T H E JOURNAL OF BIOLOGKAI. CHEMISTRY

Printedm U.S.A. Vol. 257, No. 9, I.sue of May 10, pp. 4758-4763. 19R2

Eukaryotic DNA Ligase PURIFICATION AND PROPERTIES OF THE ENZYME FROM BOVINE THYMUS, AND IMMUNOCHEMICAL STUDIES OF THE ENZYME FROM ANIMAL TISSUES*

(Received for publication, October 2, 1981)

Hirobumi TeraokaS and Kinji Tsukada From the Department of Pathological Biochemistty, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101, Japan

DNA ligase has been purified to near-homogeneity from the extract of bovine thymus with a yield of 5%. The purified enzyme catalyzed the joining of single- stranded breaks in duplex DNA at a rate of 33 nmol of phosphodiester bonds/min/mg of protein. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis and Ouchterlony double diffusion analysis. The enzyme is composed of a single poly- peptide with a molecular weight of about 130,000. The enzyme has a Stokes radius of 52 A, a sedimentation coefficient of about 5 S, and a frictional ratio of 1.6. Apparent K,,, values for ATP and M&' are 2 p~ and 0.9 m, respectively.

Antibody against bovine thymus DNA ligase was prepared by injecting a rabbit with the purified en- zyme. Immunochemical titrations revealed that the in- creased activity of DNA ligase observed after partial hepatectomy of rat and 16-fold higher activity level of mouse Ehrlich tumor cells compared with the host liver are due to a change in the enzyme quantity but not to a change in the catalytic efficiency of the enzyme mol- ecule. Wide variations in the level of DNA ligase activ- ity in extracts from various tissues of rat and mouse were accompanied by proportionate changes in the quantity of immunochemically reactive protein. "he antibody inhibited DNA ligase activity from bovine tissues with 20-fold higher efficiency, compared with the enzyme from the rodent tissues. The enzyme activ- ity from chick embryo was unaffected by the antibody.

DNA ligase (EC 6.5.1.1) that covalently joins single- stranded breaks in duplex DNA plays an important role in DNA replication and repair. The reaction of mammalian DNA ligase seems to proceed as follows (1,2).

Enzyme + ATP e enzyme-AMP + PPi (1)

Enzyme-AMP + nicked DNA e AMP-DNA. enzyme (2)

F? sealed DNA + AMP + enzyme

Escherichia coli DNA ligase that is present as a single form in vivo has been purified to homogeneity and characterized in detail (3,4). On the other hand, eukaryotic DNA ligases have been only partially purified and their characteristics are less well understood (5-16). In addition, two molecular species of

* This work was supported in part by a grant-in-aid for Cancer Research from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed.

the enzyme have been previously reported to be present in mammalian cells and tissues by several investigators including us (9, 11, 13-15). Recently, we suggested that DNA ligase of rat liver exists in a single molecular species localized in the nucleus (17). As DNA synthesis increases, the activity level of mammalian DNA ligase has been shown to increase in regen- erating rat liver (18-20), rat kidney tumor (21), rat and human hepatomas (22, 23), developing rat brain (241, and dietary- manipulated rat liver (25). The mechanism underlying the increase in the activity level of the enzyme is still unknown.

In this paper, we describe a method for the extensive purification of DNA ligase from bovine thymus and some of the catalytic and molecular properties. Furthermore, we have obtained antibodies against the purified DNA ligase. These antibodies have been used to investigate the increased expres- sion of DNA ligase activity as well as the animal species and tissue specificities.

EXPERIMENTAL PROCEDURES"~

Material*

Chemicals: l l - 3 2 P l A T P vas prepared by the method Of Walseth and Johnson

Japan). 18-3H1ATP was purchased from the Radlochemrcal Centre IRmersham.

I261 ualnq 3 2 P ~ obtalned from Japan Atomlc Energy Research Institute IIbaragl

Enqlandl. BOYlne Intestine alkallne phosphatase (type I1 and E& 8-qalactosldase Were from Boehrlnqer (Hannhelm, F R G ) . Phenylmethylsulfonyl fluoride and highly polyrnerlred calf thymus DNA (type 1) were obtalned from S l q a 1st. Lou1s USA). DEAE-cellulose lDE23) and phosphocellulose IP111 were obtained f&m Whatman lKent,Enqlandl. QAe-sephadex A-2s. Sephadex G-50, Sephadex G-150 , Sephadex G-ZOO, and Blue-Sepharose C L - 6 8 were from Pharmacla

ChemlcalS (Osaka, Japan]. Reagents far plydcrylamlde gel eleCtrOphoreSIS Iuppsala, Sweden). DNA-cellulo~e (native DNA) was purchased from Wako Pure

were obtalned from Nakaral Chemicals (Kyoto, Japan). Freund's Complete adluvant was from Dlfco (Detroit. USA]. Calclwn phosphate gel Was prepared accordlnq to the method of xeilln and Hartree (27).

Animals: Thymuses of 1- fo 3-year old castrated oxen were obtalned from T o " u n l c i p a 1 Slauqhterhouse ITokyo, Japan). Female Japanese rabblts 1 2 . 5 - 3 kg body wclqht). male W1skar rat5 1100-150 g body werqhtl, and male dd albino mlce 120-25 q body welqhtl were obtalned locally. Chicken embryos were removed from Whlte-Mqhorn-chrcken eqqs I4 days a f t e r f e r t i l i a a t l a n .

M I 6

Preparatron of DNA substrates: Nicked DNA containing 5'-32P1 and 3"OH

t e r m l n l was prepared a s descrlbed previously by uslnq I I - ~ ~ P I A T P , rat llVeT

and 3"OH termlnl was prepared according to Ichrrnura and Tsvklda 1 2 8 1 . DNA kinase, and 5';3'-OH nlcked DNA. Unlabeled nicked DNA contalnlnq 5"Pi

prevlouslyY129) on theybasis Of the method of Welss &. 1301. When necessary, the enzyme solution vas dlluted wlth 20 mM TrlS-HCl (pH 7 .51 , 1 mM DTT. and 0.1 mM EDTA. The standard reectlon mlxtnre 10.2 mll Contained 5-10

~ s s a for DNA LI ase: DNA l q a s e a c r ~ v l r y was determlned a s descrlbed

were added 0.1 ml of 0.1 M N ~ P P ~ and 3 ml of chilled 5 % cc13COOH. After

' Portions of this paper (including "Experimental Procedures") are presented in miniprint as prepared by the authors. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 81M-2424, cite authors, and include a check or money order for $1.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

* The abbreviations used are: DTT, dithiothreitol (in Miniprint); SDS, sodium dodecyl sulfate.

4758

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at 2 0 - C for 35 mln. To thc reactlo" mixture was added 0.05 ml of 2 mg/ml bovlne serum albvmln and 0.4 mi of chllled 5% CCl7COOH. fallowed by a 10-m~"

purlfzed enzyme ( 1 0 - 2 0 "91 was carried o u t at I T on a column of Sephadex Analyrlcal Gel Flltratxan: Rnalytlcal qel chromatography of the

G-150 11.5 x 6 0 cmj equlllbrated vlth 10 mM potassrum phosphate buffer IpH 7 . 5 1 conralnlng 0.5 mM OTT. 0.1 mM EDTA, and 0.5 M KC1. The apparent molecular weight and Stokes radius of the enzyme were determlned accordlnq to Rndrews I311 and 51eqel and Monty 134). respectively. Marker protelns wlth the followln~ ylecu1z.r welyhts and Stokes radli were used: catalase IMr =

albumln IMr = 6 8 . 0 0 0 . 35 a i : cytochrome c IF = 12.300. 1 7 A I . 240.000. 52 I lactate dghydroqenase IMr = 140,000. 4 3 . 5 81: bovlne serum

a s descilbed by Ouchterlony l371.

3 volumes of 50 mM Trls-HC1 (pH 7 - 5 1 . 0.5 M NaCl, 1 mM DTT. 0.1 mM EDTA. and phenylmethylsulfonyl fluoride wlth a glass-Teflon homogenlrer. Ehrllch ascites tumor c e l l s were collected from mice 8 days after the lnoculatian of

Of 0.25 M SYcrOse, 3.3 mM HqCl,, and 0.5 mM phenylmethylsulfonyl fluorlde WIth 0.05 ml of ascites fluld. washed r ~ x c e wlrh saline. and broken in 4 volumes

Anrmal tlslues examlned for umnunochemlcal analysls were homoqenlzed ln

RESULTS

Purification of DNA Ligase All operations were carried out at 0-4OC and completed

within 6 days without freezing enzyme solutions. Thymuses from castrated oxen were kept on ice after killing and used within 5 h for the purification of the enzyme. Potassium

phosphate (KPO,) buffer, adjusted to pH 7.5 at room temper- ature, contained 0.5 m~ dithiothreitol, 0.1 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride unless otherwise specified.

Step 1: Preparation of Crude Extract-The fresh thymuses (550 g) from castrated oxen were chopped into small pieces, suspended in 2200 ml of 50 mM Tris-HC1 (pH 7.5), 0.3 M KC1, 10 mM 2-mercaptoethanol, 1 mM EDTA, and 0.5 m~ phenyl- methylsulfonyl fluoride, and homogenized with a home mixer (National MX-120) at the maximum speed for 1 min. The homogenate was centrifuged at 10,OOO x g for 30 min. The supernatant solution obtained was passed through two layers of gauze to obtain crude extract (2350 d).

Step 2: Ammonium Sulfate Fractionation-To the crude extract, solid ammonium sulfate was added to 35% saturation. The solution was stirred for 30 min and the precipitate was removed by centrifugation at 10,000 X g for 20 min. The supernatant solution was brought to 60% saturation by the addition of solid ammonium sulfate, followed by centrifuga- tion at 10,000 X g for 20 min. The resulting precipitate was dissolved in 10 mM KP04 buffer containing 10 mM 2-mercap- toethanol, 0.1 m~ EDTA, and 0.5 m~ phenylmethylsulfonyl fluoride at a protein concentration of 15 mg/ml (523 ml).

Step 3: Calcium Phosphate Gel Adsorption-To the en- zyme solution, calcium phosphate gel suspended in 10 mM KPO, buffer containing 10 m~ 2-mercaptoethanol, 0.1 m~ EDTA, and 0.5 m~ phenylmethylsulfonyl fluoride (40 mg of dry gel/ml) was added with gentle stirring in a proportion of 1 mg of dry gel to 1 mg of protein, After 20-min stirring, the mixture was centrifuged at 1000 X g for 5 min. The packed gel was washed once with 800 ml of the above buffer. The enzyme fraction was eluted twice with 500 ml of 0.15 M KPO, buffer. The combined solution (1030 m l ) was brought to 60% satura- tion with solid ammonium sulfate and stirred for 20 min. The precipitate was recovered by centrifugation and dissolved in 10 mM KPO, buffer. The solution (50 m l ) was dialyzed over- night against 1500 ml of 10 mM KPO, buffer and then centri- fuged at 10,000 x g for 15 min to remove precipitate.

Step 4: Phosphocellulose Column Chromatography-The clear solution was applied to a phosphocellulose column (2.7 X 27 cm) equilibrated with 10 m~ KPO, buffer containing 20 mM KCI. After the column was washed with 180 ml of the Same buffer, the enzyme was eluted with a linear KC1 gradient (20 m~ to 0.6 M) in 700 ml of 10 mM KPO, buffer. The enzyme was eluted at 0.2 to 0.3 M KC1 concentration and the active fractions (19 &/fraction) were combined (170 ml). The com- bined solution was brought to 60% saturation with solid am- monium sulfate. The precipitate was recovered by centrifu- gation and dissolved in a small volume of 10 m~ KPO, buffer. The solution was passed through a Sephadex G-50 column (2 X 20 cm) equilibrated with 10 m~ KPO, buffer to remove ammonium sulfate.

Step 5: DEAE-cellulose Column Chromatography-The enzyme solution (9 m l ) was applied to a DEAE-cellulose (phosphate form) column (1.2 X 22 cm) equilibrated with 20 mM KPO, buffer. After the column was washed with 30 ml of the same buffer, the enzyme was eluted with a linear KPO, concentration gradient (20 m~ to 0.2 M) in 140 ml of KP0, buffer. Fractions (6.3 ml/fraction) were collected and the peak of enzyme activity was eluted with about 0.1 M KPO,. Active fractions were combined (50 ml), followed by addition of solid ammonium sulfate up to 60% saturation. The precipitate recovered by centrifugation was dissolved in 25 mM KPO, buffer. The solution (2 m l ) was dialyzed against 100 ml of 25 m~ KPO, buffer for 5 h.

Step 6: Blue Sepharose Column Chromatography-The enzyme solution was applied to a blue Sepharose column (1 X 22 cm) equilibrated with 25 m~ KPO, buffer. After the

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column was washed with 20 ml of the buffer, the enzyme was eluted with a linear gradient of KPO4 concentration (25 mM to 0.3 M) in 1 0 0 ml of KP04 buffer. Fractions (5.3 ml/fraction) were collected and the active fractions eluted at about 0.15 M KPO, were combined (37 ml). The solution was brought to 60% saturation with solid ammonium sulfate. The precipitate recovered by centrifugation was dissolved in 20 mM KPO, buffer containing 0.5 M KC].

Step 7: Sephadex G-200 Column Chromatography-The enzyme solution (1 .1 ml) was applied to a Sephadex G-200 column (1.4 X 66 cm) equilibrated with 20 mM KPO, buffer containing 0.5 M KCI. The enzyme was eluted with the same buffer and 1.7-ml fractions were collected. Active fractions were combined (6.8 ml ) and dialyzed against 250 ml of 10 mM KPO., buffer containing 20% (v/v) glycerol for 6 h.

Step 8: DNA-cellulose Column Chromatography-The so- lution was applied to a DNA-cellulose column ( 1 x 6.4 cm) equilibrated with 10 mM KPO, buffer containing 20% (v/v) glycerol. The column was washed with 10 ml of the same buffer and then the enzyme was eluted with a linear gradient of KPO, concentration (10 mM to 0.2 M ) in 40 ml of KPO, buffer containing 20% (v/v) glycerol. Active fractions (2.4 ml/ fraction) were eluted at about 50 mM KPO4.

The purified enzyme was stored at -70 “C without signifi- cant loss of the activity for at least 2 months.

The overall purification was about 4000-fold with a yield of 5R (Table I).

Purity and Molecular Properties Interfering enzymes, DNase I, DNase 11, phosphodiesterase,

and alkaline phosphatase, were not detectable in the purified DNA ligase preparation (Step 8, 0.2 to 0.5 unit).

Electrophoresis of the purified enzyme in a 5% nondenatur- ing polyacrylamide gel yielded a single band with a relative tnobility to bromphenol blue of 0.21 after staining with Coo- massie blue (not shown). The position was equal to that of DNA ligase activity that had been eluted from 2-mm slices of an identical gel. A considerable amount (about 30%) of activity and protein was always observed at the origin of the gels. This suggests that the enzyme molecule either aggregated or bound to the gel surface under the conditions described under “Methods.”

When the purified enzyme labeled with [“HIAMP (see below) was subjected to SDS-polyacrylamide gel electropho- resis, a single protein band was observed after staining for protein with Coomassie blue and the position was in coinci- dence with the position of the radioactivity (Fig. 1 ) . Different, much lower peak(s) of radioactivity with higher mobility were sometimes observed regardless of the absence of protein band, which is probably due to the released radioactivity from DNA ligase-[”HIAMP during its preparation and separation proce- dures. These results indicate that the purified DNA ligase is

TABLE I Purification of DNA ligase from bovine thymus

Protein content and enzyme activity were assayed as described under “Methods.”

Purification step Total Total Specific Yield protein activity activity

mg unik units/mg %. 1. Crude extract 24,225 205.7 0.0085 100 2. Ammonium sulfate 7,838 147.4 0.019 72 3. Calcium phosphate gel 1.228 91.0 0.074 44 4. I’hosphocellulose 147 41.7 0.284 20 5. DEAE-cellulose 25 23.5 0.94 1 1 6. Blue-Sepharose 9.6 17.4 1.81 8 7. Sephadex G-200 1.4 15.8 11.3 8 8. DNA-cellulose 0.3 9.8 32.7 5

Mr = I30 K 4.

Gel length (cm) FIG. 1. SDS-polyacrylamide gel electrophoresis of DNA li-

ga~e-[~H]AMp complex. The DNA ligase-[’HIAMP complex (2 pg. 2,900 cpm) was electrophoresed in a SDS-8C polyacrylamide gel overlaid with a 3 4 stacking gel. Detailed conditions are described under “Methods.”

TABLE I1 Identification of DNA ligase-[:’H]AMP complex

The reaction mixture contained 2,300 cpm of ligase-[”HIAMP complex. After the incubation, acid-precipitable and acid-soluble frac- tions were separated as described under “Methods.” The results were the average of two to three experiments.

‘H recovered in Addition

Acid-precipita- Acid-soluhle ble fraction fraction

w m CPm None 2077 172 Pyrophosphate (0.2 mM) 320 1724

Nicked DNA (50 pg) 550 1620 (1650)“

(1461)h “ The radioactivity was identified as ATP by QAE-Sephadex A-25

The radioactivity was identified as AMP by the chromatography. column chromatography.

homogeneous. Comparison of the electrophoretic mobility of the enzyme treated with SDS and dithiothreitol with those of marker polypeptides showed that the molecular weight of the treated enzyme was about 130,000. An apparent molecular weight of the enzyme was estimated to be 240,000 by gel filtration on Sephadex G-150. From the same gel filtration data, Stokes radius was obtained to be 52 A. The sedimenta- tion coefficient of 5 S was obtained by sucrose density gradient centrifugation. From the physical parameters, a molecular weight of 120,000 to 130,000 and a frictional ratio of about 1.6 were calculated (34). Therefore, DNA ligase from bovine thymus has a molecular weight of about 130,000 composed of a single polypeptide and seems to be highly asymmetric in shape.

Catalytic Properties Purified DNA ligase absolutely required ATP and Mg’+.

K , values for ATP and Mg2’ were obtained to be 2 PM and 0.9 mM, respectively. The enzyme was activated by polyamines at suboptimal concentrations of Mg’+ without significant ef- fect on VmaX as described previously (29). Purified enzyme catalyzed the joining of nicked DNA containing 5’-P, and 3’-

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OH termini a t a rate of 33 nmol of phosphodiester bonds/ min/mg of protein under the standard assay conditions.

DNA ligase-[,"HIAMP complex was separated by Sephadex G-150 column chromatography after incubation of the enzyme with ["HIATP. DNA ligase-AMP complex was identified by incubation with either PPi or nicked DNA containing 5'-P, and 3"OH (Table 11). After incubation, acid-soluble fractions obtained were analyzed by QAE-Sephadex A-25 chromatog- raphy. By incubation of the complex with PPi, radioactivity in the acid-soluble fraction was recovered as ATP. Radioac- tivity in the acid-soluble fraction after incubation with nicked DNA containing 5'-P, and 3'-OH was identified as AMP. These results indicate that the purified DNA ligase formed DNA ligase-AMP complex as a reaction intermediate when incubated with ATP (see the introduction).

Immunochemical Studies Fig. 2 shows an Ouchterlony double diffusion pattern of

anti(DNA 1igase)y-globulin and partially purified DNA ligase from bovine thymus. A single precipitin band was observed between DNA ligase and anti(DNA 1igase)y-globulin, further demonstrating that the purified DNA ligase is homogeneous.

FIG. 2. Ouchterlony double diffusion analysis of bovine thy- m u s DNA ligase. The agar gel ( I r ; ) contained 20 mM Tris-HCI ( p H 7.5), 0.15 M SaCI, and 0.02''; sodium azide. The plate was developed at 4 "C for 2 days before the photograph was taken. Well 1, the partially purified enzyme (Step 4) from bovine thymus (0.2 mg of protein); well 2, anti(DNA 1igase)y-globulin (0.3 mg); well 3, control y-globulin (0.25 mg).

Immunochemical titrations indicate that DNA ligase from bovine thymus was inhibited by the antibody but not by control y-globulin (Fig. 3A). The equivalence point, i.e. the extraporated point a t which enzyme activity first appeared in the supernatant fluid, was the same with crude extract, par- tially purified enzyme, and the purified enzyme. About 0.03 unit of DNA ligase from bovine thymus was equivalently inhibited by 0.1 mg of y-globulin.

Using the anti(bovine thymus DNA 1igase)y-globulin frac- tion, we have immunochemically examined the animal species specificity and tissue specificity, as well as the mechanism of the increase in the activity level of DNA ligase after partial hepatectomy. The level of DNA ligase activity increases after partial hepatectomy with increase in DNA synthesis and reaches 4- to &fold higher level 20 to 24 h after the operation (18, 19). In order to determine whether the increase in the activity level of hepatic DNA ligase is due to a change in the enzyme quantity or to a change in the catalytic efficiency per enzyme molecule, immunochemical titrations of DNA ligase activity in liver extracts from normal and 70% hepatectomized rats were conducted (Fig. 3B). In spite of the 4.5-fold differ- ence between specific activities of the two preparations, the equivalence point was essentially the same for both prepara- tions on the basis of the amount of enzyme activity added. This shows that the change in the level of enzyme activity is accompanied by a change in the amount of immunoreactive protein.

DNA ligase activity is observed to be high in tumors (21-23). We employed mouse Ehrlich ascites tumor cells to elucidate the mechanism of the increase in the enzyme activity. The tumor cells contained about 16-fold higher specific activity of DNA ligase than the host liver. Immunochemical titrations of the two preparations show the same equivalence point (Table 111), indicating that the marked difference in the level of the enzyme activity between mouse tumor cells and the host liver is due to a change in the amount of immunoreactive enzyme protein but not to a change in the catalytic efficiency of the enzyme molecule.

In Table I11 are summarized the specific activity of DNA ligase from various animal tissues and the equivalence point determined by immunochemical titration. Essentially, the same equivalence points were observed in spite of the differ- ence in the activity level of the enzyme from rat liver, brain, thymus, kidney, and lung. Similar results were obtained for these tissues of mouse (not shown). In addition, DNA ligase of bovine liver had approximately the same equivalence point as that of bovine thymus. These results suggest that tissue

- Activity added (mu) ActiGity added i"mlJ)

FIG. 3. Immunochemical analysis of levels of DNA l igase cytosols from normal (0) and 70% hepatectomized (24 h after the act ivi ty f rom bovine thymus ( A ) a n d rat liver (B). A , increasing operation) rats (0) containing DNA ligase activity indicated as milli- amounts of bovine thymus DNA ligase (Step 5) indicated as milliunits units were added to 0.3 mg of anti(DNA 1igase)y-globulin (-) or to ( m u were added to 0.15 mg of anti(DNA 1igase)y-globulin (0) or to 0.25 rng of control y-globulin (---). Equivalence point, i.e. the ex- 0.15 mg of control y-globulin (0). After the incubation at 4 "C over- trapolated point at which the enzyme activity first appeared in the night, the supernatant fluids obtained after the centrifugation were supernatant fluid, was detprmined graphically. assayed for DNA ligase activity. R. increasing amounts of liver

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TABLE I11 Level of DNA ligase activity from various animal tissues and cells,

and the activity equivalent to anti(DNA 1igase)y-globulin Immunochemical titrations of DNA ligase activity in the extracts

of the tissues and cells were carried out as described in Fig. 3. DNA ligase activity equivalent to 75 pg of anti(DNA 1igase)y-globulin was determined graphically as in Fig. 3.

DNA liease activitv Animal Tissue

- - - - _ I

Specific Equivalence activitv uoint

Rat Liver-normal Liver-regenerating Brain Thymus Kidney Lung

Mouse Liver Ehrlich cells

Bovine Thymus Liver

milliunits/mg milliunits ~ ~ ~~

protein

0.721 3.3

24.6 0.83} 0.7-1.0 0.301 0.63 0.50 0.7 8.1 0.7

10.5 20 0.4 15

Chicken Embryo 2.5 0“

a This means that the antibody did not inhibit DNA ligase activity from chick embryo.

specificity of DNA ligase is fairly low in mammals. The activity of DNA ligase isolated from chick embryo was

not affected by anti(bovine thymus DNA 1igase)y-globulin (Table 111). Bovine thymus DNA ligase in the crude extracts as well as the purified enzyme was inhibited by the antibody with about 20-fold higher efficiency than the rodent enzyme (Table 111). Ouchterlony double diffusion analysis of DNA ligase partially purified from rat liver cytosol (15) shows no or very faint precipitin band even in the presence of more DNA ligase activity than that of bovine thymus enzyme (not shown).

DIscussIaN A mammalian DNA ligase was obtained as a homogeneous

preparation for the first time, as evidenced by SDS-polyacryl- amide gel electrophoresis as well as by Ouchterlony double diffusion analysis. The specific activity of 32.7 units/mg of protein a t 37 “C of the purified DNA ligase is at least 10- to 100-fold higher than that of mammalian DNA ligases hitherto purified, such as DNA ligase from calf thymus (0.19 unit/mg of protein at 20 “C (14)), the enzyme from rat liver cytosol (0.09 unit/mg of protein at 37 “C (X)), and the enzyme from rat liver nuclear extract (0.5 to 0.9 unit/mg of protein a t 37 “c (12, 15)). The specific activity of the purified DNA ligase from bovine thymus is approximately similar to that of the homo- geneous enzyme from E. coli (3) under the standard assay conditions.

Apparent molecular weights of mammalian DNA ligases have been variably estimated to fall within a wide range of 320,000 to 50,000 (5-16). This is probably due to the highly asymmetric molecular structure of mammalian DNA ligase. For example, bovine thymus DNA ligase has a Stokes radius of 52 A, a sedimentation coefficient of 5 S, and a frictional ratio of 1.6. In addition, partial proteolysis during isolation and purification procedures results in the appearance of more than one molecular species of DNA ligase (see below).

Several investigators including us have previously reported that two species of DNA ligase exist in mammalian cells (9, 11, 13-15, see Ref. 40). DNA ligase I or “cytoplasmic DNA ligase” has a higher molecular weight and a lower K,,, for ATP

to M). On the other hand, DNA ligase I1 or “nuclear DNA ligase” has a lower molecular weight and a higher K,,, for ATP ( W 5 to M). The activity of DNA ligase I is

reported to be high in actively growing cells, whereas the activity of DNA ligase I1 seems to be essentially constant (14). Soderhiill(20) has reported that rat liver contains two species of DNA ligase, one of which is increased after partial hepatec- tomy. We have recently suggested that nuclear DNA ligase or DNA ligase I1 is a product of partial proteolysis of DNA ligase localized in nucleus as a single molecular species (15, 17). DNA ligase was mainly distributed in the nuclei rapidly isolated from rat liver and found to be almost all “cytoplasmic- type enzyme.” Nuclear DNA ligase appeared by prolonged incubation of the nuclei or nuclear extract, the appearance of which was partially inhibited by phenylmethylsulfonyl fluo- ride, a serine-proteinase inhibitor. Yeast conditional lethal mutants defective in both DNA replication and repair have been found to contain no detectable activity of DNA ligase (41, 42), implying that in this eukaryotic organism, a single form of the enzyme is responsible for essential ligation in replication and repair. In general, DNA ligase in contrast to DNA polymerase seems to exist in vivo as a single molecular species.

Pedrali-Noy et al. (11) and SoderhZiU and Lindahl(l4) have previously indicated that DNA ligase I converted to the active enzyme with about one-half the molecular weight of DNA ligase I during tbe storage of crude enzyme preparations or after extensive purification. In our purification procedures of bovine thymus DNA ligase, the enzyme with a smaller molec- ular weight sometimes appeared by using frozen thymuses or by prolonging the duration of purification, which showed one or two bands with molecular weight between 90,000 to 50,000 in SDS-polyacrylamide gel electrophoresis. To detect smaller DNA ligase molecules or DNA ligase 11-type enzyme by exogeneous proteolysis, we treated the purified enzyme from bovine thymus with trypsin and chymotrypsin. The enzyme was inactivated according to pseudo-first order kinetics by the proteolysis at 25 “C and 0 “C, but neither the active species of the enzyme with smaller molecular weight nor DNA ligase II- type enzyme was d e t e ~ t e d . ~

DNA ligase purified from bovine thymus seems to be the same enzyme as calf thymus DNA ligase I reported by Sod- erhiill and Lindahl (14). They have obtained antibodies against DNA ligase I, partially purified from calf thymus, by repeated injections of the enzyme preparations into rabbits. In contrast, we report that only two injections to rabbits were required to induce antibodies against our homogeneous DNA ligase preparation. Anti(DNA 1igase)y-globulin fraction that we have obtained is likely to be a useful tool to investigate the role of DNA ligase in mammalian DNA replication in isolated nuclei and in reconstituted systems. Preliminary experiments indicate that the antibody against DNA ligase did not affect [3H]dTMP incorporation into the acid-precipitable fraction in a DNA-synthesizing system containing isolated nuclei from ascites tumor cells.3

Immunochemical titration (Table 111) and Ouchterlony double diffusion analysis indicate that DNA ligases from bovine, rodent, and aves are immunochemically different from one another. Analogously, rabbit antibodies to calf thymus DNA polymerase-n and -p have also shown a gradation of cross-reactivity with antigens from various species of animals (43). If the structure at catalytic site of animal DNA ligase is conserved during evolution, the sites other than the catalytic site seem to be predominantly recognized as antigenic deter- minants. In order to analyze precisely molecular structure of animal DNA ligase as well as species specificity of the enzyme, monoclonal antibodies are considered to be preferable to conventional antibodies.

H. Teraoka, unpublished results.

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Acknowledgments-We thank Norikazu Okamoto and Kazuo Ko- zuki for technical assistance in a part of this work. We also thank Dr. Ronald T. Hay for critical reading of this manuscript.

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H Teraoka and K Tsukadathymus, and immunochemical studies of the enzyme from animal tissues.

Eukaryotic DNA ligase. Purification and properties of the enzyme from bovine

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