Vol. 265, No. 21, Issue of July 25, PP. 12611-12617, 1990 Printed in U.S. A.

THE JOURNAL OF B~~.~ccu. CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Mammalian DNA Ligases CATALYTIC DOMAIN AND SIZE OF DNA LIGASE I*

(Received for publication, March 26, 1990)

Alan E. Tomkinson, Dana D. LaskoS, Graham Daly, and Tomas Lindahl From the Imperial Cancer Research Fund, Clnre Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, United Kingdom

DNA ligase I is the major DNA ligase activity in proliferating mammalian cells. The protein has been purified to apparent homogeneity from calf thymus. It has a monomeric structure and a blocked N-terminal residue. DNA ligase I is a 125-kDa polypeptide as estimated by sodium dodecyl sulfate-gel electrophore- sis and by gel chromatography under denaturing con- ditions, whereas hydrodynamic measurements indicate that the enzyme is an asymmetric 9%kDa protein. Immunoblotting with rabbit polyclonal antibodies to the enzyme revealed a single polypeptide of 125 kDa in freshly prepared crude cell extracts of calf thymus. Limited digestion of the purified DNA ligase I with several reagent proteolytic enzymes generated a rela- tively protease-resistant 85-kDa fragment. This do- main retained full catalytic activity. Similar results were obtained with partially purified human DNA li- gase I. The active large fragment represents the C- terminal part of the intact protein, and contains an epitope conserved between mammalian DNA ligase I and yeast and vaccinia virus DNA ligases. The function of the N-terminal region of DNA Iigase I is unknown.

Two distinct DNA ligases have been detected in extracts of mammalian cells and Drosophila melanogaster embryos (l-8). The two mammalian enzymes do not cross-react serologically (2, 4) and exhibit different substrate specificities (5). More- over, only one of these ligases is induced on cell proliferation (9, 10). This enzyme, DNA ligase I, is the dominant ligase activity in rapidly dividing cells and tissues (2). It acts by a mechanism apparently identical to that established previously for Escherichiu coli DNA ligase (ll), with the formation of covalent ligase-AMP and DNA-AMP reaction intermediates, except that the mammalian enzyme, in common with bacte- riophage T4 DNA ligase, uses ATP and not NAD as the source of the AMP group (12-15).

The increase in DNA ligase I activity in response to cellular proliferation indicates that the enzyme may be involved in DNA replication. Recently, the observation that altered forms of DNA ligase I are present in cell lines representative of the human inherited disease Bloom’s syndrome has drawn in- creased attention to the enzyme (7,16-U). Bloom’s syndrome cells exhibit a reduced rate of progression of replication forks with delayed joining of DNA replicative intermediates (19-

* This work was supported by the Imperial Cancer Research Fund and by a grant from the Jane Coffin Childs Memorial Fund for Medical Research (to D. D. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Ludwig Institute for Cancer Research, Montreal, Quebec H3A lA1, Canada.

21) and show diminished ability to convert linearized plasmid DNA to a circular form after transfection (22).

DNA ligase I is sensitive to proteolysis in cell extracts, and the protein apparently has an asymmetric structure. More- over, it has been proposed (6, 23,24) that the enzyme may be derived from a very labile 200-kDa precursor molecule. For these reasons, widely different estimates have been made for the size and heterogeneity of the enzyme by different research groups (1, 15, 17, 18, 23-26). Here, we have investigated this problem by characterizing the physical properties of the in- tact, purified enzyme and then following its conversion by limited proteolysis to an active large fragment.

EXPERIMENTAL PROCEDURES

Cells and Tissues-Calf thymus glands were obtained from newly slaughtered calves (less than 6 months old) at the local abattoir. The tissues were packed in ice and used for initial enzyme preparation within 3 h.

A human lymphoblastoid cell line derived from a healthy individ- ual, GM1953, was obtained from the Human Genetic Mutant Cell Repository (Camden, NJ). These cells were grown in suspension culture at 37 “C in RPM1 1640 medium supplemented with 15% fetal bovine serum.

Purification of Calf Thymus DNA Ligase Z-All procedures were carried out at O-4 “C and centrifugations were at 10,000 X g for 30 min when not otherwise stated. Calf thymus glands (2 kg) were disrupted in aliquots by homogenization for 3 x 30 s in a Waring blendor with 4 liters of a buffer containing 0.1 M NaCl, 50 mM Tris- HCl, pH 7.5,l mM EDTA, 0.5 mM DTT,’ 1 mM phenylmethylsulfonyl fluoride, 1.9 pg/ml aprotinin, and 0.5 fig/ml each of leupeptin, pep- statin, chymostatin, and TLCK. After gentle stirring for 1 h, cellular debris was removed by centrifugation. The supernatant (Fraction I, crude extract) was diluted to a final NaCl concentration of 20 mM by the addition of 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM DTT and then batch adsorbed to 3.5 liters of a thick slurry (500 g dry weight) of Pll phosphocellulose (Whatman), which had been pre- equilibrated with 20 mM NaCl, 50 mM Tris-HCI, pH 7.5,1 mM EDTA, 0.5 mM DTT (buffer A). After washing the phosphocellulose with 10 liters of buffer A, adsorbed proteins were eluted with 3 liters of buffer A containing 0.5 M NaCl. The eluate (Fraction II) was supplemented with 231 g/liter ammonium sulfate, and 1 M Tris base was added intermittently to keep the pH at 7.1-7.5. After 30 min of gentle stirring, the precipitate was removed by centrifugation. Additional ammonium sulfate (160 g/liter) was added to the supernatant, and the resulting suspension was again neutralized and stirred gently for 30 min. The precipitate was collected by centrifugation, divided into four equal aliquots, and quickly frozen. The ammonium sulfate pre- cipitates could be stored at -70 “C with no significant loss of DNA ligase activity after 3 months. By this procedure, we avoided the use of frozen thymus glands which yield extracts containing higher levels of protease activity than fresh glands.

One quarter of the ammonium sulfate precipitate (material from 500 g of tissue) was diluted to a thick slurry and dialyzed for 4 h against 1 M NaCl, 50 mM Tris-HCI, pH 7.5, 1 mM EDTA, 0.5 mM DTT (buffer B). Insoluble material was removed by centrifugation

1 The abbreviations used are: DTT, dithiothreitol; SDS, sodium dodecyl sulfate; TLCK, No-p-tosyl-L-lysine chloromethyl ketone; FPLC, fast protein liquid chromatography.

12611

12612 Structure of DNA Ligase I

(Fraction III, 22 ml). Fraction III was loaded onto a 2.5 X 100~cm Ultrogel AcA 34 (Pharmacia LKB Biotechnology Inc.) column, equil- ibrated with buffer B. Proteins were eluted with buffer B and fractions were assayed both for DNA ligation activity and formation of enzyme- AMP complex. Due to its large size and asymmetric conformation, DNA ligase I eluted before the major protein peak. Active fractions were pooled and supplemented with 1 mM K,HPO, (Fraction IV, 60 ml). Fraction IV was loaded onto a 1.6 x 29-cm hydroxylapatite (Bio- Rad HT) column, which had been equilibrated with 1 M NaCl, 50 mM Tris-HCl, pH 7.5, 0.5 mM DTT, 1 mM K,HPO,. Efficient sepa- ration was only achieved if the hydroxylapatite was equilibrated with this low phosphate buffer immediately before use. Proteins were eluted by steps of 50, 150, and 400 mM potassium phosphate, pH 7.5, 0.5 mM DTT. The 150-mM phosphate eluate contained the DNA ligase I activity (Fraction V, 60 ml) and was dialyzed against 30 mM NaCl, 50 mM Tris-HCl, pH 7.5,l mM EDTA, 0.5 mM DTT, and 10% glycerol (buffer C).

The dialyzed Fraction V was loaded onto a 1.6 x 5-cm double- stranded DNA-cellulose (Sigma) column, pre-equilibrated with buffer C. After washing with buffer C, DNA ligase I activity was eluted with buffer C containing 0.3 M NaCl. Active fractions were pooled (Frac- tion VI, 4 ml) and dialyzed against buffer C. This material was loaded onto a FPLC Mono Q column (Pharmacia), pre-equilibrated with buffer C. Proteins were eluted with a 30-ml linear gradient of O-l M NaCl in buffer C. Fractions containina DNA liaase I activitv (Fraction VII, 1.0 ml) did not significantly lose activityafter storage for up to 2 weeks on ice. For longer term storage, the fractions were pooled and either dialyzed against buffer C containing 50% glycerol and stored at -20 “C, or frozen in liquid nitrogen before storage at -70 “C. The activity in fractions kept at -20 “C was stable for 2-3 months. The fractions stored at -70 “C lost activity on freezing and were used for amino acid sequencing studies.

Partial Purification. of Human DNA Liguse I-A 20-liter culture (8 x lo” cells/ml) of the human cell line GM1953 was harvested by centrifugation and washed with isotonic phosphate-buffered saline. The cells were then washed in 20 mM Tris-HCI, pH 7.5, 1.5 mM MgClr, 5 mM KCl, 1 mM DTT (buffer D) containing 250 mM sucrose, resuspended in 140 ml of buffer D (without sucrose) and allowed to swell in this hypotonic buffer for 20 min on ice. Following the addition of the protease inhibitors, leupeptin, chymostatin, pepstatin, TLCK (each to 0.5 kg/ml), aprotinin (1.9 Kg/ml), and 1 mM phenylmethyl- sulfonyl fluoride, the cells were lysed by Dounce homogenization and NaCl was added to a final concentration of 0.1 M NaCl. After 1 h on ice, cellular debris was removed by centrifugation. Human DNA ligase I was approximately 500-fold purified from this crude cell extract by ammonium sulfate fractionation, gel filtration, hydroxylapatite chro- matography, and DNA-cellulose chromatography as described for the calf thvmus enzyme, with a final yield of 0.2 mg of protein.

DNA Ligase Assays-Reaction-mixtures (6O;l) contained 60 mM Tris-HCl. nH 8.0. 10 mM M&l,. 5 mM DTT. 1 mM ATP. 50 uelml nuclease-free bovine serum albumin, polynucldotide substrate (2o:bOO cpm), and a limiting amount of DNA ligase. Incubations were at 16 “C for 15 min. For the preparation of substrate, oligo(dT)so was synthesized on a commercial DNA synthesizer, radioactively labeled (10 pg of oligonucleotide, 100 KCi of [r-“‘P]ATP (>5,000 Ci/mmol, Amersham Coin.). 10 umol of unlabeled ATP) using T4 nolvnucleo- tide kinase (P-L Biochemicals), and mixed with an equimolar”amount of poly(dA) (P-L Biochemicals). The conversion of 5’-32P-labeled phosphomonoesters to alkaline phosphatase-resistant diesters was measured (27, 28). One unit of DNA ligase activity catalyzes the conversion of 1 nmol of terminal phosphate residues to a phosphatase resistant form in 15 min at 16 “C.

Formation of DNA Ligase-adenylate-Reaction mixtures (10 ~1) contained 60 mM Tris-HCl, pH 8.0, 10 mM MgC12, 5 mM DTT, 50 pg/ml bovine serum albumin, 0.5 pCi of [a-32P]ATP (3000 Ci/mmol, Amersham Corp.), and DNA ligase I. Incubations were at room temperature for 15 min. After the addition of 5 ~1 of SDS sample buffer, reaction mixtures were heated at 90 “C for 10 min. Proteins were separated by electrophoresis through a 7.5% SDS-polyacryl- amide gel. Gels were fixed for 10 min in 10% acetic acid, dried, and adenylated proteins detected by autoradiography.

Guanidine Hydrochloride Gel Filtration-Following adenylation, DNA ligase I protein (Fraction VII) was dialyzed at room temperature against 5 M guanidine hydrochloride, 50 mM Tris-HCl, pH 7.5, 10 mM DTT, 1 mM EDTA, and then applied to a 1 X 72-cm Sephacryl S-200 column, pre-equilibrated with the same buffer. The column was calibrated with /3-galactosidase (116 kDa), bovine serum albumin (69 kDa), and P-amylase (45 kDa).

Sucrose Density Gradient Sedimentation-DNA ligase I (Fraction VII) was sedimented through 5 ml of 5-20% (w/v) linear sucrose gradients in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM DTT containing 0.15, 0.2, 0.5, or 1.0 M NaCl. Gradients were centrifuged at 40,000 rpm in a Beckman SW 50.1 rotor for 16 h at 4 “C. Fractions were collected from the bottom of the tube and DNA ligase I activity detected by the formation of polypeptide-AMP complex. In some cases DNA ligase I was adenylated prior to centrifugation, and adenylated polypeptides were detected by liquid scintillation count- ing. [Methyl-‘4C]bovine serum albumin (4.4sz0,w) (Du Pont-New Eng- land Nuclear) was used as internal molecular weight marker and was also centrifuged in a separate tube with r-globulin (7.3sg0.w), oval- bumin (3.66&w), and myoglobin (1.97s20.w) (Bio-Rad). The s20.w value for DNA liease I was calculated according to Martin and Ames (29).

Analytical Gel Filtration-Gel filtration of DNA ligase I (Fraction VII) was carried out at 4 “C on an FPLC Sunerose 12 column (Pharmacia), equilibrated with 50 mM Tris-HCl, pH 7.5,l mM EDTA, 0.5 mM DTT plus 0.2 or 1.0 M NaCl. Fractions containing DNA ligase I protein were detected by measuring absorbance at 280 nm and by the formation of polypeptide-AMP complex. The column was cali- brated with thyroglobulin (670 kDa), y-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin D (1.4 kDa) (Bio-Rad). [Methyl-i4C]bovine serum albumin (69 kDa) (Du Pont-New England Nuclear) was used as an internal and external molecular mass stand- ard. The Stokes radius of DNA ligase I was calculated according to Siegel and Monty (30).

Amino Acid Sequencing-N-terminal sequences of fragments and peptides derived from DNA ligase I were sequenced on an Applied Biosystems model 475a sequenator. Material from SDS-polyacryl- amide gels was first transferred to an Immohilon Transfer membrane (Millipore) and visualized by brief staining with Coomassie Blue.

Immunization Procedures-DNA ligase I (Fraction VII, >95% pure) was further purified by SDS-polyacrylamide gel electrophoresis. A gel slice containing DNA ligase I protein (approximately 100 pg) stained with Coomassie Blue was excised, homogenized in the gel running buffer, and then emulsified with Freund’s complete adjuvant. This material was injected in multiple small aliquots subcutaneously into a rabbit. The rabbit received four further injections of 100 pg each of active DNA ligase I (Fraction VII) emulsified with Freund’s incomplete adjuvant,-with an interval of 4 weeks between each iniection. The rabbit was bled 10 davs after the last iniection.

“A 17-mer peptide, (H,N)-CGISLRFPRFTRIREDK-(COOH), highly conserved (31) between yeast DNA ligases and vaccinia virus DNA ligase (except for the N-terminal cysteine linker residue) was made with a commercial peptide synthesizer. The peptide was coupled to keyhole limpet hemocyanin and bovine serum albumin using the glutaraldehyde method (32). Rabbit antibodies were raised against the peptides coupled to hemocyanin by following a similar immuni- zation schedule to the one described above. Antibodies specific for the peptide were purified on a Bio-Rad Affi-Gel-10 column to which peptide-conjugated bovine serum albumin had been coupled.

Zmmunoblots-Proteins were separated by SDS-polyacrylamide gel electrophoresis (33) and then transferred to nitrocellulose filters (Schleicher & Schuell) in 25 mM Tris-HCl, 192 mM glycine, 20% methanol, pH 8.3. After transfer, proteins were visualized by staining with Ponceau S and destained by washing in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl (TBS). After incubation for 2 h at room tempera- ture in TBS containing 20% fetal bovine serum (blocking solution), nitrocellulose strips were incubated for 12-16 h at 4 “C with antibody diluted in blocking solution. All subsequent steps were carried out at room temperature. The nitrocellulose strips were washed four times with TBS containing 0.05% Tween 20 to remove unbound antibody. After incubation for 5 min in blocking solution, ““I-protein A (0.1 &X/ml, Amersham Corp.) was added and incubation continued for 1 h. The nitrocellulose strips were then washed with TBS, dried, and exposed to preflashed x-ray film (Kodak) at -70 “C with an intensi- fying screen. When quantitation was required, a set of standards of known amounts of DNA ligase I were run on the same gel as the unknown sample. The intensity of the 125-kDa band on the autora- diogram was determined by densitometry scanning (LKB Ultrascan XL).

Digestion by Carborypeptidase Y-DNA ligase I (5 rg, Fraction VII) was dialyzed against 50 mM sodium acetate, pH 5.5, 5 M urea (34) for 4 h at room temperature. The final volume was adjusted to 75 ~1, and an aliquot (15 ~1) was taken. 500 ng of carboxypeptidase Y (Boehringer Mannheim) was then added and the reaction mixture was incubated at 37 “C. Aliquots were taken after 3, 10, 30, and 90

Structure of DNA Ligase I 12613

TABLE I Purification of DNA &use I from calf thymus

The purification of DNA ligase I from 500 g of calf thymus is shown. The first three steps were usually performed at a four times larger scale, as described under “Experimental Procedures.” The determination of DNA ligase activity in Fractions IV-VII was based on DNA ligation assays and also on the formation of an enzyme- AMP complex. Ligase assays on Fractions I-III gave underestimates due to the presence of interfering factors, and the numbers shown have been estimated by comparing immunoblotting data for these fractions and Fraction VII, and also by semi-quantitative enzyme assays. Protein concentrations were determined by the Bradford method (35).

Fraction Protein

I. Crude extract II. Phosphocellulose

III. Ammonium sulfate IV. Gel filtration

V. Hydroxylapatite VI. DNA-cellulose

w 13,400

1,500 450

30 6 0.9

Total activity

units 6.1 5.8 4.8 3.5 2.2 1.5

Specific activity

unitslmg 0.5 x 10-l

4 x lo-” 11 x lo-.’

0.12 0.37 1.7

VII. FPLC, Mono Q 0.4 1.0 2.5

min, and the reactions were stopped by the addition of 7.5 ~1 of SDS sample buffer followed by heating at 90 “C for 10 min. The samples were split into two, and the digestion products were separated on two 7.5% SDS-polacrylamide gels. The digestion products were detected on one gel by silver staining (Bio-Rad) and on the other gel by immunoblotting with the antiserum raised against the peptide highly conserved between yeast and vaccinia DNA ligases. The proportion of high molecular mass polypeptide (-125 kDa) detected by silver staining or immunoblotting was quantitated by scanning densitome- try.

RESULTS

Purification and Size of Calf Thynus DNA Ligase I-The enzyme purification procedure is summarized in Table I. The final yield of DNA ligase I was 15% and the enzyme was 5000- fold purified. Based on estimates of the number of cells/g of calf thymus (36, 37) there are 5000 molecules of DNA ligase I/cell. DNA ligase II, which only accounts for 510% of the total ligase activity in the crude cell extract, copurified with ligase I through the first three steps, but was removed in the subsequent gel filtration and hydroxylapatite chromatography steps (5). After DNA-cellulose chromatography, two major bands of 125 and 50 kDa and traces of other proteins were observed following SDS-polyacrylamide gel electrophoresis. The 50-kDa material did not adsorb to Mono-Q, whereas the 125-kDa protein, the DNA ligase activity, and the enzyme- adenylate forming activity coeluted at 0.26 M NaCl during gradient chromatography (Fig. 1). Analysis of the latter ma- terial (Fraction VII) by SDS-polyacrylamide gel electropho- resis (Fig. 2) indicated that DNA ligase I has a molecular mass of 125 kDa and that the preparation was greater than 95% homogeneous.

Several attempts to determine the N-terminal sequence of 50-100 pg amounts of two different preparations of the 125- kDa DNA ligase I by microsequencing yielded no clear positive signal. However, the amino acid sequences of several tryptic peptides could be determined from equivalent amounts of DNA ligase I by the same procedures.* We conclude that the N terminus of the 125-kDa polypeptide is blocked. Amino acid analysis of DNA ligase I revealed an unusually high proline content of 8-9% but otherwise no unusual features (data not shown).

In addition to the SDS-polyacrylamide gel electrophoresis

‘A. E. Tomkinson, D. D. Lasko, and T. Lindahl, unpublished observations.

1.0

0.5

0.0 k2 . . . . . ..I An- *NJ-..-----.,.I . .

0 20 40 60 80 Fraction No.

0.6

0.4

0.2

0.0

FIG. 1. Purification of calf thymus DNA ligase I by FPLC Mono Q chromatography. Fractions containing DNA ligase I ac- tivity following DNA cellulose chromatography (Fraction VI) were loaded by two injections of 2 ml each onto the Mono Q column, and bound proteins were gradient eluted. Protein was detected by absorb- ance at 280 nm (-). Assays for formation of enzyme-adenylate complex (inset) and DNA ligation (lY) were performed as described under “Experimental Procedures.” Size markers labeled with iJC were 200-kDa myosin, 92.5-kDa phosphorylase b, 69-kDa bovine serum albumin, 46-kDa ovalbumin, and 30-kDa carbonic anhydrase.

1 2 3 4 5

i -125 -125 116- 116-

97.5- 97.5-

69- 69-

FIG. 2. Size of DNA ligase I from calf thymus as measured by SDS-polyacrylamide gel electrophoresis. DNA ligase I pro- tein (Fraction VII) was subjected to electrophoresis in a 7.5% SDS- polyacrylamide gel. Lane I, 1 pg of DNA ligase I; lane 2, 2 pg of DNA ligase I; lane 3, 5 pg of DNA ligase I. Proteins were detected by staining with Coomassie Brilliant Blue. Lane 4, 0.5 rg of DNA ligase I; lane 5, SDS sample buffer alone. Proteins were detected by silver staining (Bio-Rad). The molecular mass markers were; myosin, 200 kDa; P-galactosidase, 116 kDa; phosphorylase b, 97.5 kDa; bovine serum albumin, 69 kDa; and ovalbumin, 43 kDa.

experiments (Fig. 2), the molecular weight of the DNA ligase I polypeptide was also determined by gel filtration chroma- tography in the presence of 5 M guanidine hydrochloride. A covalent DNA ligase I-[“‘PIAMP complex eluted immediately before the 116-kDa /3-galactosidase reference protein (data not shown). The estimated mass of DNA ligase I by this procedure was 125-130 kDa, in good agreement with the data obtained by SDS-polyacrylamide gel electrophoresis.

12614 Structure of DNA Ligase I

A rabbit antiserum against bovine DNA ligase I was raised by repeated injections of the homogeneous enzyme. When freshly prepared crude cell extracts of calf thymus were ana- lyzed by immunoblotting with the antibody, a single protein of 125 kDa was observed (Fig. 3, lane B). No corresponding material was detected with preimmune serum from the same rabbit (Fig. 3, lane A). There was no indication, in any of several immunoblotting experiments with the DNA ligase I antiserum, of the presence of the 200-kDa form of DNA ligase I reported by Teraoka and Tsukada (6, 23). These results suggest that the 125-kDa polypeptide is the primary transla- tion product and that this is the form of the enzyme acting in Go. A more detailed study with a bovine cell line yielded similar results (38).

Hydrodynamic Properties of Native DNA Ligase I-Gel filtration of the purified, intact enzyme on a column calibrated with several globular proteins yielded a Stokes radius of 53 A. The sedimentation coefficient of the enzyme, as determined by sucrose density gradient centrifugation together with ref- erence proteins, was 4.4 S. There was no evidence for degra- dation of the DNA ligase I protein during the gel filtration and sedimentation experiments, since peak fractions of en- zyme activity contained the 125-kDa polypeptide detected by Coomassie Blue staining following separation by SDS-poly- acrylamide gel electrophoresis. In addition the 125-kDa en- zyme-adenylate intermediate also had a sedimentation coef- ficient of 4.4 S, indicating that the enzyme does not undergo major conformational change following adenylation. Assum- ing a partial specific volume of 0.73 g/ml, DNA ligase I protein has a molecular mass of 98 kDa and frictional coefficient of 1.9 (30). These results indicate that the enzyme is a monomer of 98 kDa with a markedly asymmetric shape.

Generation of an Active Fragment by Partial Proteolysis- During the preparation of DNA ligase I, delays in the purifi- cation procedure resulted in partial conversion of the 125- kDa form to an 85-kDa form, as measured by SDS-polyacryl- amide gel electrophoresis. This conversion could be partly counteracted by the inclusion of several protease inhibitors in the buffers employed and is attributed to the effect of endogenous proteases. The same discontinuous conversion from a 125-kDa form to one or two forms of -85 kDa was

A B

69-

FIG. 3. Immunoblotting of DNA ligase I in a calf thymus crude extract. Proteins extracted from calf thymus (Fraction I, 80 rg/lane) were electrophoresed through a 7.5% SDS-polyacrylamide gel and then transferred to nitrocellulose. The nitrocellulose was cut into strips and incubated with either a I:200 dilution of rabbit preimmune serum (lane A) or a 1:200 dilution of serum following immunization with homogeneous bovine DNA ligase I (lane B). Polypeptides recognized by the antisera were detected as described under “Experimental Procedures.” Molecular weight markers were as for Fig. 2.

also observed when purified DNA ligase I was incubated with low concentrations of subtilisin, trypsin, chymotrypsin, and V8 protease. The data on subtilisin cleavage are shown in Figs. 4 and 5. When most of the 125-kDa form had been fragmented (Fig. 4, 5 ng of subtihin), an 85kDa form was the major species observed, with only traces of fragments of a size intermediary between 125 and 85 kDa. No smaller polypeptide could be detected as being simultaneously gener- ated in the 125-85 kDa conversion by several proteolytic enzymes. These data show that the 125-kDa DNA ligase I contains a relatively protease-resistant 85-kDa core and a more protease-sensitive terminal region(s). The 85-kDa poly- peptide was subsequently degraded mainly into polypeptides of -45 and -36 kDa although there were minor bands of 60- 70 kDa.

In order to assess the residual catalytic activity of the various proteolytic fragments, aliquots of subtilisin-treated samples were investigated for biological activity by the stand- ard ligation assay. Treatment of DNA ligase I with 5 ng of

0 2 5 lo 20 50 so "g BubIllloin ,25- b c*..1.- *Q.

I.%&. . , .,, -116 -97.5

65- ; ymt6 -abe -.+v-+.

I x-,ul,e~ 1

..p : ,-66 \ 8; , , p

45- - ,.T .ww..m. - -43

FIG. 4. Subtilisin digestion of bovine DNA ligase I. Assay mixtures (20 ~1) contained 50 mM Tris-HCl, pH 8.0, 10 mM MgCl,, 5 mM DTT, 100 ng of DNA ligase I and subtilisin as indicated. After 20 min at 37 “C, the reactions were stopped by the addition 3.8 pg of aprotinin and 5 ,ul of SDS sample buffer. Proteins were separated by electrophoresis in a 7.5% SDS-polyacrylamide gel and then visualized by silver staining. Molecular weight markers were as for Fig. 2.

12 3 4 5 6 7 8 9 10

125-

-m-b-- -92.5

05- -69

45- -46

-30 2:

FIG. 5. Proteolytic digestion of bovine DNA ligase I-AMP complex versus formation of polypeptide-AMP complexes by proteolytic fragments of bovine DNA ligase I. DNA ligase I (Fraction VII, 500 ng) was incubated with 20 pCi of [~I-~*P]ATP in the presence of 50 mM Tris-HCI, pH 8.0, 10 mM MgCb, and 5 mM DTT for 15 min at room temperature. The reaction was stopped by the addition of 2 ~1 of 0.5 M EDTA. Protein-adenylate (100 ng) was incubated with subtilisin for 15 min at 37 “C as follows; lane I, no addition; lane 2, 2 ng; lane 3, 5 ng; lane 4, 10 ng; lane 5, 20 ng. Reactions were stopped by the addition of 3.8 pg of aprotinin and 5 ~1 of SDS sample buffer. Alternatively, DNA ligase I (Fraction VII, 100 ng) was incubated with subtilisin as indicated for 15 min at 37 “C in 50 mM Tris-HCl, pH 8.0,10 mM MgClz, and 5 mM DTT. Reactions were stopped by the addition of 3.8 pg of aprotinin. 4 PCi of [a-32P] ATP was added to each reaction, and after incubation for 15 min at room temperature, the reactions were terminated by the addition of 5 ~1 of SDS sample buffer. Polypeptide-adenylate complexes were separated by electrophoresis through a 7.5% SDS-polyacrylamide gel, which was fixed in 10% acetic acid, dried down, and exposed to x-ray film. Lane 6, no addition; lane 7, 2 ng; lane 8, 5 ng; lane 9, 10 ng; lane 10, 20 ng. Molecular weight markers were as for Fig. 1.

Structure of DNA Ligate I 12615

subtilisin (Fig. 4) converted >90% of the 125-kDa protein to approximately equal amounts of an 85-kDa form, and several smaller fragments. This material retained 53% of its original DNA ligase activity. After exposure to 20 ng of subtilisin (Fig. 4), only IO-15% of the material remained as an 85-kDa fragment, with multiple smaller fragments accounting for the majority of the material. This aliquot had retained 11% of its original DNA ligase activity. The data strongly indicated that the 85-kDa fragment retained its enzymatic function, whereas smaller fragments were inactive. Occasionally during purifi- cation, an 85-kDa form of the enzyme, which had also retained ligase activity, was separated from the intact enzyme by gel filtration (data not shown). Thus, the 85-kDa form represents a biologically active domain of DNA ligase I.

Similar results were obtained when the formation of an enzyme-adenylate complex was investigated. As expected, this DNA ligase I-adenylate complex could be completely disso- ciated by incubation with nicked DNA, or PP,, under standard assay conditions (data not shown). The fragment pattern produced by partial proteolysis with subtilisin (Fig. 5) strongly resembled that shown in Fig. 4. When a preformed ligase- AMP complex was digested with subtilisin, a discontinuous conversion of the 125-kDa form to a radioactively labeled 85- kDa form was observed. The latter could then be degraded to a major radioactively labeled fragment of about 45 kDa (Fig. 5, lanes 2-5). When DNA ligase I was treated with subtilisin prior to the adenylation assays, the 85-kDa fragment appeared fully active (Fig. 5, lanes 7-10). The smaller fragments seemed inactive, although a 70.kDa peptide might have retained a trace of activity.

A preparation of DNA ligase I which contained similar amounts of active 125 and 85 kDa entities due to proteolysis during purification, and storage was blotted onto a polyvinyl- idene difluoride membrane and analyzed by amino acid se- quencing. In agreement with the solution studies described above, the 125 kDa form (25-150 pmol in different experi- ments) had a blocked N terminus. However, the 85-kDa form (25 pmol) yielded the N-terminal sequence (Ser)-Gln-Ala- Gln-Pro-Pro-(Trp)-Lys-Thr-Pro-Lys-Thr-Leu. The results show that the blocked N terminus had been removed during the discontinuous conversion of the 125-kDa DNA ligase I to an 85-kDa active fragment, indicating that the latter entity was derived from the C-terminal part of the enzyme. A precise location of the cleavage site will be possible when a complete DNA ligase I cDNA sequence becomes available.

A Conserved Epitope in Yeast and Vaccinia Virus DNA Ligases Is Also Present in Mammalian DNA Ligose I-The sequences of the DNA ligases of Saccharomyces cerevisiae and Schizosaccharomyces pombe exhibit several homologous ar- rays, in particular in the C-terminal regions of the proteins (39). Recently, the sequence of a vaccinia virus-encoded DNA ligase was determined (31). This enzyme only shows limited homology to the yeast ligases but, as noted by Smith et al. (31), all three enzymes contain a highly conserved arginine- rich stretch of 16 amino acids close to the C terminus. We have prepared antibodies against a synthetic peptide corre- sponding to this sequence and used it to analyze bovine DNA ligase I by immunoblotting. The antiserum specifically rec- ognizes the 125-kDa DNA ligase I (Fig. 6, lane I). After conversion of the 125-kDa enzyme to the 85-kDa active frag- ment by subtilisin treatment, the anti-peptide antiserum also recognized this fragment (Fig. 6, lanes 2-6). Thus, the 85-kDa domain of DNA ligase I contains an epitope present very close to the C terminus in other DNA ligases.

Treatment of the 125-kDa DNA ligase I with carboxypep- tidase Y caused a rapid disappearance of the epitope from the

1 2 3 456

-200 ‘;;~-,,-92.5

125-w-116 -69 -97.5

-46

-69

-30

FIG. 6. Bovine DNA ligase I contains a C-terminal epitope conserved between yeast and vaccinia virus DNA ligases. Partially purified bovine DNA ligase I (Fraction III, 50 pg of protein) was electrophoresed through a 7.5% SDS-polyacrylamide gel and transferred to a nitrocellulose filter (lane 1). Molecular weight mark- ers were as for Fig. 2. In a separate experiment, reaction mixtures (20 11) containing 500 ng of purified DNA ligase I (Fraction VII) in 50 mM Tris-HCl. DH 8.0. 10 mM M&l,. and 5 mM DTT were incubated

. y _ .

with subtilisin as indicated for 20 min at 37 “C. Reactions were stopped by the addition of 3.8 pg of aprotinin and 5 ~1 of SDS sample buffer. After separation on a 12% SDS-polyacrylamide gel, proteins were transferred to a nitrocellulose filter. Lane 2, no addition; lane 3, 10 ng; lane 4, 25 ng; lane 5, 50 ng; lane 6, 100 ng. Molecular weight markers were as for Fig. 5. Polypeptides recognized by a 1:lOO dilution of rabbit antiserum raised against the synthetic peptide were detected as described under “Experimental Procedures.”

enzyme. After digestion of DNA ligase I with carboxypepti- dase Y for 10,30, and 60 min, >90,67, and 17% of the protein remained as 120-125-kDa material, as estimated by SDS- polyacrylamide gel electrophoresis. However, on immunoblot- ting with the antipeptide antiserum, only 45, 12, and <2% of the antigenic material remained. These data strongly indicate that mammalian DNA ligase I resembles yeast and vaccinia virus DNA ligases in having a conserved arginine-rich se- quence near the C terminus. Since this epitope is also present in the 85-kDa active fragment of DNA ligase I, it may be concluded that this domain represents the C-terminal region of the 125-kDa enzyme.

Properties of Human DNA Ligase I-A 500-fold purified preparation of human DNA ligase I was prepared from a lymphoblastoid cell line. Due to the difficulties in obtaining large quantities of proliferating human cells, no attempts were made to isolate homogeneous human DNA ligase I. However, the partly purified human enzyme clearly shared several key features with bovine DNA ligase I. The human enzyme was recognized on immunoblots by the rabbit antiserum against bovine DNA ligase I (Fig. 7, lane 1) and also by the antipeptide antiserum (data not shown). By comparison of the intensity of the bands in Fig. 7, lane 1 and data from ligase assays, it was apparent that the DNA ligase I antiserum employed gave a 5-10 times weaker signal with human than with bovine DNA ligase I. The size of the 125-kDa human enzyme was indistinguishable from that of the bovine enzyme, and no larger form of the human enzyme was detected. After aden- ylation of human DNA ligase I with [“‘PIAMP, and subse- quent subtilisin treatment, the 125-kDa enzyme was first degraded to a radioactively labeled 85-kDa fragment, and then to smaller fragments (Fig. 7, lanes 2-6) as also observed for the bovine enzyme (Fig. 5). Thus, it may be concluded that bovine and human DNA ligase I are very closely related in their structural features.

DISCUSSION

A simple model for mammalian DNA ligase I summarizing the results of this study is given in Fig. 8. When measured under denaturing conditions, the enzyme has an apparent molecular mass of 125 kDa whereas the native enzyme has an apparent molecular mass of 98 kDa. DNA ligase I has a high proline content (8-g%), and it has been observed previously

12616 Structure of DNA Ligme I

1 2 3 4 5 6 no indication of the existence of such a precursor form (Fig. 3). Similarly in a parallel study (38), we did not detect a 200- kDa form of DNA ligase I after rapid SDS lysis of proliferating bovine tissue culture cells. Instead, the present observation that the N terminus of the 125kDa form of DNA ligase I is blocked to Edman degradation suggests that this molecule is the initial translation product, since about 80% of the soluble proteins in mammalian cell extracts have an N-terminal amino acid residue blocked by acetylation (43).

125-- -92.5

-69 -69

FIG. 7. Immunological cross-reaction between bovine and human DNA ligase I. Human DNA ligase I (6 ua of nrotein). partially purifiedby ammonium sulfate precipitation; and gel filtra- tion was electrophoresed through a 7.5% SDS-polyacrylamide gel and transferred to nitrocellulose. Polypeptides recognized by a 1:200 di- lution of the antiserum raised against bovine DNA ligase I were detected as described under “Experimental Procedures” (lane I). Molecular weight markers were as for Fig. 5. A fraction (1.5 fig) containing human DNA ligase I activity that had been further purified by hydroxylapatite and DNA cellulose chromatography was incubated with 10 pCi of [(u-“‘P]ATP in the presence of 50 mM Tris-HCl, pH 8.0, 10 mM MgCl,, 5 mM DTT, and 50 fig/ml bovine serum albumin for 15 min at room temperature. The reaction was stopped by the addition of 2 ~1 of 0.5 M EDTA. The enzyme-adenylate containing fraction (250 ng of protein) was incubated with subtilisin for 15 min at 37 “C as follows; lane 2, no addition; lane 3, 0.5 ng; lane 4, 1.5 ng; lane 5, 5 ng; lane 6, 15 ng; lane 7, 50 ng. The reactions were stopped by the addition of 3.8 pg of aprotinin and 5 ~1 of SDS sample buffer. Polypeptide-adenylate complexes were separated by electrophoresis through a 7.5% SDS-polyacrylamide gel, which was fixed in 10% acetic acid, dried down, and exposed to x-ray film. Molecular weight markers were as for Fig. 5.

Blocked N-terminus

C-terminus

Protease sensitive site

I ’ A Conserved basic peptide

intact enzyme

c

Catalytic . domain

.

FIG. 8. Model of mammalian DNA ligase I. The molecular mass of the intact enzyme has been estimated to be 98 kDa by hydrodynamic methods and 125 kDa by SDS-polyacrylamide gel electrophoresis, indicating a discrepancy between these techniques.

that proline-rich proteins give anomalously high molecular weights when estimated by denaturing gel electrophoresis (40- 42). Alternatively, or in addition, the calculation from the combined sedimentation velocity and gel filtration data might be an underestimate, although this size determination should be essentially correct even for a highly asymmetric protein (30). Since many of the results presented in this work were obtained by SDS-polyacrylamide gel electrophoresis, we refer to the protein band observed as a 125kDa polypeptide. How- ever, sequencing studies will be required to ascertain which of the two estimates of relative molecular weight is closest to the authentic molecular mass of DNA ligase I.

A 120-130-kDa form of DNA ligase I has been purified previously from calf thymus by Teraoka and Tsukada (15). However, these authors concluded that this form of the en- zyme was derived by proteolysis from a labile 200-kDa pre- cursor polypeptide (6, 23). In the present work we have had

Although DNA ligase I is present intracellularly as a 125 kDa polypeptide, it can be converted to a 85-kDa fragment, which is relatively protease resistant, as well as traces of other fragments on exposure to endogenous proteases in cell ex- tracts or reagent proteolytic enzymes. The 85kDa fragment retains full activity in a standard DNA ligase assay. This catalytic domain represents the C-terminal region of the protein, as judged by several criteria: (i) the intact enzyme has a blocked N terminus, whereas the 85-kDa fragment provides a unique N-terminal amino acid sequence; (ii) the 85-kDa fragment contains an epitope located very close to the C terminus of S. pombe, S. cereuisiae, and vaccinia virus DNA ligases; (iii) this epitope disappears after limited digestion of DNA ligase I with carboxypeptidase Y.

Both the E. coli and bacteriophage T4 DNA ligases have lower sedimentation coefficients than would be expected from their molecular weights and this suggests that the enzymes have an asymmetric shape (12). The hydrodynamic properties of DNA ligase I indicate that this protein also has a highly asymmetric structure. Gel filtration data on DNA ligase I would suggest that the protein has a molecular mass of 200- 260 kDa if the erroneous assumption is made that the enzyme is a globular protein, and such reasoning has caused consid- erable confusion in the literature (10, 25). However, there is general agreement that intact DNA ligase I has a Stokes radius, as determined by gel filtration in 0.2-l M NaCl, of 50- 53A (13, 15, 26). Measurements of sedimentation coefficients have encountered the additional problem that determinations made on crude enzyme fractions have given estimates of 8.0- 8.5 S (1, 26), possibly because of weak interaction of DNA ligase I with other protein(s). In contrast, the homogeneous enzyme has a sedimentation coefficient of 4.4 S. From these values, it may be estimated that DNA ligase I protein has a frictional ratio of 1.9 which is indicative of a highly asym- metric shape (30).

The DNA ligases of S. cereuisiae and S. pombe have been shown by Barker et al. (39) to exhibit partial homology throughout their sequences except for the N-terminal 150 amino acids, which are completely different. The authors speculated that the N-terminal regions of the yeast DNA ligases might function in species-specific events such as nu- clear translocation or interaction with other DNA replication factors. In apparent agreement with such a model, it was observed that removal of -100 terminal amino acids from S. cereuisiae DNA ligase by proteolysis occurred without loss of ligase activity in an in vitro assay (44). In addition, an 83-86- kDa form of DNA ligase I from D. melunogaster could be converted to a similarly active 75-kDa form by proteolysis (8, 45). In the present work, we have observed that an N-terminal region of mammalian DNA ligase I can be removed by pro- teolysis, causing a reduction in mass from 125 to 85 kDa as judged by SDS-polyacrylamide gel electrophoresis, without an accompanying loss of ligase activity. Moreover, this N-ter- minal stretch of DNA ligase I appeared to have an extended structure, since it was degraded by proteolytic enzymes and could not be detected as a separate domain. It seems likely that the N-terminal region of mammalian DNA ligase I has a

Structure of DNA Ligase 1 12617

function similar to that suggested for the N-terminal region of yeast DNA ligases. The part of the ligase molecule not required for enzymatic activity is larger in the mammalian enzyme than in the yeast enzymes, and this provides an explanation for the higher molecular weight of the former protein. In these studies on limited proteolysis of mammalian DNA ligase I, we have not detected significant generation of smaller proteolytic fragments of the enzyme similar to those described for E. coli DNA ligase (46), which retained the ability to form enzyme-adenylate complexes but had no DNA ligation activity.

The presence of a C-terminal catalytic domain in DNA ligase I has several counterparts among other mammalian nuclear enzymes. A catalytically active fragment of the 90- lOO-kDa DNA topoisomerase I corresponds to the C-terminal two-thirds of the enzyme (47). It was proposed that the N- terminal region might serve to interact with other nuclear proteins, or regulate the topoisomerase activity. Similarly, in the 190-kDa mammalian DNA methylase the methyltransfer- ase domain is located in the C-terminal half of the protein (48).

The availability of an antiserum that specifically recognizes human DNA ligase I will allow a more rigorous characteriza- tion of the altered forms of DNA ligase I detected in Bloom’s syndrome cells. The isolation of several tryptic peptides from DNA ligase I has facilitated our molecular cloning studies. The sequence of a cDNA molecule will provide information complementary to the present data.

Acknowledgments-We thank R. Brown and K. Perks for their help with amino acid analysis and protein sequencing, D. Hancock and G. Evan for peptide synthesis, and G. R. Smith for communicat- ing the vaccinia virus DNA ligase sequence prior to publication.

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