Immunochemical analysis of molecular forms of mammalian DNA ligases I and II

Biochimica et Biophysica Acta 873 (1986) 297-303 Elsevier

297

BBA32626

I m m u n o c h e m i c a l a n a l y s i s of m o l e c u l a r f o r m s of m a m m a l i a n D N A ligases I a n d II Hirobumi Teraoka and Kinji Tsukada Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101 (Japan) (Received 28 April 1986)

Key words: Western blotting; DNA ligase; Immunochemistry; (Calf thymus)

Using specific antibodies against calf thymus DNA iigases I and II (EC 6.5.1.1), we have investigated the polypeptide structures of DNA ligases I and II present in the impure enzyme preparations, and estimated the polypeptides of DNA ligases I and II present in vivo. Immunoblot analysis of DNA ligase I after sodium dodecyl sulfate-polyacrylamide gel electrophoresis "revealed a 130-kDa polypeptide as a major one in the enzyme preparations from calf thymus throughout the purification. In addition to the 130-kDa polypeptide, a 200-kDa polypeptide was detected in the enzyme preparations at the earlier steps of the purification, and a 90-kDa polypeptide was observed as a minor one in the enzyme preparations at the later steps of the purification. The polypeptides with molecular weight of 130000 and 90000 were detected by SDS-polyacrylamide gel electrol~horesis of DNA ligase I-[3H]AMP complex. These results suggest that a 200-kDa polypeptide of DNA ligase I present in vivo is degraded to a 130-kDa polypeptide and then to a 90-kDa polypeptide during the isolation and purification procedures. On the other hand, the monospecific antibody against calf thymus DNA ligase II cross-reacted with only a 68 kDa polypeptide in the enzyme preparations throughout the purification, suggesting that the 68-kDa polypeptide is a single form of calf thymus DNA ligase II present in vivo as well as in vitro.

Introduction

DNA ligase (EC 6.5.1.1), which covalently joins single-stranded breaks in duplex DNA plays an essential role in DNA replication and repair [1]. The molecular weight of mammalian DNA ligases has been variably estimated to fall within the wide range of 50000 to 480000 [2-18]. This Seems to be due to the following reasons: mammalian DNA ligase has a highly asymmetric molecular structure, so that a relatively higher M r on gel-filtration Correspondence address: Dr. H. Teraoka, Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental [Jniversity, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101, Japan.

and lower s20,w value on sucrose density gradient centrifugation have been obtained (see Refs. 18 and 19); DNA ligase is degraded to lower-Mr forms during the isolation and purification procedures [8,18,19]; two distinct forms of DNA ligase are likely to exist in mammalian cells [6,8,10-12, 17,181. Recently we have purified DNA ligases I (M r 130000) [19] and II (M r 68000) [20] from calf thymus and obtained monospecific antibodies against DNA ligases I and II. The antibody against DNA ligase I does not cross-react with DNA ligase II, or vice versa, indicating that DNA ligases I and II are immunochemically different from each other [20]. In this work, we detected immunoreactive polypeptides of calf thymus DNA

0167-4838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

298 ligases I and II that are present in the impure enzyme preparations from the purification steps directly on a nitrocellulose membrane after SDSPAGE, and estimated molecular forms present in vivo. Materials and Methods

Chemicals. Rabbit muscle myosin and egg white trypsin inhibitor were obtained from Sigma. Escherichia coli R N A polymerase was from Boehringer-Mannheim. Protein A was from UCB-Bioproducts S.A. Na125I was purchased from New England Nuclear. Chloramine T was from Nakarai Chemicals (Kyoto). The immunoblot-assay kit was purchased from Bio-Rad. All other chemicals were as described previously [19-22]. Calf thymus. Fresh calf thymuses were obtained from Tokyo Municipal Slaughterhause and the frozen tissue was from Mitsubishi Kasei (Tokyo). Preparation of lgG fraction from rabbit antisera. IgG fractions were prepared from antisera to calf thymus DNA ligases I [19] and II [20]. The rabbit antisera were treated with 35%-saturated ammonium sulfate and the precipitate was dissolved in 10 mM NaPO 4, p H 8, and dialyzed against the buffer. The dialysate was applied to a DEAE-cellulose column equilibrated with 10 mM NaPO 4, pH 8, and washed with the buffer. The protein in the pass-through fraction was precipitated with 50%-saturated ammonium sulfate. The precipitate was dissolved in and dialyzed against 10 mM NaPO 4, pH 7.5, containing 0.14 M NaC1. Iodination of protein A. Protein A was radioiodinated with 1251 by a modification of the chloramine T method [23]. Protein A (200/~g), Na225I (225/~Ci) and chloramine T (20 #g) were mixed in a total volume of 65 #l in 0.15 M NaPO 4 buffer, pH 7.5, and incubated at 25°C for 15 rain. After successive additions of 20/~l NaHSO 3 (1 mg/ml), 20 #l KI (10 m g / m l ) and 0.4 ml of 0.5% bovine serum albumin in phosphate-buffered saline, 1251labeled Protein A was isolated on a Sephadex G-50 column equilibrated with phosphate-buffered saline containing 0.5% bovine serum albumin. Preparation of calf thymus DNA ligase. Enzyme fractions (steps 1 to 6) from a typical purification of DNA ligase I from calf thymus were obtained as described previously [19] except that the calf

thymus frozen tissue was homogenized in 10 mM KPO 4 buffer, pH 7.5, containing 0.3 M KC1, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM sodium bisulfite, 0.05 m g / m l egg white trypsin inhibitor and 7 mM 2-mercaptoethanol. D N A ligase II preparations were obtained as described previously [20]. SDS-PAGE and protein transfer. SDS-PAGE (slab-gel) was carried out as described by King and Laemmli [24]. Proteins in the gel were electrophoretically transferred to a nitrocellulose membrane (Schleicher and Schi~ll) at 15 V for 8 h and then at 30 V for 1 h with an electrophoretic transfer apparatus (Marysol, Tokyo) in 25 mM T r i s / 1 9 2 mM glycine buffer (pH 8.3) containing 20% methanol [25]. Marker polypeptides on the nitrocellulose membrane after electrophoretic blots from the SDS-polyacrylamide gel were stained with Amido black. Immunoreactive polypeptides were detected with either 125I-labeled Protein A or goat anti-rabbit IgG-peroxidase conjugate (BioRad). For the radioimmunoassay, nitrocellulose membrane was rinsed overnight in phosphatebuffered saline containing 0.5% Nonidet P-40 and then washed three times for 15 min each with phosphate-buffered saline containing 0.05% Tween 20. The membrane was incubated with 3% bovine serum albumin in phosphate-buffered saline at 37°C for 1.5 h, and then washed three times for 15 min each with phosphate-buffered saline containing 0.05% Tween 20. The membrane was mounted with IgG (16/~g p r o t e i n / m l phosphatebuffered saline containing 3% bovine serum albumin), incubated at 37°C for 1.5 h, and then washed six times with phosphate-buffered saline/ 0.05% Tween 20 for 15 min each. The membrane was incubated with 125I-labeled Protein A (2.7 • 105 c p m / m l phosphate-buffered saline containing 3% bovine serum albumin) at 37°C for 1 h and then washed 10 times for 15 min each with phosphatebuffered saline/0.05% Tween 20. The membrane was subjected to air-dried and autoradiography was carried out at - 7 0 ° C with an intensifying screen. The enzyme immunoassay was carried out essentially by the protocol recommended by BioRad as described previously [20].

Gel electrophoresis of DNA ligase-[3H]AMP complex. The preparations of DNA ligases I and II were incubated with [3H]ATP to obtain DNA

299

ligase-[3H]AMP complex [19,20]. More than 95% of 3H-radioactivity was recovered as [3H]ATP after incubation with PPi, and more than 90% of the radioactivity as [3H]AMP after incubation with 5'-Pi,3'-OH nicked DNA [19,20]. The DNA ligase I-[3H]AMP complex was subjected to electrophoresis in an 8% SDS-polyacrylamide gel by the method of Weber and Osborn [26]. The cylindrical gel (0.5 × 9 cm long) was cut into 2-ram section and counted as described previously [19]. The recovery of radioactivity was more than 90%. DNA ligase II-[3H]AMP was subjected to SDS-PAGE (10% polyacrylamide slab gel) [24] and fluoropgraphy was carried out as described previously [22].

Gel filtration of DNA ligase I-[3H]AMP complex. DNA ligase I-[3H]AMP was subjected to gel filtration on a Sephadex G-150 column (1.2 x 90 cm) equilibrated with 25 mM KPO4, pH 7.5/0.5 M KC1/0.5 mM dithiothreitol/0.1 mM EDTA. Void volume was determined with blue dextran. Catalase (M r 240 000), lactate dehydrogenase (M r 140000) and ovalbumin (M r 45 000) were used as external standard proteins. Assay of DNA ligase. DNA ligase activity was determined as described previously [19-22]. DNA ligase I was incubated at 37°C for 10 min in reaction mixture (0,2 ml) containing 75 mM TrisHC1, pH 7.8/10 mM MgC12/0.2 mM ATP/5'-32p nicked DNA ((1-5). 104 cpm/3-20 pmol)/2 mM dithiothreitol/20/ag bovine serum albumin. DNA ligase II was incubated at 30°C for 20 min in the reaction mixture containing 0.1 M KC1. One unit (U) of the enzyme was defined as the amount converting 1 nmol 32p/rain to an alkaline phosphatase-resistant form under the standard assay conditions. Protein was determined by the method of Bradford [27]. Results

Using anti(DNA ligase I)IgG, we detected immunoreactive polypeptides during the purification of calf thymus DNA ligase I by Western blot/ radioimmunoassay (Fig. 1). Aliquots containing 23-94/~g protein in the fractions at the purification steps (see steps 1 to 6 in Table I of Ref. 19) were subjected to SDS-PAGE and electrophoretically transferred to the nitrocellulose membrane. After incubation with anti(DNA ligase I)IgG and

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Fig. 1. Detection of imrnunoreactive polypeptides of DNA ligase I by radioimmunoassay after Western blot. The preparations of enzyme fractions (purification steps 1 to 6) were subjected to SDS-PAGE in an 8% polyacrylamide slab gel [24]. Electrophoretic blotting and immunological detection with 125I-Protein A were carried out as described under Materials and Methods. Lane 1, step 1, crude extract (0.6 mU, 94 btg protein); lane 2, step 2, ammonium sulfate fractionation (35-60% saturation) (0.6 mU, 75 /~g protein); lane 3, step 3, calcium phosphate gel (0.9 mU, 50/tg protein); lane 4, step 4, phosphocellulose (5.6 mU, 30 /tg protein); lane 5, step 5, DEAE-cellulose (12.6 mU, 31 /zg protein); lane 6, step 6, Blue-Sepharose (21.5 mU, 23 /~g protein). X-ray films were developed 10 days after exposure for (A) and 1 day for (B). Marker polypeptides used were myosin ( M r 200000), fl-galactosidase (M r 116000), phosphorylase b (M r 94000), bovine serum albumin ( M r 67 000), ovalbumin ( M r 43 000) and lactate dehydrogenase (M r 35 000).

then with 125I-labeled Protein A, the membrane was autoradiographed for 10 days (Fig. 1A) and for 1 day (Fig. 1B). A 130-kDa polypeptide was observed throughout the purification steps (lanes 1 to 6). The intensity of the polypeptide bands in the autoradiogram almost corresponded to the activity level present in the enzyme preparations at the purification steps. In addition, a faint band corresponding to the position of M r 200 000 was detected at the earlier steps (lanes 1 and 2) of the purification. Enzyme-linked immunoassay was also employed to determine immunoreactive polypeptides of DNA ligase I (Fig. 2). Essentially identical results were obtained: a 130-kDa polypeptide was observed throughout the purification (steps 1 to 4); a 200-kDa polypeptide was present in steps 1 and 2 in the purification, and disappeared at step 3. These results suggest that the 130-kDa polypeptide is derived from the 200-kDa polypeptide by limited degradation during the iso-

300

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calf thymocytes that had been metabolically labeled with [3H]leucine [21]. A minor polypeptide of M r 90000 was observed at the later steps of the purification (Fig. 1, lanes 4 to 6, and Fig. 2, lane 4), indicating that the 90-kDa polypeptide is likely a degradative product of the 130-kDa polypeptide during the purification procedure. Some of the purified preparations of DNA ligase I from Ehrlich ascites tumor cells [22] contained a polypeptide of M r about 90000 in addition to a 130-120-kDa polypeptide (unpublished data). DNA ligase I in the step 6 preparation (Fig. 1, lane 6) was labeled with [3H]AMP as DNA ligaseAMP complex, a stable reaction intermediate, and then subjected to electrophoresis on SDS-polyacrylamide gel (Fig. 3B). DNA ligase-[3H]AMP complex migrated as polypeptides of Mr 130000 and 90000. When the complex was subjected to chromatography on a Sephadex G-150 column, DNA ligase activity and 3H-radioactivity were recovered at identical positions corresponding to apparent Mr 240000 and 120 000 (Fig. 4B). Since the 130-kDa polypeptide is derived from an enzyme form of apparent Mr 240000 on gel filtration (see Fig. 4A and Ref. 19), the 90-kDa polypeptide seems to correspond to an enzyme form of apparent Mr 120000 on gel-filtration. In some enzyme preparations from frozen materials and a prolonged purification procedure, only a 90-kDa polypeptide was observed in the DNA ligase[3H]AMP complex as the major one (Fig. 3C).

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front-Fig. 2. Detection of immunoreactive polypeptide of D N A ligase 1 by enzyme-immunoassay after Werstern blot. SDSPAGE (8% polyacrylamide slab gel) and Western blot/enzyme immunoassay were carried out as described under Materials and Methods. Lane 1, step 1, crude extract (0.1 mU, 20 #g); lane 2, step 2, ammonium sulfate fractionation (35-75% saturation) (0.2 mU, 25 #g); lane 3, step 3, calcium phosphate gel (0.5 mU, 30 #g); lane 4, step 4, phosphoceUulose (1.3 mU, 20/~g). Marker polypeptides were as described in the legend to Fig. 1.

lation and purification procedures. In biosynthetic experiments with DNA ligase I, a 200-kDa polypeptide was precipitated with the antibody from detergent-lysates of Ehrlich ascites tumor cells and

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Fig. 3. SDS-PAGE of DNA ligase I-[3H]AMP complex. DNA ligase I labeled with [3H]AMP was subjected to SDS-PAGE (8% cylindrical gel) as described under Materials and Methods. (A) D N A ligase I was purified up to step 7 (Sephadex G-200 column chromatography) in Ref. 19 from fresh calf thymus without storage during the purification procedure. (B) The preparation of DNA ligase I corresponding to the step 6 preparation of Fig. 1 was obtained from frozen materials (see Materials and Methods). (C) DNA ligase II was purified up to step 7 from frozen materials with prolonged storage during the purification procedure. Bovine serum albumin (Mr 67000) and E. coli RNA polymerase fl ( M r 160000), fl' ( M r 150000), o ( M r 82000) and a ( M r 40000) subunits were used as marker polypeptides. The positions of marker polypeptides and bromophenol blue (BPB) are indicated by vertical lines.

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Fig. 4. Sephadex G-150 column chromatogram of DNA ligase I-[3H]AMP complex. The enzyme preparation of step 7 in Ref. 19 from fresh calf thymus (A) and the step 6 enzyme of Fig. 1 (B) were labeled by incubation with [3H]ATP. The DNA ligase-[3H]AMP was applied to a Sephadex G-150 colunm (1.2X90 cm). Fractions of 1.5 ml (A) and 1.35 ml (B) were collected. The elution positions are expressed as elution volume versus void volume, e, enzyme activity; x , 3H radioactivity.

With fresh thymuses, DNA ligase I-[3H]AMP from the highly purified preparation consisted almost entirely of a 130-kDa polypeptide (Fig. 3A), which showed apparent M r 240 000 on gel-filtration (Fig. 4A). As can be seen in Figs. 1 and 2, an immunoreactive polypeptide with M r about 50000 was also detected throughout the purification of calf thymus DNA ligase I. Such a polypeptide of M r about 50000 was also observed as a minor component of DNA ligase I-[3H]AMP on the SDS-polyacrylamide gel (Fig. 3). Furthermore, a minor peak of DNA ligase I activity of apparent M e 60 000-70 000 was observed on gel-filtration of some enzyme-preparations from calf thymus (not shown). There is no conclusive evidence, however, that an enzyme form of M r about 50 000 is one of the sequential degradative products of DNA ligase I present in vivo. Most recently we succeeded in obtaining monospecific antibody against calf thymus DNA ligase II [20], which does not cross-react with DNA ligase I at all. Thus we intended to detect im-

munoreactive polypeptides of DNA ligase II present in several steps of the purification procedure. The enzyme-immunoblot assay revealed that anti(DNA ligase II)IgG reacted with a single 68kDa polypeptide throughout the purification (steps 1 to 4) (Fig. 5). DNA ligases I and II can be separated from each other at the calcium phosphate gel step (step 3) during the purification procedure [6,10,19,20]. From the data from hydroxyapatite column chromatography separating DNA ligases I and II in the preparations at step 1 or 2 (data not shown), DNA ligase II activity in steps 1 and 2 is estimated to be about one-third the activity determined under the standard assay conditions for DNA ligase II. On the basis of this correction, the intensity of the band corresponded roughly to the activity level applied. The step 4 enzyme was labeled with [3H]AMP and the DNA ligase II-[3H]AMP complex was subjected to SDS-PAGE. After fluorography, a single band corresponding to a 68-kDa polypeptide was observed (data not shown; see Ref. 20). In contrast to DNA ligase I, DNA ligase II seems to be present as a single 68-kDa polypeptide in the impure preparations of the enzyme and in vivo.

top-

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4535frontF i g . 5. Detection of immunoreactive polypeptides of DNA ligase II by enzyme-immunoassay after Western blot. The preparations of enzyme purification steps (steps 1 to 4) were subjected to SDS-PAGE (10% polyacrylamide slab-gel). Lane 1, step 1, crude extract (0.16 mU, 33 #g); lane 2, step 2, ammonium sulfate fractionation (0.21 mU, 34 #g); lane 3, step 3, calcium phosphate gel (0.11 mU, 20 #g); lane 4, step 4, phosphocellulose (0.25 mU, 5 #g). Marker polypeptides used were as described in the legend to Fig. 1.

302

Discussion

Several investigators, including us, have reported that two distinct enzyme forms of DNA ligase (DNA ligase I or cytoplasmic DNA ligase, and DNA ligase II or nuclear DNA ligase) exist in mammalian cells and tissues [6,10-14,16-18,2830]. In general, DNA ligase I has a higher M r and lower K m for ATP and DNA ligase II has a lower M r and higher K m for ATP. In this paper, multiple forms of DNA ligase I are detected with anti(DNA ligase I)IgG in the crude extract and the impure enzyme preparations from calf thymus. A 200-kDa polypeptide was detected at the earlier steps of the purification of DNA ligase I. A similar or identical polypeptide of M r 200000 was precipitated with the anti(DNA ligase I)IgG from detergent-lysates of mouse Ehrlich ascites tumor cells and calf thymocytes in the biosynthetic experiments with DNA ligase I [21]. In gel-filtration experiments, a species of DNA ligase I of apparent M r about 500000 was observed in the crude or partially purified preparations from rat liver [11,14], rat brain [13], Chinese hamster ovary cells [17,28] and calf thymus (unpublished data). The species of DNA ligase I of higher M r disappeared during storage or purification procedures. Therefore, the species of DNA ligase I of apparent M r about 500 000 on gel-filtration may correspond to a 200-kDa polypeptide detected in the protein blotting (this work) and biosynthetic experiments [21]. The 200-kDa polypeptide is most probably a native form of DNA ligase I that is extraordinarily susceptible to limited proteolysis during the isolation and purification procedures. Anti(DNA ligase II)IgG cross-reacted with a 68-kDa polypeptide throughout the purification (steps 1 to 4), and DNA ligase II-[3H]AMP complex from the impure enzyme preparations showed a single band corresponding to M r 68000. It is likely that DNA ligase II exists as a 68-kDa polypeptide in vivo. U sing the 'activity gel method' (in situ assay of enzyme activity after SDS-PAGE), Mezzina and co-workers [16,29] have detected multiple forms of catalytically active DNA ligase in the impure enzyme preparations of CV1-P monkey kidney cells. DNA ligase I that had been separated from DNA ligase II on a hydroxyapatite column showed three or four catalytically

active polypeptides of M r 60000 to 200 0()0, and the DNA ligase II fraction revealed two major polypeptides of M r 60 000 to 70 000. The lower-M r form of DNA ligase II may be a degradation product. In addition, they said that a common peptide of M r 58 000 is present in DNA ligases I and II [29]. However, our immunochemical works on calf thymus DNA ligases I and II suggested that these two forms are independent of each other [20]. Although the activity gel method is an excellent technique to detect active polypeptides in crude extract or impure enzyme preparations, the activity detected by this method seems to be qualitative but not quantitative [31]. For example, it seems likely that it is more difficult to restore the activity of higher-Mr polypeptides in the gel compared with lower-M r polypeptides. Although a variety of molecular weights of mammalian DNA ligase have been reported hitherto, we now consider that DNA ligase I and DNA ligase II, two distinct molecular forms of mammalian D N A ligase, are likely to be present in mammalian cells as a 200-kDa polypeptide and a 68-kDa polypeptide, respectively. We have been investigating the expression and regulation of mammalian DNA ligase. In yeast cells containing a single species of DNA ligase [32,33], mRNA levels of the enzyme seem to be regulated independently in correlation with DNA replication and repair [34]. Similarly, the activity of mammalian DNA ligase has been reported to be enhanced in the DNA replication process [13,14,28,35-43] and in the DNA repair process [43-45]. As for DNA ligase I, or cytoplasmic DNA ligase, which seems to be involved in DNA replication, we have found that the increase in the enzyme activity in correlation with DNA replication can be ascribed to the change in the enzyme quantity by the method of immunochemical titration [19]. Using antibody against calf thymus DNA ligase II, we intended to determine whether the change in the enzyme activity during the DNA repair process, if present, is due to the change in enzyme quantity or the alteration of catalytic efficiency per enzyme molecule. Unfortunately, the animal species-specificity of the anti(calf thymus DNA ligase II)IgG is so high that it is probably difficult to analyse rodent and human DNA ligase IIs immunochemically (unpublished data). A pre-

303 l i m i n a r y e x p e r i m e n t has d e m o n s t r a t e d t h a t c a l f thymus DNA l i g a s e II is i n h i b i t e d b y a p o l y ( A D P - r i b o s y l ) a t i o n r e a c t i o n in v i t r o [46,47]. T h i s o b s e r v a t i o n is c o n s i s t e n t w i t h the r e p o r t t h a t t h e i n h i b i t i o n of p o l y ( A D P - r i b o s e ) s y n t h e s i s b y 3 - a m i n o b e n z a m i d i n e facilitates D N A l i g a t i o n in h u m a n l y m p h o b l a s t o i d W I L - 2 cells [48]. I n add i t i o n to t h e e n z y m o l o g i c a l studies, w e are n o w in the process of investigating the gene expression a n d r e g u l a t i o n o f the t w o f o r m s of m a m m a l i a n D N A ligase.

Acknowledgements T h i s w o r k was s u p p o r t e d in p a r t b y a G r a n t - i n A i d 58580133 f o r S c i e n t i f i c R e s e a r c h f r o m the Ministry of Education, Science and Culture of J a p a n . W e t h a n k M a s a j i Sawai, S o h S u m i a n d T a k a o S u m i k a w a for t e c h n i c a l assistance,

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