Eur. J. Biochem. 162, 325-332 (1987) 0 FEBS 1987

Mammalian DNA ligase Structure and function in rat-liver tissues

Mauro MEZZINA', Jean-Michel ROSSIGNOL' , Michel PHILIPPE', Raffaella IZZO ', Umberto BERTAZZONI and Alain SARASIN

' Groupe de Biologie et GCnetique Molkculaires du Centre National de la Recherche Scientifique, Equipe de Recherche 272, Villejuif Istituto di Genetica Biochimica Evoluzionistica del Consiglio Nazionale delle Ricerche, Pavia

(Received August 2, 1986) - EJB 86 0850

DNA ligase was partially purified from normal and regenerating rat liver. Its structure was studied using the activity gel procedure that identifies the functional polypeptides. Two slightly different purification procedures were followed, leading to the isolation of one or two peaks (fractions A and B) of DNA ligase by hydroxyapatite chromatography. When analyzed on activity gels, all these enzyme fractions corresponded to a single active 130- kDa polypeptide both in normal and regenerating liver. A limited trypsin digestion of ligase fractions A and B gave rise to an identical pattern of smaller polypeptides of 110 kDa, 100 kDa and 75 kDa. Also storage at 4°C of fractions A and B produced smaller polypeptides of 110 kDa, 100 kDa, 85 kDa and 60 kDa, which were identical for the two fractions. Our results indicate that the same ligase polypeptide of 130 kDa can be isolated from stationary or regenerating rat liver cells. However, physiological or artifactual proteolysis during various purification procedures can lead to the isolation of two enzyme fractions with different chromatographic behaviour but with the same molecular mass.

DNA ligase promotes the formation of a phosphodiester bond between 3'-hydroxyl and 5'-phosphoryl termini in a double-stranded DNA molecule and its activity is essential in joining DNA chains during replication, repair and recombina- tion processes [I]. To fulfill these different functions a unique enzyme form has been found in T4 bacteriophage [2], E. coli [3] and yeast [4, 51. In mammalian cells two enzyme forms have been reported, DNA ligases I and 11, which seem to be involved in DNA replication and in DNA repair respectively [6]. Since these two activities can be distinguished from each other by their physical properties of molecular mass and sedi- mentation coefficient, ligases I and I1 activities can be separated in certain conditions either after chromatography or after sedimentation in velocity gradients of cellular extracts [7 - 101. In fact after gel filtration analysis or velocity gradient sedimentations DNA ligase I has been described as a large enzyme with an apparent molecular mass ranging from 175 kDa to 220 kDa and a sedimentation coefficient of about 8 S, whereas ligase I1 has been described as a smaller enzyme of about 85 kDa and 4.5 S [6,7,10]. More recently it has been shown that, after NaDodS04/polyacrylamide gel electropho- resis, purified DNA ligases I and I1 from calf thymus present a single polypeptide with a molecular mass of 130 kDa and 67 kDa respectively [II, 121. DNA ligase I is supposed to be

Correspondence to M. Mezzina, Laboratoire de Mutagenese Moleculaire, ER 272 du CNRS, Boite postale 8, F-94802 Villejuif Cedex, France

Abbreviation. NaDodSO+, sodium dodecyl sulphate. Enzymes. Polynucleotide ligase (EC 6.5.1 .l); polynucleotide

kinase (EC 2.7.1.78); alkaline phosphatase (EC 3.1.3.1); trypsin (EC 3.4.21.4); P-galactosidase (EC 3.2.1.23); phosphorylase (EC 2.4.1.1).

involved in DNA replication since its activity is the major form in rapidly dividing cells of regenerating rat liver [13] or monkey cells infected with simian virus 40 [14]. DNA ligase I1 is possibly related to the DNA repair process, since its activity appears to be the major form in resting cells of normal rat liver [13] and increases in cells treated with various DNA- damaging agents [I4 - 161. Moreover, antibodies directed against calf thymus ligase I do not seem to affect ligase I1 activity [8] and vice versa [12, 171, thus providing serological evidence that these two enzyme fractions could be two sepa- rate proteins.

However, using different purification procedures, only one ligase form was isolated from rabbit, rat and calf tissues. This activity usually presented the physical properties of ligase I [18 -211. Therefore, it has been suggested that a proteolytic process, occurring during purification or storage of the high- molecular-mass enzyme from human cells or rat liver, could generate a lower form, which could correspond to ligase 11

We have recently developed an activity gel method allowing the identification of the functional ligase poly- peptides found in crude extracts or in purified enzyme fractions after NaDodS04/polyacrylamide gel electrophore- sis. Using this method we showed that the ligases I and 11, obtained from cultured simian cells, were heterogeneous, con- taining several active polypeptides ranging from 58 kDa to 200 kDa for ligase I and 58 kDa and 65 kDa for the ligase I1 fraction. In addition we have found that a proteolytic degradation process, possibly associated with the repair of mitomycin-C-induced DNA damage, converted the higher- molecular-mass polypeptides of both DNA ligases I and I1 into smaller species [24, 251.

[21-231.

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Table 1. Partial purification of DNA ligase from regenerating or normal rat liver

Fraction Regenerating rat liver Normal rat liver

volume proteins total specific volume proteins total specific activity activity activity activity

~~

ml mg U U/mg ml mg U U/mg SlOO 198 2356 29 923 12.7 200 3880 4556 1.2 Phosphocellulose 600 156 22932 147 225 60.8 1417 20.3 HA-125 15 12.5 3 338 268 7.5 '2.4 790 84 HA-250 5.8 6.2 75 12 7.5 5.2 12.5 2.4

In the present study we used the rat liver tissues to in- vestigate the activity and the structure of DNA ligase in both normal and regenerating conditions, since they have been found very useful for studying the variations of enzyme activi- ties in resting and proliferating tissues [26, 271. The results we have obtained indicate that a single ligase protein of 130 kDa is present in rat liver cells. A slight variation in the purification conditions has led to the isolation of two different ligase fractions, which exhibited different chromatographic be- haviour but appeared to derive from polypeptide structures of the same molecular mass.

EXPERIMENTAL PROCEDURE

Chemicals and enzymes

(dT)16 and (dA)500 were obtained from PL-Biochemicals. Acrylamide, N,N'-methylenebisacrylamide, NaDodS04 were from Bio-Rad; [Y-~~PIATP (3000 Ci/mmol), purified T4 polynucleotide kinase and ligase were from New England Nuclear. Escherichia coli alkaline phosphatase and sodium metabisulfite were from Sigma. Protein molecular mass markers were from Pharmacia. Trypsin was from Choay- Chimie. Phenylmethylsulphonyl fluoride, leupeptin and calf intestinal alkaline phosphatase were from Boehringer.

Chromatography and buffers

Phosphocellulose (PI 1) and hydroxyapatite Bio-Gel HTP DNA grade were purchased from Whatman and Bio-Rad, respectively, and prepared according the manufacturers' in- structions. All potassium phosphate buffers were at pH 7.6 and contained 1 mM 2-mercaptoethanol, 0.2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium metabisulfite, 2 pg/ml leupeptin and 10% glycerol.

Animals

Regenerating livers were obtained from male WAG rats 40 h after partial hepatectomy as previously described [28]. Normal livers were removed from adult male WAG rats.

In vitro standard and activity gel ligase assays

For both the standard and the activity gel ligase assays in vitro the (dA)500 . [32P](dT)16 (1 : 1) was used as substrate to detect the enzyme activity. This substrate has been prepared by labeling the (dT)16 with polynucleotide kinase and [ y -

32P]ATP and annealing the [32P]oligo(dT) with poly(dA) (1 : 1). The ligase reaction was carried out at 37°C; the substrate was boiled and afterwards incubated at 85°C in the presence of bacterial alkaline phosphatase as already described [29]. 1 unit of DNA ligase is defined as the amount of enzyme converting 1 nmol [32P]oligo(dT) into an alkaline-phosphatase-resistant form in 1 h. Ligase activity was quantified by initial velocity measurements and expressed as i*nits/mg protein. In the crude extracts the enzyme reaction is linear within the first 9 min whereas in purified fractions it is linear for at least 30 min. In the activity gel method, crude cellular extracts or partially purified enzyme fractions were electrophoresed in NaDodS04/polyacrylamide gels containing 4 - 16 pM poly(dA) . [32P]oligo(dT), (4- 8) x lo8 cpm, as previously de- scribed [24]. After renaturation of proteins, the ligase reaction was carried out in the intact gel in the presence of ATP and Mg2+ at 20°C. After treatment of the gel with calf intestinal alkaline phosphatase at 37 "C to remove the unreacted sub- strate, in situ detection of active ligase bands was achieved by autoradiography. The size of [32P]oligo(dT) extracted from radioactive bands was analyzed on DNA sequencing gels [24].

Partial purification of DNA ligase

All operations were carried out at 2-4°C. The cytosol postmicrosomal supernatant fraction (100 000 x g superna- tant; S100) was prepared as previously described [28]. The pellet or nuclear fraction was suspended in 5 volumes of the same buffer used for the SlOO fraction. This fraction was homogenized in a glasslteflon homogenizer, then sonicated three times at 50 W for 30 s using a Branson sonifier type B-12 (Daubury, CT), and centrifuged as for the SlOO fraction. The cytosol or the nuclear fraction was loaded onto a phosphocellulose column (30 -- 40 mg proteins/ml packed phosphocellulose), previously equilibrated with 50 mM potassium phosphate buffer at a flow rate of one column volume/h. The column was washed with the same buffer, until no proteins could be detected in the effluent. By this procedure, 43% of the loaded proteins were eluted in the 50 mM potassium phosphate wash. Enzyme activity was eluted with the 125 mM potassium phosphate buffer. No ligase activity was recovered using 250 mM or 500 mM potassium phosphate buffers. Active fractions were pooled and 1 volume of saturated ammonium sulfate solution, buffered at pH 7.5, was added. The suspension was stirred for 2 hand the precipitate collected by centrifugation at 23000 x g for 1 h. The pellet was resuspended and dialyzed against 20 mM potassium phosphate buffer. The dialysate was loaded onto a hydroxyapatite column equilibrated with the same

327

with 3 vol. saturated ammonium sulfate solution buffered at pH 7.5. After stirring for 2 h the suspension was centrifuged at 39000 x g for 2 h and the pellet dialyzed against 20 mM potassium phosphate buffer, containing 50% glycerol. Dialyzed enzyme fractions, after hydroxyapatite chromatog- raphy and ammonium sulfate precipitation, were kept at - 20°C and under these conditions were stable for several months.

In vitro proteolysis of ligase fractions Trypsin digestion was carried out using various protease

concentrations at 15°C for 15 min. At the end of the reaction, samples were immediately processed for activity gel analysis or for in vitro standard ligase assay. In the latter case, bo- vine serum albumin was added at the final concentration of 4 mg/ml.

Fig. 1. Activity gel analysis of DNA ligasefrorn cytoplasmic and nuclear extract of regenerating rat liver ( A ) and detection of the ligated sub- strate by DNA sequencing gel (B) . (A) The activity gel was loaded with 200 pg proteins of the postmicrosomal supernatant fraction (SIOO) from regenerating liver prepared in the presence (lane 1) or in the absence (lane 2) of protease inhibitors (see Experimental Proce- dure), with 220 pg proteins from nuclear extracts (lane 3) and with 0.2 Weiss unit [33] of commercial T4 DNA ligase (lane 4). The gel was prepared and processed as described [24]. (B) The radioactive bands in (A) were cut from the gel, the [32P]oligo(dT) extracted as described [24] and electrophoresed on a 20% acrylamide gel contain- ing 8 M urea. Lanes: 1, control unligated [32P](dT)16; 2 and 3, 60- kDa bands of lanes 1 , 2 (A); 4 and 5,130-kDa bands of lanes 1,2 (A); 6 and 7, 60-kDa and 70-kDa bands from T4 DNA ligase. n corresponds to 16 nucleotides. Protein markers were myosin (204 kDa), P-galactosidase (1 16 kDa), phosphorylase (94 kDa), bo- vine serum albumin (67 kDa) and egg albumin (43 kDa)

buffer (5 mg protein/ml packed hydroxyapatite). Following extensive washing with 20 mM potassium phosphate buffer, the column was washed with 125 mM potassium phosphate buffer until no more proteins could be detected in the effluent. Finally the enzyme was eluted with a 250mM potassium phosphate buffer. In preliminary experiments no ligase activi- ty has been detected after washing with 500 mM potassium phosphate buffer. Active fractions were pooled and mixed

RESULTS

Correlation of DNA ligase activity with a polypeptide of high molecular mass

Cytoplasmic postmicrosomal supernatant (S100) from re- generating liver exhibited DNA ligase activity (see Table 1). When this fraction was loaded on activity gel (Fig. 1 A, lane 1) two bands of 130 kDa and 60 kDa were observed. The absence of protease inhibitors during the preparation of cellular extracts did not modify the polypeptide pattern (Fig. 1 A, lane 2). For comparison, the activity gel of T4 DNA ligase is shown in lane 4, exhibiting the usual doublet of 60 kDa and 70 kDa [24,25].

Radioactive material, detected on activity gel, was extract- ed from each single band of lanes 1, 2 and 4 and run on a DNA sequencing gel as previously described [24]. (dT),, is totally converted into ligated polymers in the bands cor- responding to the doublet of T4 phage DNA ligase (Fig. 1 B, lanes 6 and 7) and in the 130-kDa bands from rat liver extracts (Fig. 1 B, lanes 4 and 5). Thus, the 130-kDa bands correspond to DNA ligase activity. However, the radioactive material of the 60-kDa bands of regenerating liver extracts corresponds only to unligated and unmodified (dT)16 (Fig. 1 B, lanes 2 and 3). This could result from the tight binding of an unidentified protein of the crude extract to the 5’-32P-labelled substrate, leading to its protection from phosphatase action, and therefore it does not contain ligase activity.

Purification of DNA ligase was achieved from both re- generating and normal rat liver, as described in Experimental Procedure. The results, which are summarized in Table 1, confirmed that ligase-specific activity is about 10 times higher in regenerating liver. Although ligases I and I1 have been often separated after a hydroxyapatite chromatography, in our case all ligase activity was eluted in a single peak with 125 mM potassium phosphate buffer step (HA-125). No activity was recovered with the 50 mM potassium phosphate buffer wash nor with buffers of molarity higher than 125 mM potassium phosphate (Fig.2A and B).

Active fractions of each purification step were pooled, concentrated by ammonium sulfate precipitation (75% saturation) and analyzed on activity gel (Fig. 3). Fractions from regenerating liver are shown in lanes 1 -4. It is evident that in the SlOO extract (lane 1) both 60-kDa and 130-kDa bands are present while after chromatography on phos- phocellulose and hydroxyapatite only the 130-kDa active

328

20 i 0 SO 20 LO 60 0

. . . , : h

I b.

j k~

-L-%> /--

20 LO 60 20 LO 60 C fractions

125 mM 250mM KPOL

1.5

1

0.5

P \ *p-q p ' :

-1.5 , ! , I f ' s

1- ; b\

2- -1

3- -0.5

20 LO 60 80 fractions

Fig.2. Hydroxyapatite chromatography o f D N A ligase from regenerating ( A and C ) and normal ( B ) liver. (A, B) The purification procedure is as described in Experimental Procedure; (C) the purification procedure has been modified for the phosphocellulose chromatography step as described in Results. Stepwise elution was carried out in all cases using 125 mM and 250 mM potassium phosphate buffers. Ligase activity (0-0) was determined in 5-pl aliquot of each indicated fraction after 15 min incubation at 37 "C. Protein concentration (0 - - ~ - 0)

band is visible (lanes 2 and 3). Proteins eluted with the 250 mM potassium phosphate step on hydroxyapatite chro- matography (HA-250) did not show any detectable activity bands (lane 4). In lane 5 the results obtained with the crude extract (SIOO) from normal liver indicated the presence of a strong band of 60 kDa whereas the 130-kDa polypeptide could not be easily detected possibly because of its weak activity. The 60-kDa band did not correspond to a ligation event of the [32P]oligo(dT) when analyzed on the DNA sequencing gel (not shown), indicating that, as previously shown for the regenerating liver, it did not contain ligase

activity. After the phosphocellulose chromatography, the 130- kDa polypeptide became visible (lane 6), although an additional band of lower molecular mass appeared (53 kDa). Proteins eluted with a 125 mM potassium phosphate step on hydroxyapatite chromatography show a smaller (100-kDa) polypeptide, which could be a degradative product (see Iane 7). As for the regenerating liver, the proteins eluted with the 250 mM potassium phosphate step did not exhibit detectable ligase activity bands (not shown).

To ascertain that a lower-molecular-mass form of enzyme, corresponding to ligase 11, which has been described as being

329

Fig. 3 . Activity gel analysis of DNA ligase after partial purification of the enzyme from regenerating and normal liver. Lanes 3 and 5: 200 pg proteins of postmicrosomal supernatant fraction (S100) from re- generating and normal liver, respectively. Lanes 2 and 6: enzyme fraction obtained after phosphocellulose chromatography and ammo- nium sulfate precipitation from regenerating and normal liver (100 pg and 165 pg proteins respectively). Lanes 3 and 4: hydroxyapatite fractions from regenerating liver (see Fig.2A) eluted with 125 mM and 250 mM potassium phosphate buffer (40 pg and 50 pg proteins respectively); lane 7, 160 pg protein fraction from normal liver (see Fig. 2B) eluted from hydroxyapatite with 125 mM potassium phosphate buffer. Protein markers were as in legend of Fig. 1

Fig.4. Detection of rat liver DNA ligase polypeptides in activity gel after limited trypsin degradation. The enzyme fractions (40 pg) after hydroxyapatite chromatography (125 rnM potassium phosphate buf- fer elution step of the regenerating liver sample, Fig.2A) were in- cubated with 0,O.l pg/ml, 0.5 pg/ml and 1 pg/ml trypsin (lanes 1-4) for 15 min at 15°C before loading onto the gel. Protein markers were as in legend of Fig. 1

associated with the rat liver nuclear fraction [9, 301, was not lost in our nuclear pellet of the SlOO fraction, this pellet was processed as described in Experimental Procedure and analyzed for ligase activity. Although it contained a significant amount of proteins (32% of total), less than 0.5% of the total ligase activity was found in it. When this fraction was loaded on an activity gel (Fig. 1 A, lane 3) no bands were detected even after overexposure of the gel. Thus it appears that in our case only one ligase species was present in the extracts of both regenerating and normal liver presenting chromatographic properties and molecular mass similar to those described for the ligase I [8, 111.

Fig. 5. Detection of catalytic polypeptides on activify gel after limited trypsin treatment of DNA ligase fractions A and B. Fractions A and B of the ligase were prepared by using the modified purification protocol described in Results (see Fig.2C). 45 pg ligase fraction A (odd-numbered lanes) and B (even-numbered lanes) were incubated in the same conditions as in legend of Fig.4, with 0 (lanes 1 , 2), 0.5 (lanes 3 , 4), 1 (lanes 5, 6) and 5 pg/ml (lanes 7, 8) trypsin, and then processed for activity gel as described in Experimental Procedure. Protein markers were as in legend of Fig. 1

Proteolytic degradation of ligase generates smaller active fragments

In order to detect smaller ligase polypeptides and, if any, those corresponding to the ligase-11-type enzyme we have analyzed the effect of proteolytic degradation of the 130-kDa ligase polypeptide after a limited trypsin treatment carried out on the HA-125 enzyme fraction from regenerating livers. Contrary to what has been observed in similar experiments, carried out on purified calf thymus ligase I [ll], the ligase activity measured in the standard in vitro assay was almost unaffected after 15 min treatment at 15°C with 0.1 pg/ml, 0.5 pg/ml and 1 pg/ml trypsin whereas most of it was lost at trypsin concentration of 5 pg/ml (not shown). The ligase samples treated with trypsin were also analyzed on activity gels. As shown in Fig. 4 (lanes 2 - 4) active polypeptides of 110 kDa, 100 kDa, 85 kDa and 75 kDa were progressively generated with increasing amount of trypsin. All these polypeptides contain ligase activity after the analysis of the size of oligo(dT) recovered from the bands on DNA sequencing gels (not shown). At a trypsin concentration of 1 pg/ml the initial 130-kDa active band disappears comple- tely and no accumulation of polypeptides with molecular mass lower than 75 kDa occurs (Fig. 4, lane 4). As has been already suggested for the calf thymus enzyme [ll], the trypsin diges- tion of the 130-kDa polypeptide does not produce active polypeptides of smaller molecular mass resembling the ligase I1 enzyme.

A slight modification of enzyme purification procedure allows the separation of two ligase fractions

As has been previously shown in our laboratory and in others, the ligase I1 fraction can be isolated on hydroxyapatite when the phosphocellulose chromatography is carried .out with a single-step elution, using buffers of high ionic strength [7, 8, 16, 241. To ascertain whether in our conditions, using a similar protocol, a second ligase fraction could be obtained on the hydroxyapatite column, a slight modification of the purification procedure has been carried out in the phosphocellulose chromatography step. Therefore, the S100 was loaded on a phosphocellulose column equilibrated with 50 mM potassium phosphate buffer and ligase activity was directly eluted with 125 mM potassium phosphate buffer

330

Fig.6. Activity gel analysis of degradative products of regenerating liver DNA ligase polypeptides during storage at 4°C ( A ) and detection of ligated substrate by DNA sequencing gel (B) . (A) 25 pg ligase fractions A and B, obtained by hydroxyapatite chromatography (see Fig.2C), were analyzed on activity gel immediately (lanes 1 and 2) and after storage at 4°C for 2 weeks (lanes 3 and 4), 10 weeks (lanes 5 and 6) or 15 weeks (lanes 7, 8). 10% (for lanes 1-6) and 8% (for lanes 7 and 8) acrylamide gels were used. (B) The radioactive bands in (A) were cut out from the gel and the [3ZP]oligo(dT) extracted and electrophoresed on 20% acrylamide gel containing 8 M urea as already described [24]. Lanes: a, control unligated [32P](dT)16; b and c, 130-kDa bands from lanes 3 and 4 of (A) respectively; d, 13 5-kDa band from lane 5; e and f, 100-kDa and 85-kDa bands from lane 7; g and h, 60-kDa bands from lanes 7 and 8 of (A) respectively. Protein markers were as in legend of Fig. 1

without previous washing with the equilibrating buffer. The 125 mM salt-wash, which contained 50% of the total proteins, was then processed as described in Experimental Procedure, by ammonium sulfate precipitation, and hydroxyapatite chro- matography. In this case two peaks of ligase activity were eluted on hydroxyapatite, at 125 mM (fraction A) and 250 mM potassium phosphate (fraction B) (see Fig. 2C). These two fractions are not distinguishable by their catalytic properties, i. e. pH dependence, NHf; concentration optimum and K,,, for ATP (not shown) nor they can be distinguished from the single rat liver ligase, obtained using the common phosphocellulose chromatography procedure. When ana- lyzed on activity gel, they presented a single major active polypeptide of 130 kDa (Fig. 5, lanes 1, 2).

Although fractions A and B present polypeptides of identi- cal molecular mass we wanted to analyze whether, after

controlled proteolysis of these two fractions, the 130-kDa polypeptides exhibit similar patterns of degradation. After treatment with increasing concentrations of trypsin, fraction A yielded bands of 110 kDa, 100 kDa, 85 kDa and 75 kDa (Fig. 5, lanes 3,5,7) on activity gel, as observed before for the single ligase enzyme (see Fig.4). The same result was obtained for fraction B except that the degradation occurred more rapidly at the same trypsin concentration (Fig. 5, compare lanes 5 and 6).

After storage of fractions A arid B at 4°C for several weeks the appearance of smaller active polypeptides was revealed by activity gel analysis (Fig.6). The same polypeptides of 110 kDa, 100 kDa, 85 kDa and 60 kDa appeared in both cases, with the first three corresponding to those observed during the limited tryptic digestion. Again ligase fraction B seemed to be more rapidly degraded than ligase fraction A

331

(compare Fig. 6A, lanes 5 and 6, lanes 7 and 8). Ligase activity was found in the polypeptides of 130 kDa, 110 kDa, 100 kDa and 85 kDa from fraction A and in 130 kDa from fraction B, as determined by the size of oligo(dT) measured on the DNA sequencing gel (Fig. 6 B, lanes b - f). Again this activity was not found in the case of the 60-kDa bands from both fractions A and B (Fig.6B, lanes g- h). These results suggest that DNA ligases of fractions A and B are composed of similar active polypeptides.

DISCUSSION

In this paper we have presented evidence that only one ligase form is detected in both normal and regenerating rat liver cells when using a certain purification procedure. We have also confirmed that ligase activity is increased of at least tenfold in regenerating rat liver [13, 311, suggesting that the low amount of ligase present in resting cells is not sufficient for completing the DNA synthesis process.

The combined use of activity and DNA sequencing gels has allowed us to determine the molecular mass of the ligase catalytic polypeptides, and also to follow their persistance during the different steps of purification. In the activity gel the DNA ligase purified from regenerating liver presents a catalytic polypeptide of 130 kDa. This catalytic activity could be partially degraded by trypsin treatment or storage at 4 "C giving rise to smaller polypeptides of 110 kDa, 100 kDa, 85 kDa and 75 kDa, which are still active. The same inter- mediate species are obtained from fractions isolated from normal rat liver. However, in the latter the sensitivity of the enzyme to degradation seems to be higher than in the re- generating liver (see Fig.3). This could be due to different proteolytic activities in normal liver compared to regenerating tissue.

The detection on activity gel of radioactive bands equal to or smaller than 60 kDa, which are devoid of ligase activity [as demonstrated by the lack of oligo(dT) ligation], can be explained by the specific binding of unknown proteins of crude extract to the 3'-OH, 5'-32P termini, thus hindering phosphatase action. We have previously shown that some DNA-binding proteins, such as rat liver HMGl or histone H1, are able to protect the substrate from the phosphatase action in our activity gel conditions [24]. Since a similar protec- tion to the phosphatase action has been revealed for the 60-kDa polypeptide generated after DNA ligase degradation, it is possible that this polypeptide contains the DNA-binding domain of DNA ligase.

By a slight modification of the purification protocol for the regenerating liver DNA ligase, we have been able to sepa- rate on hydroxyapatite two distinct enzyme fractions (A and B), which could correspond to the ligases I and I1 already described, in term of their chromatographic properties [7]. However, when analyzed on activity gel these fractions present polypeptides with the same molecular mass. After proteolytic treatment they present an identical degradation pattern. We suggest, therefore, that they derive from the same precursor polypeptide of 130 kDa. The reason for their different chro- matographic behaviour is not yet understood but it could be related to uncontrolled proteolytic events occurring after the phosphocellulose column, and/or to the association with other cellular proteins. In fact, since with this modified purification procedure almost 50% of the total proteins are found in the active ligase fraction while only 5% are found with the usual procedure, it is plausible that this different protein environ-

ment could partially modify the chromatographic properties of the DNA ligase activity.

Our results suggest that the major form of DNA ligase of rat liver cells corresponds to a species of 130 kDa. As has been previously shown in mouse cells [32] it is likely that the native form of the ligase could be a polypeptide of higher molecular mass (200 kDa), which is rapidly degraded into a 130-kDa form. Also in some of our preparations of rat liver enzyme, polypeptides of higher molecular mass (1 50 kDa and 200 kDa) have been detected by activity gel but were rapidly converted into the 130-kDa species (not shown).

In conclusion we propose that this 130-kDa protein corre- sponds to the unique enzyme having DNA ligase activity in rat liver cells. In certain conditions this enzyme is degraded into polypeptides of smaller molecular mass, which are still active except for the 60-kDa fragment. Therefore the identification of a rat liver DNA-ligase-11-type enzyme, using the activity gel technique, remains to be established.

This work is supported by grants from Commission of European Communities nos B16-163 F and BI6-158-1. U. Bertazzoni is a scien- tific official of Commission of the European Communities (Contribu- tion no 2366). We thank C. Lavenot for expert technical assistance in some parts of this work. We are also grateful to Dr R. Elder for the critical reading of this paper.

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