from 46BR- lGl and control MRC5V1 cells after60 s of pulse-labelling. These fragments were initially chasedinto high-molecular-weight DNA at indistinguishable rates,with 70 to 75% conversion after a 60-s chase period (Fig. 8).Thus, there was no discernible retardation in the initial rateof joining of most Okazaki fragments by the DNA ligaseI-defective 46BR. lGl cells. However, a clear differencewas seen at chase times beyond 2 min, in that 25 to 30% ofthe newly synthesized DNA remained in low-molecular-weight form for long time periods in the permeabilized46BR. lGl cells, whereas all such DNA was converted tohigh-molecular-weight form in control MRC5V1 cells (Fig.8).The persistence of a minor proportion of the newly syn-

thesized DNA in low-molecular-weight form could not beascribed to retarded joining of fragments of newly synthe-sized DNA in 46BR. lGl cells following incision at occa-sionally incorporated dUMP residues by uracil-DNA glyco-sylase (21). Highly purified dCTP, essentially free from

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00

60

(U 20

e0 00 30 60 90 120

Time (min)FIG. 6. DNA repair of a plasmid containing a single uracil

residue by cell extracts. Uracil-containing plasmid (0.3 ,ug) wasincubated with cell extract (50 p1g of protein) from 46BR (filledsymbols) and MRC5 (open symbols) cells, either with (triangles) orwithout (circles) 2 mM NAD, in 50-,ul reaction mixtures at 30°C forvarious times. At each time point, the relative proportions of closedcircular DNA and open circular DNA were determined by agarosegel electrophoresis in the presence of EtBr and by densitometry.Consistent results were obtained in several experiments, and stan-dard errors were as described previously (25).

contaminating dUTP, was used in these DNA synthesisexperiments, and deliberate addition of dUTP did not in-crease the proportion of low-molecular-weight fragments.Conversely, when reaction mixtures were supplementedwith 2 mM uracil to inhibit uracil-DNA glycosylase, theselow-molecular-weight fragments were still generated in46BR. lGl cells.

DISCUSSION

The reaction catalyzed by the malfunctioning DNA ligaseI from 46BR. lGl cells is shown in Fig. 5. The DNA ligaseI defect in the human cell line described here appearsanalogous to the molecular defect present in S. cerevisiaecdc9, Schizosaccharomyces pombe cdc17, and E. coli ligmutants. Biochemical characterization of the altered enzyme

MRC5

r-1 2 3 4 5 6

1 1 1 1 1 1A

B

46BR

7 8 9 10

1 1 1 1

- Gc

1-- cc

*CC- Ge

- Ccc

FIG. 7. Reduction of the DNA repair patch size generated in46BR cell extract by complementation with purified DNA ligase I.Plasmid (0.3 pLg) was incubated with cell extract (50 ,ug of protein)from MRC5 (lanes 1 to 5) or 46BR (lanes 6 to 10) cells in a 50-ilrepair reaction mixture containing [a-32P]dGTP and 2 mM NAD at30°C for 60 min. Lanes 1 and 6, control plasmid without uracil; lanes2 to 5 and 7 to 10, plasmid containing one uracil residue. Reactionmixtures were supplemented with purified calf thymus DNA ligase Ias follows: lanes 1, 2, 6, and 7, no added DNA ligase I; lanes 3 and8, 2.5 U; lanes 4 and 9, 5 U; lanes 5 and 10, 10 U. Reaction productswere separated by agarose gel electrophoresis and visualized withEtBr (A). DNA repair replication was determined by autoradiogra-phy of agarose gels (B). CC, closed circular DNA; OC, open circularDNA.

11

316 PRIGENT ET AL.

I--,

101

6(

CZg 4(El

,.< 2(

FIG. 8. Joireplication.and incubated[a-32P]dATP.allow for conhigh-moleculalysis. DNA w

rated by gel e

percentage ofments (60 tomated by deexperiments ('MRC5V1 (0)

has been caresidual catamolecular dreaction, an(ticularly renmutant (8, 9ligases in m,ligase I is amutations mgene.

Defectivebeen investilthe presentiWith regardsystem for bvivo studiesjoining of D'DNA repairbe ascribedrepair patchin the in vitrDNA ligasethe anomalotioning DN)expression46BR cells csulfonate anidata providcexcision repThe effec

cellular DN)vivo studies

of the newly synthesized DNA was retarded, and the sim-0o plest interpretation would be a general delay in the rate of

joining of Okazaki fragments into high-molecular-weightDNA. The more detailed studies with permeabilized cells

0 performed here did not really confirm this notion, since mostof the newly synthesized Okazaki fragments were joined atan apparently normal rate (Fig. 8). However, as 25 to 30% of

0 the newly synthesized DNA persisted as low-molecular-weight fragments in the permeabilized 46BR system, there isno experimental discrepancy between the present results

0 L and the earlier in vivo data. DNA ligase I is the only DNAligase induced upon cell proliferation, and the enzyme has

Dbeen detected in cellular replication complexes containingseveral other factors required for lagging-strand DNA syn-thesis (16, 18). Furthermore, DNA ligase I has been directlyimplicated in the completion of mammalian lagging-strand

0 5 1 0 4 DNA replication (31). If DNA ligase I is not the rate-limitingfactor in normal lagging-strand replication, the reduced

Time (min) activity of the 46BR enzyme may be sufficient for joiningining.o. Oka

Time . . most, but not all, fragments at the replication fork. Frag-ining~~~~~~~~~~~~ ~ofOaaifamnsdriglgigsrn.N ments that remain in low-molecular-weight form beyond the

'ells synchronized in early S phase were permeabilized .vfor a 60-s pulse with a replication mixture containing replisome may subsequently be joined only slowly in a DNAThe reaction was chased with nonradioactive dATP to repair-like reaction. Alternatively, many of the persistentversion of newly synthesized DNA fragments to a small fragments may no longer be hydrogen bonded to their-weight form and stopped after various times by cell template strand but be generated by DNA strand displace-ras isolated, and replication intermediates were sepa- ment of nonligated Okazaki fragments. The present datalectrophoresis and detected by autoradiography. The could also be explained by another DNA ligase being in-replication intermediates the size of Okazaki frag- volved in joining of Okazaki fragments, with the enzymes

250 nucleotides) remaining after the chase was esti- . . .s

,nsitometry of autoradiograms from three different being able to substitute inefficiently for each other.SE = +10%) carried out with both 46BR- lGl (@) and In 46BR cells, the persistence of part of the newly synthe-cells. sized DNA in low-molecular-weight form for much longer

time periods than normal may explain one of the mostcharacteristic phenotypes of these cells, i.e., a marked

Lrried out with several E. coli lig mutants. The hypersensitivity to 3-aminobenzamide (17, 28) that interferesalytic activity of the DNA ligase I in 46BR, the with poly(ADP-ribose) synthesis in cell nuclei. In a currentlefect in the latter stages of the DNA joining model (24), poly(ADP-ribose) polymerase binds tightly tod the corresponding cellular phenotype are par- DNA strand interruptions and is released by automodifica-niniscent of the properties of the E. coli fig4 tion; 3-aminobenzamide prevents this release from taking, 12). In spite of the presence of multiple DNA place and freezes poly(ADP-ribose) polymerase at strandammalian cell nuclei, it seems likely that DNA breaks, where it interferes with DNA repair. DNA replica-in essential enzyme, so that only leaky point tion forks with continuous leading-strand and discontinuousiay be found in patients with a defective LIG1 lagging-strand synthesis are protected by the replisome from

deleterious effects of poly(ADP-ribose) polymerase on lag-DNA repair and replication in 46BR cells have ging-strand synthesis. It is noteworthy that a simpler in vitrogated previously in vivo (10, 15, 17, 27, 28), and replication system, employing discontinuous DNA synthesisin vitro experiments extend these observations. by DNA polymerase a on both template strands, is suscep-to DNA repair, our data with a 46BR cell-free tible to inhibition by poly(ADP-ribose) polymerase (6). Inase excision repair are in agreement with the in the case of 46BR, newly synthesized DNA fragments escap-

; of Teo et al. (28); in both systems, delayed ing from the replisome without having been joined may beINA strand breaks and an increased amount of targets for binding of poly(ADP-ribose) polymerase, whichsynthesis were observed. The latter effect could would delay their conversion to a high-molecular-weighthere to an increase in size of individual DNA form; this effect would be greatly aggravated in the presencees. Moreover, the altered pattern of DNA repair of 3-aminobenzamide.o system was normalized by addition of purified Insights into DNA repair processes in human cells haveI to reaction mixtures, providing evidence that been facilitated by the occurrence of mutant human cell linesbus DNA repair could be ascribed to the malfunc- hypersensitive to DNA damage and derived from patientsA ligase I in 46BR cells. In separate studies, with inherited clinical syndromes. In contrast, inheritedof a cDNA encoding normal DNA ligase I in defects in enzymes involved in DNA replication or recom-:orrected the hypersensitivity to ethyl methane- bination have not been reported for human cells, presumablyd 3-aminobenzamide (26). Taken together, these because most mutations would lead to loss of viability.D evidence for a direct role of DNA ligase I in Studies on these processes in mammalian cells have insteadair in human cells. relied on detailed biochemical experimentation and tentativet of the DNA ligase I mutation in 46BR on parallels with genetically defined microbial model systems.A replication appears more complex. Earlier in The existence of a mutant cell line, with a defect in an(10, 15, 17) showed that joining of at least part extensively investigated enzyme acting in DNA repair and

MOL. CELL. BIOL.

DEFECTIVE HUMAN DNA LIGASE I 317

replication, provides a more direct tool for evaluating theroles of human DNA ligase I in DNA metabolism.

ACKNOWLEDGMENTS

We thank Julian Gannon for isolating monoclonal antibodiesagainst DNA ligase I.This work was supported by the Imperial Cancer Research Fund.

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VOL. 14, 1994