Analysis of the most tightly bound proteins in eukaryotic DNA

390

Biochimica et Biophysica Acta, 564 (1979) 390--401 © Elsevier/North-Holland Biomedical Press

BBA 99541

ANALYSIS OF THE MOST T I G H T L Y BOUND PROTEINS IN E U K A R Y O T I C DNA

WANDA KRAUTH and DIETER WERNER

Institute for Cell Research, German Cancer Research Center, D-6900 Heidelberg (F.R.G.) (Received October 16th, 1978) (Revised manuscript received March 15th, 1979)

Key words: DNA-protein complex; Eukaryotic DNA, Protein binding

Summary DNA isolated by procedures generally considered to be most efficient for purifying DNA still contains detectable peptide components. The characteristics of this material and the stability of its linkage to DNA were investigated: DNA released from [3SS]methionine-labelled cells by SDS in the presence of proteases contains a significant amount of 3sS label which is n o t removed by additional treatment with proteases and phenol and which cosediments and cobands together with DNA on alkaline gradients. Furthermore, some peptide material which is copurified with native DNA and which remains complexed with DNA after alkali treatment can be labelled with ~2sI and analyzed on SDSpolyacrylamide-gels. The amino acid analysis of hydrolysates of purified DNA gives a rough estimate of the a m o u n t of the peptide material which is copurifled with DNA. The results indicate that distinct proteins between 54 000 and 68 000 daltons in size are n o t removed from DNA by phenol, proteases, alkali or by any combination of these treatments. They can only be isolated by degradation of DNA. This extreme stability of the DNA-protein linkage indicates that these proteins are not merely contaminants which are difficult to eliminate b u t are rather covalently or otherwise b o u n d (alkali-stable) to DNA. The size of these proteins and the stability of their linkage to DNA suggests that they are related to the class of non-histone proteins which are thought to be involved in chromatin structure e.g. by keeping DNA in a supercoiled state. Other possible functions are discussed. Introduction

Following treatment of mammalian cells in lysis buffer (SDS, salt, EDTA), DNA is released in form of a 'DNA-protein complex' which is free of histones Abbreviation:

SDS, sodium dodecyl sulphate.

391 [1] and in which DNA is present in a superhelical conformation [2--4]. It is highly probable that the 'clamps' which restrain the ends of the superhelical loops of these complexes consist of a peptide material because partial digestion with proteases results in more relaxed structures [ 1 ] and after extensive digestion of cell lysates with proteases and ribonucleases high molecular weight DNA (40--500 • 106 daltons) is completely linear [5]. In this paper we demonstrate that both completely relaxed native DNA and alkali-denatured DNA still contains tightly bound and distinct proteins which are 54 000--68 000 daltons in size. Similar proteins are also present in nuclear ghosts [6--8], histonedepleted metaphase chromosomes [9] and in salt-treated polytene chromosomes [10]. Materials and Methods

Labelling of cellular proteins with L-[aSS]methionine, cobanding and cosedimentation of the 3SS-label with DNA Five days after inoculation of Ehrlich ascites tumor cells two mice received 100 ~Ci of [3H]thymidine (20 Ci/mmol) and 125 /~Ci of L-[3SS]methionine (800 Ci/mmol) by intraperitoneal injection. 24 h later the injections were repeated and after another 24 h period the tumor cells were harvested, collected by centrifugation and lysed in 40 ml of the following lysis buffer: 10 mM Tris, 10 mM EDTA, 100 mM NaC1, 0.5% SDS, 50 ~zg/ml of proteinase K (Merck, Darmstadt, F.R.G.). Cell lysis was completed at 37°C for 24 h which was followed by dialysis (10 mM Tris, 2.5 mM EDTA, 10 mM NaC1, pH 8) for 48 h. Portions of this lysate were additionally treated with proteases (6 mg/ml) for 24 h at 37°C. I ml aliquots of these additionally proteasetreated lysates were mixed with 0.5 ml I N NaOH, underlayered with alkaline (0.33 N NaOH, 0.7 M NaC1) 5--20% sucrose gradients and centrifuged in polyallomer tubes of the SW27 rotor at 5°C; 20 000 rev./min; 13 h. Fractions were taken from the top (Autodensiflow IIC, Buchler, Fort Lee, U.S.A.). DNA was precipitated by 10% trichloroacetic acid and filtered through Millipore BSWP 02500 filters, which were washed with trichloroacetic acid, ethanol and counted in a Nuclear Chicago scintillation spectrometer (Mark III) with the appropriate program for double-label counting for 3H/3SS. Furthermore, 2.4 g amounts of Cs2SO4 were mixed in polyallomer tubes of the Ti50 rotor with 0.5 ml of I N NaOH, 1 ml portions of the lysate, phenolized lysate, or lysate which was additionally treated with proteases (6 mg/ml), 24 h, 37°C. The volume was made up to 4 ml with distilled water and the DNA was banded at 20°C; 35 000 rev./min; 48 h. Fractions were taken from the top and counted as described for the fractions of the sucrose gradients. Isolation of DNA by means of proteases and phenol DNA from Ehrlich ascites cells was isolated according to the method described by Gross-Bellard et al. [5] with minor modifications. This method includes cell lysis (2 • 10 s cells/300 ml of lysis buffer (10 mM Tris, 10 mM EDTA, 100 mM NaC1, 0.5% SDS, pH 8) in the presence of proteinase K (50 ~zg/ml) or pronase (6mg/ml, Serva, Heidelberg) careful phenolisation, dialysis, treatment with pancreatic and T1 ribonucleases (Boehringer, Mannheim, F.R.G.), second treatment with proteinase K in the presence of 0.5%

392 SDS, careful phenolisation and, finally, dialysis. DNA prepared aceording to this procedure showed A26o/A28o ratios in the order of 1.65--1.70. These DNA preparations still contained a large a m o u n t of peptide components. Therefore, t h e y were vigorously shaken (twice) with equal volumes of phenol equilibrated with SDS-buffer (500 mM Tris, 10 mM EDTA, 10 mM NaC1, 0.5% SDS, pH 8). Finally the DNA was dialysed against a dialysis buffer of the following composition: 10 mM Tris, 2.5 mM EDTA, 10 mM NaC1, pH 8. Further purification o f this DNA was achieved by sedimentation through alkaline sucrose gradients. DNA prepared as described above was denatured by mixing 10 ml portions with 5 ml of 1 N NaOH in SW27 polyallomer tubes. After storage at 20°C for 1 h the solution of the denatured DNA was underlayered with 22 ml of 20% sucrose in 0.33 N NaOH. Centrifugation was at 5°C; 25 000 rev./min; 13 h. The supernatant was discarded and the bottom-fractions (6 ml) were pooled and dialysed against bidistilled water. DNA from sea urchin sperm was isolated according to the m e t h o d described above as well as DNA from rat liver. In the latter case the tissue was first homogenized in liquid nitrogen [11]. However, both, liver DNA and sea urchin sperm DNA were not further purified by alkali treatment.

Isolation o f DNA by direct alkaline cell lysis For isolation of DNA in the absence of proteases Ehrlich ascites cells (2 • 10 s) were suspended in 100 ml of a buffer with the following composition: 50 mM Tris, 20 mM EDTA and 100 mM NaC1, pH 8. The suspension was added under vigorous shaking to a mixture of 100 ml of 1 N NaOH and 100 ml of the following lysis buffer: 10 mM Tris, 10 mM EDTA, 100 mM NaC1, 0.5% SDS, pH 8. After 2 h at room temperature the lysate was shaken three times with equal volumes of phenol. Finally the DNA solution was dialysed against 10 mM Tris, 2.5 mM EDTA, 10 mM NaC1, pH 8.

Amino acid analysis Several hundred micrograms of DNA prepared by the proteinase K/phenol m e t h o d as described above and DNA prepared according to the same m e t h o d but additionally sedimented through alkaline sucrose were dialysed against bidistilled water and mixed with 10 N hydrochloric acid to a final concentration of 6 N. After hydrolysis in a sealed glass tube at l l 0 ° C for 24 h the hydrochloric acid was evaporated and the residue was dissolved in 1 ml of bidistilled water. Some undissolved material was removed by centrifugation and the clear supernatant was absorbed at a DOWEX 50 WX2 column (10 ml) which was washed first with 2 N NH3 and then with bidistilled water until the pH was neutral. The amino acids were eluted from this column with 10 ml of 2 N NH3. The NH3 was evaporated and the residue was dissolved in 0.5 ml of sodium citrate buffer (0.2 N, pH 2.2). The samples were analysed with an amino acid analyser (Beckman, Multichrom) attached with a computing peak integrator (Autolab).

Labelling o f DNA with Na12SI/chloramine T Carrierfree Na12SI was bought from the Radiochemical Center, Amersham (U.K.). Alcohol-precipitated and dried DNA samples were dissolved in a solu-

393 tion which was composed of 3 g of urea, 100 mg of SDS and 3.5 ml of 0.5 M Tris buffer, pH 7.6. 0.1 ml aliquots containing DNA in the order of 100--200 pg were heated to 95°C for 20 min, cooled to 20°C and mixed with 1--2 mCi of Na'2SI. ~2sI was liberated by addition of 50 ~zl of chloramine T (12 mg/ml, Merck Darmstadt, F.R.G.). After 2 min 100 gl of a 10% solution of mercaptoethanol were added. The whole reaction mixture was layered on a Sephadex G25 column (1 ml, disposeable syringe) which had been equilibrated with the solution described above and centrifuged to dryness. The sample was then collected by centrifugation under identical conditions. 0.1 ml portions were mixed with equal volumes of 60 mM Tris, 2% SDS, 10% glycerol, 10% mercaptoethanol, pH 6.8, heated to 95°C for 30 min and submitted to polyacrylamide gel electrophoresis [12].

Enzymatic degradation o f DNA DNA prepared by means of proteinase K or pronase followed by phenolisation was precipitated by 2 vol. of ethanol and collected by centrifugation. 5--10 mg were suspended in 2--3 ml of acetate buffer (75 mM) containing 10 mM MgSO4, pH 5, by pressing the highly viscous solution through a hyperdermic needle. After dialysis in Ultra Thimbles (Schleicher and Schuell, Dassel, F.R.G.) against the same buffer to remove traces of ethanol 40 units (20 ug) of desoxyribonuclease I, grade 1 (Boehringer Mannheim, F.R.G.) were added and dialysis was continued at 20°C. After 1 h some material precipitated which was collected, denatured in SDS-buffer (90°C) and submitted to SDS-polyacrylamide gel electrophoresis [12]. Other methods DNA concentrations were estimated by absorbance at 260 nm assuming that the A of a 1% DNA solution is 200. Labelled proteins and proteins precipitated after degradation of large amounts of DNA in small volumes were analysed on one-dimensional slab-gels [12]. Gels were either stained by coomassie or dried at 90°C and exposed to X-ray films (Kodak Royal X-Omat) for various lengths of time. Molecular weights of proteins were calculated on the basis of standardproteins (Boehringer) run on parallel slots. The filter-binding assay of Coombs and Pearson was used without modifications [13]. Densitometer tracings were recorded by a Vernon densitometer. Results

Labelling o f the proteins most tightly bound to DNA with L-[aSS]methionine Prolonged treatment at high pH {>12) is considered to cause the complete dissociation of protein and RNA from cellular DNA and to separate DNA into its single strands [14,15]. Therefore, cell lysis followed by sedimentation of DNA through alkaline sucrose gradients or followed by banding of DNA in alkaline Cs2SO4 gradients are some of the best methods to isolate pure denatured DNA. Earlier efforts to label peptide material which is not released by these methods failed because the applied double labelling technique ([3H]thymidine for DNA and '4C-labelled amino-acids for peptide-material) resulted in nonsignificant cosedimenting ~4C label [14]. Furthermore, the possible

394

2O •

A

16- ] ~

8!

16q

J

4 8 12 16 20 24 Fractiol~s from top Fig. 1. C o s e d i m e n t a t i o n of (o) [ 3 H ] D N A a n d ( e ) [ 3 5 S ] p r o t e i n o n alkaline 5 - - 2 0 % s u c r o s e gradients. D N A was r e l e a s e d f r o m [ 3 H ] t h y m i d i n e - a n d L - [ 3 S S ] m e t h i o n i n e - l a b e n e d cells b y cell lysis in the p r e s e n c e of 50 /~g/ml of p r o t e i n a s e K. P o r t i o n s o f this l y s a t e w e r e a d d i t i o n a l l y t r e a t e d at 3 7 ° C f o r 24 h w i t h 6 m g / m l o f ( A ) p r o n a s e , (B) p r o t e i n a s e K.

utilization of 14C-labelled amino-acids for the de novo synthesis of pyrimidine and purine bases makes this technique very uncertain. In order to eliminate this artefact we labelled the alkali-stable b o u n d peptide material in DNA with L-[3SS]methionine. By labelling of exponentially growing Ehrlich ascites cells during two cell cycles with [3H]thymidine and L-[3SS]methionine (of high specific activity) we observed a significant 3sS label (150 cpm per A260) cosedimenting together with DNA through alkaline sucrose gradients (Fig. 1) and cobanding with DNA on alkaline Cs2SO4 gradients (Fig. 2). In addition to this DNA-bound 3sS label some 3sS label was found banding independently of DNA on the top of the alkaline Cs2SO4 gradient (Fig. 2A). Additional treatment of these lysates with higher protease concentrations (6 mg/ml) does not remove the material which is bound to DNA (Fig. 2C and D). In contrast, part of the 3SS-labelled material on the top of the gradient disappears under these conditions. Furthermore, this ~SS-labelled material which is n o t bound to DNA is completely removed by phenol (Fig. 2B). The distribution between DNAb o u n d and u n b o u n d 3sS label observed on the gradients is consistent with the results of the filter-binding assay of Coombs and Pearson [13] designed to retain protein which is covalently b o u n d to DNA on glass fibre filters. DNAand protein-molecules which are n o t coupled by a covalent linkage are thought to pass the filter. The lysate which was treated with 50/~g/ml of proteinase K and which contained, as judged from the gradient demonstrated in Fig. 2A, roughly one third of DNA°bound 3sS label and two thirds of DNA-unbound 3sS label, was first extensively sheared by sonification, then brought to 1 M

395 20-

24-

C

20-

16-

16" 1212" 8-

84-

40 24.

o 24L

-

B

D

20-

o20-

A

"6 16-

g~ 12-

12 g

8

."4'=~,-:: ; , 4

8

, , ~ . 12

16

r

,

,

4 2Q 24 Fractions from top

,

i

8

,

i

12

,

i

16

,

,

20

J

24

,

Fig. 2. Cobanding of (o) [ 3 H ] D N A and (o) [ 3 $ S ] p r o t e i n on alkaline Cs2SO 4 gradients, D N A was released from [ 3 H ] t h y m i d i n e - and L-[3SS]methionine-labelled cells by cell lysis in the presence of 50 # g / m l of proteinase K. (A) Original lysate, (B) phenol-treated lysate, (C) lysate after additional t r e a t m e n t with pronase or (D) with proteinase K.

NaC1 or 1 M guanidine hydrochloride respective, passed through the glass fibre filters and washed as described by Coombs and Pearson [13]. In the presence of 1 M NaC1 39.1 _+9.3% (n = 10) and in the presence of 1 M guanidine hydrochloride 35.1 _+ 12.9% (n = 10) of the 3ss label were retained on the filters. This is in good agreement with the portion of the peptide-material which cobands together with D N A and which seems to be tightly if not covalently bound.

Labelling of the proteins most tightly bound to DNA with Na12SI/chloramine T Initial experiments using the standard procedure of Bolton and Hunter [16] for 12SI-labelling of the peptide-material which is most tightly bound to isolated D N A resulted in low yields of ~2SI-labelled material indicating that under these conditions the accessibility of free amino-groups in D N A was very low. Accordingly, in the following experiments the D N A samples were first denatured and then treated with the ~2SI-labelled reagent. In this case more radioactivity was incorporated into the material which was eluted in the exclusionvolume after chromatography of reaction-mixtures on Sephadex columns ( 1 - - 2 . 1 0 7 cpm/2 A200). The analysis of this labelled material from D N A isolated by the various procedures on SDS polyacrylamide gels revealed that a portion of the radioactivity migrated with the buffer front indicating that this

396

68 000

54 000

a

b

c

d

e

f

Fig. 3. T h e a u t o r a d i o g r a p h s of S D S - p o l y a c r y l a m i d e gels s h o w a set of p r o t e i n s (54 0 0 0 - - 6 8 0 0 0 d a l t o n s ) in D N A i s o l a t e d f r o m v a r i o u s s o u r c e s a n d b y v a r i o u s p r o c e d u r e s . P r o t e i n s p r e s e n t in i s o l a t e d D N A w e r e labelled b y 125 I / c h l o r a m i n e T a n d s u b m i t t e d to e l e c t r o p h o r e s i s . ( a ) - - ( c ) P r o t e i n s p r e s e n t in i s o l a t e d D N A f r o m E h r l i e h ascites cells. (a) D N A i s o l a t e d b y p r o n a s e / p h e n o l , (b) D N A i s o l a t e d b y p r o t e i n a s e K / p h e n o l , (c) D N A i s o l a t e d b y alkali. ( d ) - - ( f ) P r o t e i n s p r e s e n t in D N A f r o m o t h e r s o u r c e s (d) r a t liver, (e) sea u r c h i n s p e r m , (f) calf t h y m u s .

radioactivity was associated with low molecular weight material. In addition to this low molecular weight material other radioactive material was observed which migrated between the buffer front and a protein marker (trypsininhibitor, 21 500 daltons) run on a parallel slot (not shown). Larger ~2~I-labelled proteins, 30 000 and 54 000--68 000 daltons in size, were observed when the electrophoresis was carried out for a time which allowed the small peptide material to leave the gel. Thus the gel could be exposed to the X-ray film for a longer time without running into the problem t h a t labelled proteins with higher molecular weight were over-shadowed by the highly labelled peptide-material which migrated together with the buffer-front or just behind. These larger proteins are still better revealed by the Na~2SI/chloramine T procedure. Fig. 3 shows the distribution of the radioactivity on SDS-polyacrylamide gels after electrophoresis of iodinated proteins released from DNA isolated by various methods and from various sources. Fig. 4 demonstrates densitometer tracings of some of these gels. DNA from Ehrlich ascites cells isolated by alkali, proteinase K or pronase as well as DNA from rat liver, sea urchin sperm (both isolated by proteinase K) and calf t h y m u s (commercial product) showed a number of labelled bands in the region of 54 000-68 000 daltons. Despite minor differences it is obvious that DNA from various sources and isolated by various methods contains a very similar pattern of proteins with molecular weights between 54 000 and 68 000 daltons. DNA pre-

o 68000

397

54000 (a)

~(b) c) Distance from top

Fig. 4. Densitometer tracings of the gels d e m o n s t r a t e d in Fig. 3(a)--(c).

pared only by means of proteases/phenol contains in addition to the 54 000-68 000 dalton proteins some other protein-material which is about 30 000 in size. In contrast, DNA released by alkaline cell lysis in the absence of proteases or DNA isolated first by proteases/phenol followed by sedimentation through alkaline sucrose gradients shows no bands in the 30 000 dalton region. As the positions of these 3 0 0 0 0 dalton proteins on SDS-polyacrylamide gels resembles those of the main bands of pronase and proteinase K (not shown) it is unclear whether these 30 000 dalton proteins represent protease-molecules which were firmly attached to DNA during the isolation procedure or whether these 30 000 dalton proteins are cellular proteins which are not or not completely removed from DNA by proteases/phenol. However, in any case these 30 000 dalton proteins are not alkali-stable bound to DNA. DNA isolated by any procedure in which alkali-treatment is included does not contain these 30 000 dalton proteins. That means that these 30 000 dalton proteins do not belong to the class of the most tightly bound proteins which are cobanding and cosedimenting with DNA under alkaline conditions.

Amino acid analysis of acid-hydrolysates of isolated DNA DNA solutions prepared by proteinase K/phenol contain relatively large amounts of peptide-material. The presence of this material is due to the fact that proteins are readily degraded by proteinase K in the presence of SDS, however, peptides large enough to be retained in dialysis bags prove to be

398

TABLE

I

AMINO

ACID

ANALYSIS

OF

DNA

HYDROLYSATES

*

a: D N A i s o l a t e d b y m e a n s o f p r o t e i n a s e K f o l l o w e d by v i g o r o u s shaking w i t h p h e n o l ; b: p r e p a r a t i o n as d e s c r i b e d u n d e r (a) b u t d e n a t u r e d and c e n t r i f u g e d t h r o u g h alkaline sucrose, b ( 1 ) - - b ( 3 ) r e p r e s e n t i d e n t i c a l repeats. R e s u l t s s h o w p e r c e n t o f m a t e r i a l analysed. A m i n o acid

DNA preparation

A260/A280: Asp Thr Ser Glu Pro Ala Cys

a

b(1)

b(2)

b(3)

1.72

1.77

1.89

1.90

0.62

**

**

**

0.19

**

**

**

0.33

**

**

**

0.82 0.19

0.08 **

0.09 **

0.06 **

0.15

0.18

0.23

0.20

0.08

**

**

**

Val

0.15

0.23

0.23

0.25

Met Ile Leu Tyr Phe His Lys Arg Total

0.06

**

**

**

0.13

0.17

0.17

0.13

0.18

0.10

0.11

0.13

0.85

**

**

**

0.21

**

**

**

0.14

**

**

**

0.36

0.33

0.40

0.48

0.07

**

**

**

4.53

1.09

1.23

1.24

* T r y p t o p h a n w h i c h is d e s t r o y e d by acid h y d r o l y s i s and g l y c i n e w h i c h is o v e r - s h a d o w e d by the degradat i o n o f D N A purines have n o t b e e n d e t e r m i n e d . * * A m i n o acid p r e s e n t b u t i n t e g r a t i o n n o t p o s s i b l e b e c a u s e o f l o w c o n c e n t r a t i o n .

rather resistant to further degradation under these conditions [17]. Therefore, most of this material can be easily removed by further purification steps indicating that the bulk of this material is not tightly attached to D N A and represents an unspecific contamination. For example phenolisation under vigorous shaking removes all but about four to five percent of the peptide material. According to the amino acid analysis the purest D N A preparation was obtained after denaturation of D N A and its sedimentation through alkaline sucrose (Table I, b(1)-b(3)). This is in agreement with the results obtained after 12sIlabelling of D N A preparations and SDS-polyacrylamide gel electrophoresis of the released proteins (Fig. 3). According to the gels alkali-treated D N A mainly contains the 54 0 0 0 - - 6 8 000 dalton proteins whereas D N A which is isolated by proteases/phenol contains, in addition, the proteins of the 30 000 dalton region. Although all natural amino acids could be detected in the alkali-treated D N A preparation only six of them could be estimated quantitatively. The amount of the amino acids analysed quantitatively was still 1.1--1.3% of the total material analysed. It is highly probable that the total amount of the amino acids which were not quantitatively detected was in the same order. Therefore, we suggest that roughly 1--2% of the purest D N A isolated by means o f proteinase K and further purified by alkali-denaturation and sedimentation through alkaline sucrose consists of peptide material. It seems that an amount

399 of peptide material in this order cannot be removed from DNA by any method leaving DNA undegraded.

Isolation o f the proteins most tightly bound to DNA Our results indicate that the amount of specific protein which is co-purified with DNA and which is not removed from DNA despite of vigorous attempts is roughly in the order of 1--2% of the DNA. This material can be isolated by degradation of DNA. 5--10 mg amounts of DNA were digested in small volumes of buffer by deoxyribonuclease I. The material which precipitated during the degradation of DNA was collected by centrifugation and further analysed. It proved to be completely insoluble in all buffers which did not contain detergents. However, it was dissolved in buffers containing both, SDS and mercaptoethanol. SDS-polyacrylamide gel electrophoresis of this material revealed a protein-pattern (Fig. 5) which was very similar to the pattern observed after ~2SI-labelling of purified DNA followed by SDS-polyacrylamide gel electrophoresis of the radioactively labelled material (Fig. 3). Distinct protein bands can be observed indicating the presence of proteins which are 54 000--68 000 daltons in size. Furthermore, after degradation of DNA which was isolated from cells which were highly labelled (25 000 cpm/A26o) in their DNA by [3H]thymidine the precipitating material contained a significant amount of the 3H label (0.04% of the total 3H counts before degradation) which could not be removed by washings with buffers. This is a preliminary evidence suggesting that the residual proteins remain bound to nucleotides or oligonu-

~"'--68000 ~-"'-54000

Fig. 5. S D S - p o l y a c r y l a m i d e gel electrophoresis of proteins w h i c h precipitate during digestion o f large a m o u n t s o f D N A from Ehrlich ascites cells b y d e o x y r i h o n u c l e a s e I. D N A w a s isolated b y m e a n s o f proteinase K/phenol.

400 cleotides which are produced during the degradation of DNA by deoxyribonuclease I. Discussion

A number of authors have collected evidences indicating that a small but persistent amount of peptide material is co-purified with native eukaryotic D N A [18--23]. As this peptide material could n o t be removed from DNA despite of vigorous attempts it was suggested that this material plays a role in DNA-structure and/or function [23]. In connection with our work on proteaseinduced alkali lability of DNA [24] we characterized the trace amounts of peptide material in highly purified DNA preparations by sensitive techniques. Since alkali-denatured DNA behaves as a long molecule alkali-stability is a prerequisite for possible peptide-linkers joining adjacent single-stranded DNA subunits [24]. We found that some peptide material which is not removed from DNA by proteases and phenol is indeed alkali-stable b o u n d to DNA. This tightly bound non-deoxynucleotide material consists of a set of proteins which are between 54 000 and 68 000 daltons in size. From this it must be concluded that the DNA-protein complex is specific and quite unusually strong or the protein must even be covalently attached to DNA. In addition to these most tightly b o u n d proteins we observed other proteins which have strong affinities to DNA, however, these proteins are not alkali-stable bound to DNA and it is n o t clear whether these proteins are cellular proteins or protease molecules which bind to DNA during the isolation procedure. At present we have no direct experimental evidence for the biological significance of the alkali-stable DNA-protein linkage. However, according to their properties (size and stability) proteins involved in this linkage could play a role in maintaining the stability of superhelical loops and three-dimensional structure of chromatin. Furthermore, this alkali-stable linkage could also be involved in non-deoxynucleotide linkers between adjacent DNA single strands [24]. The proteins involved in such a linkage have characteristics which are prerequisites for protein-linkers suggested earlier, e.g. they could join DNA strands by alkali-stable linkages. Finally, beside of their possible function in DNA structure and/or chromatin structure the proteins could also be involved in nucleic acid synthesis. In procaryotic systems other authors observed proteins which are covalently b o u n d to DNA in the vicinity of the origin of replication [13,25,26]. Accordingly these proteins were considered to be involved in nucleic acid synthesis. It is possible that the proteins demonstrated in this paper play a similar role in eukaryotic DNA synthesis. References 1 2 3 4 5 6 7 8

N a k a n e , M., Ide, T., Anzai, K., O h a r a , S. a n d A n d o h , T. ( 1 9 7 8 ) J. B i o c h e m . 84, 1 4 5 - - 1 5 7 C o o k , P.R. a n d Brazell, J.A. ( 1 9 7 5 ) J. Cell Sci. 19, 2 6 1 - - 2 7 9 I d e , T., N a k a n e , M., Anzai, K. a n d A n d o h , T. ( 1 9 7 5 ) N a t u r e 258, 4 4 5 - - 4 4 7 B e n y a j a t i , C. a n d Worcel, A. ( 1 9 7 6 ) Cell 9, 2 9 3 - - 4 0 8 Gross-Bellard, M., O u d e t , P. a n d C h a m b o n , P. ( 1 9 7 3 ) Eur. J. B i o c h e m . 36, 3 2 - - 3 8 R i l e y , D.E., Keller, J.M. a n d Byers, B. ( 1 9 7 5 ) B i o c h e m i s t r y 14, 3 0 0 5 - - 3 0 1 3 Keller, J.M. a n d Riley, D.E. ( 1 9 7 6 ) Science 193, 3 9 9 - - 4 0 0 R i l e y , D.E. a n d Keller, J.M. ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 444, 8 9 9 - - 9 1 1

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9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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