Internucleotide protein linkers in Ehrlich ascites cell DNA

243

Biochimica et Biophysica Acta, 608 (1980) 243--258 © Elsevier/North-Holland Biomedical Press

BBA 99696

I N T E R N U C L E O T I D E P R O T E I N L I N K E R S IN E H R L I C H ASCITES CELL DNA

DIETER WERNER, WANDA KRAUTH and HOWARD V. HERSHEY

Institute for Cell Research, German Cancer Research Center, Heidelberg (F.R.G.) (Received January 2nd, 1980)

Key words: DNA nick; Peptide/protein linker; DNA structure; Nuclease sensitivity; Protease sensitivity; (Ehrlich ascites tumor cell)

Summary DNA from Ehrlich ascites t u m o r cells is nicked or gapped by a reaction which is induced b y proteases such as autodigested pronase, proteinase K, trypsin, chymotrypsin and subtilisin. The cleavage of the protease-sensitive sites is inhibited by protease inhibitors. The nicks or gaps induced by proteases can be demonstrated by nuclease S1 sensitivity of native DNA and by a change of the sedimentation rate of alkali-denatured DNA. The limit size of denatured DNA released after optimal protease treatment is 8.5" 104 daltons (27 kilo bases). The molecular weight of the native DNA pieces released after nuclease S1 degradation of DNA containing the protease-induced nicks or gaps is in the same order indicating that the protease-sensitive sites are alternatively arranged on the opposite DNA strands at an average distance of 13.5 kilo base pairs. Since the protease-induced nicks or gaps in phosphatase-treated DNA are n o t attacked by Escherichia coli polymerase I, one or both ends liberated by the protease treatment must be blocked by a material other than phosphate groups. The results are most compatible with peptide/protein linkers joining adjacent single-strand DNA subunits. Alternative explanations such as alkali-stable RNA linkers, protein-protected R N A linkers, site-specific nuclease contaminations in the protease preparations or cellular nucleases activated by the protease treatm e n t are eliminated b y the results presented in this paper.

Introduction

Although it is n o t possible to isolate 'chromosome-sized' DNA molecules from eukaryotic cells [1] by any presently available method, there is evidence SDS, sodium dodecyl sulfate; TLCK, L-l-chloro-3-p-tosylamido-7-amino-2-heptanon: PMSF, phenylrnethylsulfonylfluoride. Abbreviations:

244 indicating that the DNA of one chromosome consists of only one continuous DNA duplex molecule [1--3]. However, this does not predict that each of the antiparallel DNA strands must be continuous throughout the full length of the duplex molecule. The antiparallel strands could be subdivided into much smaller units by permanent or transient discontinuities such as nicks, gaps or linkers of material other than DNA. Native DNA containing such discontinuities could still behave as a long and continuous molecule as long as these discontinuities in the antiparallel strands are not arranged opposite or close to each other [1,4]. According to our previous work [5] denatured DNA which can be isolated from Ehrlich ascites cells by alkaline cell lysis and sucrose gradient centrifugation is in the order of 100 • 106 daltons or heavier indicating that discontinuities, if present, are either distanced 1 0 0 . 1 0 6 daltons or they must be alkali stable for hours. In contrast, alkali-denatured DNA from Ehrlich ascites t u m o r cells is much shorter than 1 0 0 . 1 0 6 daltons after pretreatment with high concentrations of autodigested pronase. However, in contrast to cellular DNA similarly treated phage DNA is not changed under these conditions indicating that the change in size of cellular DNA induced by pronase is n o t due to a soluble and nonspecific nuclease acting during the proteasetreatment. Other nucleases such as site-specific restriction nucleases which might cleave cellular DNA b u t n o t viral DNA or a chromatin-bound nuclease possibly activated by proteases were n o t eliminated explicitely by our previous work. Thus, the protease-inducible alkali lability of DNA could be explained by site-specific nucleases which can work under the conditions of cell lysis (SDS, EDTA). Alternatively this p h e n o m e n o n could be explained by alkali-stable special structural sites of non-deoxynucleotide nature which are changed by the protease during the lysis procedure in such a way that they become alkali labile. Possible candidates for such protease-sensitive sites include: (a) modified and alkali-resistant ribonucleotides susceptible to a ribonuclease activity present as a contaminating activity in pronase preparations or activated in the cells by pronase; ( b ) a n R N A linker between DNA strands protected from alkaline degradation by a protein which becomes susceptible to alkali after pronase treatment, and finally (c) a protein or peptide linker in DNA. The present paper presents evidence indicating that the protease-induced alkali lability of DNA is due to the presence of protein or peptide linkers in DNA. We are able to demonstrate directly that DNA is nicked or gapped by a protease-catalyzed reaction during treatment with high concentrations of proteases. Thus, the present paper supplies stronger evidence for peptide/protein linkers in DNA than was previously available. Materials and Methods Cells and cell lysates Ehrlich ascites t u m o r cells growing in vivo were labelled at day seven after inoculation by intraperitoneal injection of 100 ~Ci of [3H]thymidine (20 Ci/ mmol) or 40 pCi of [14C]thymidine (61 Ci/mol). 24 h later the cells were harvested and collected b y centrifugation. Ehrlich ascites t u m o r cells growing in vitro [6] were labelled during 24 h in the presence of 0.5 ~Ci of [3H]thymidine/ml. Suspensions of radioactively labelled cells in 50 mM Tris, 20 mM

245 EDTA, 100 mM NaC1, pH 8, were used for the lysis procedures. All lysates were prepared in polyallomer tubes of the SW27 rotor. Cell lysis without proteases. 2 • 106 cells were lysed in a final volume of 1 ml containing 0.25% SDS, 20 mM EDTA, 150 mM NaHCO3, pH 8 (lysis buffer). For alkaline cell lysis 2 • 106 cells were lysed in 1 ml of the lysis buffer and 0.5 ml of 1 N NaOH. Cell lysis in the presence o f proteases. 2 • 106 cells were lysed in lysis buffer which was mixed with varying amounts of autodigested pronase (Serva, Heidelberg, F.R.G.). Autodigestion of pronase was accomplished by dissolving 30 mg of the enzyme in 1 ml of 50 mM Tris, 20 mM EDTA, 100 mM NaC1, pH 8, and heating the enzyme for 10 min at 80°C followed by incubation for 1 h at 3 7°C. In some experiments pronase was replaced by other proteases which were n o t autodigested prior to use: Proteinase K, trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1; Merck, Darmstadt, F.R.G.) subtilisin (EC 3.4.21.14), thermolysin (EC 3.4.24.4; Boehringer, Mannheim, F.R.G.). L-l-chloro-3-ptosylamido-7-amino-2-heptanon (TLCK)-inactivated trypsin was prepared according to Ref. 7. In some experiments indicated in the text the cell lysates contained the protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and p-aminobenzamidine. These inhibitors as well as TLCK were products of Serva, Heidelberg, F.R.G. Cell lysis in the presence o f pronase and excess unlabelled DNA. 6 mg amounts of autodigested pronase were mixed in 1 ml of lysis buffer with pronase-pretreated and extensively sheared unlabelled DNA (300 /~g, 600 ~g and 1200 ~g) before 2 • 104 cells labelled in vitro and representing 0.5 ~zg of 3H-labelled DNA were added. Unless otherwise stated in the text cell lysis was completed at 20°C within 24 h followed by denaturation of DNA by addition of 0.5 ml of 1 N NaOH.

Isolation o f high molecular weight D N A DNA was isolated from Ehrlich ascites t u m o r cells according to the m e t h o d of Gross-Bellard et al. [8]. This m e t h o d includes t w o digestion steps with low levels of proteinase K (50 /~g/ml), phenolisation and treatment with pancreatic and T1 ribonucleases (Boehringer, Mannheim, F.R.G.). For the preparation of pronase-treated DNA the proteinase K was replaced by 6 mg/ml pronase: Labelled DNA was isolated from Ehrlich ascites cells which were treated in vivo with [3H]thymidine as described above. Treatment o f isolated native and denatured D N A with pronase Native DNA. 0.5 A260 unit of DNA isolated in the presence of low levels of proteinase K (equivalent to 106 cells) were dialysed against lysis buffer and carefully transferred to a polyallomer SW27 tube. After incubation with 6 mg/ml of autodigested pronase for 16 h at 37°C the DNA was denatured by addition of 1 N NaOH to a final concentration of 0.33 N. Similarly treated DNA which was incubated under identical conditions but w i t h o u t pronase served as control. Denatured DNA. 0.5 A260 unit of DNA isolated in the presence of low levels o f proteinase K were denatured by addition of 1 N NaOH to a final concentration of 0.3 for 15 min at r o o m temperature, redialysed against lysis buffer and

246

carefully transferred to a polyallomer SW27 tube. 6 mg/ml of autodigested pronase was added, and after an incubation period of 24 h at room temperature 1 N NaOH was added to a final concentration of 0.33 N. Similarly treated DNA which was incubated under identical conditions without pronase served as control.

Nick translation DNA treated with high concentrations of pronase (6 mg/ml) and DNA additionally treated with alkaline phosphatase [9] (Boehringer, Mannheim, F.R.G.) was incubated in 1 ml of 50 mM Tris, pH 8.3, 50 mM KC1, 5 mM MgSO4 and 5 mM mercaptoethanol with 1.38 U Escherichla coli polymerase I, 5 pCi (44 Ci/mmol) [3H]dTTP and 0.01 mM of each of the other three deoxynucleotide triphosphates. Aliquots were taken at the times indicated and analysed for trichloroacetic acid-insoluble material. After 30 min 1 ~g of deoxyribonuclease I (grade I, Boehringer, Mannheim, F.R.G.) was added and the analysis was continued.

Gradient centrifugation To avoid mechanical shear all gradients were formed beneath the lysates and incubates. Alkaline 5--20% sucrose gradients contained 0.7 M NaC1, 0.3 N NaOH, 1 mM EDTA. Neutral 5--20% sucrose gradients contained 1 M NaCl, 50 mM Tris, 1 mM EDTA, pH 8. If not otherwise stated in the text centrifugation was at 5°C for 13 h at 20 000 rev./min. Fractions were taken from the top (Autodensiflow IIC, Buchler, Fort Lee, U.S.A.). DNA was precipitated by 10% trichloroacetic acid and filtered through MiUipore BSWP 02500 filters, washed with trichloroacetic acid, C2HsOH, and counted.

Agarose gel electrophoresis of high molecular weight DNA after nuclease $1 digestion 10A:60 units of high molecular weight native [3H]DNA isolated in the presence of 50 ~g/ml proteinase K or 10 A2~0 units of high molecular weight native [3H]DNA isolated in the presence of 6 mg/ml pronase were dialyzed against 3 mM sodium acetate, 100 mM NaC1, pH 4.5. The dialyzed DNA solution was adjusted to 3 ml. After addition of 0.03 ~mol of ZnC12 and 5000 units of nuclease S l (Miles-Biochemicals) the mixtures were incubated at 37°C. At the times indicated 0.2 ml samples were taken from the incubates with a wide mouth pipette and carefully mixed with 20 ~1 of 200 mM EDTA, pH 8, and 50 pl of glycerol and submitted to 0.6% agarose electrophoresis (Biomedical Division Maine Colloids Inc., Rockland, U.S.A.). Electrophoresis buffer was 4 mM Tris, 5 mM sodium acetate and 1 mM EDTA, pH 7.5. After electrophoresis at 20 mA for 16 h, the gels were dried, sliced and counted.

Calculations of molecular weights The SW27 rotor was calibrated for runs at 20 000 rev./min, 13 h with 3Hlabelled SV40 marker (from G. Sauer, Institute of Virology, German Cancer Research Center, Heidelberg, F.R.G.) or ~4C-labelled T4 marker (from H.W. Thielman, Institute of Biochemistry, German Cancer Research Center, Heidelberg, F.R.G.). Agarose gels were calibrated by EcoRI digestion products of

247 phage (from H.F. Lauppe, Institute of Biochemistry, German Cancer Research Center). Results

Limit size o f denatured DNA after cell lysis in the presence o f autodigested pronase Most of the DNA released by alkaline cell lysis or by neutral cell lysis in the presence of detergents followed by alkali denaturation is 100" 106 daltons or larger indicating that single-stranded DNA of this size is alkali stable for hours. Pronase-treated native DNA is at least 2 0 0 . 1 0 6 daltons in size or larger (Fig. 1). However, when the pronase-treated DNA is alkali denatured the long double-stranded DNA molecules fall apart into single strands which are much shorter than the original native DNA molecules, indicating that pronase-treated DNA contains alkali-labile sites. This change in size depends on the pronase

33S 16-

A

12-

8-

:>, ~'~

4o °

5

,R~,

oru

:s

351

0

30-

_

12

__

16

20

24

16

17 S

20-

8

A

1©-

® 5g

8

~ ~2

25N

. . . . . . . . . . . 4

-'

i

4

,

r

0_ 53S

,~1

8

Fractions

,

12 from

r

16

/t 4.

,

~,

20 To~)

i

24

,

~ ~ j

~jr

4

j

8

,

i

12

Fractions

,

i

,

J

16

20

from

Top

,

i

,

24

Fig. 1. S e d i m e n t a t i o n a n a l y s i s o f u n i f o r m l y l a b e l l e d D N A a f t e r cell lysis in a l k a l i n e lysis b u f f e r (B o ) ; cell lysis i n n e u t r a l lysis b u f f e r f o l l o w e d b y alkali d e n a t t t r a t i o n (o o); SV40 markez (e e ) ; a l k a l i n e 5 - - 2 0 % s u c r o s e g r a d i e n t s , 5 ° C ; 2 0 0 0 0 r e v . / m i n , 13 h . N a t i v e D N A i s o l a t e d in t h e p r e s e n c e o f 6 m g / m l p r o n a s e (A A); n e u t r a l 5 - - 2 0 % s u c r o s e g r a d i e n t . 5 ° C , 2 0 0 0 0 r e v . / m i n , 1 3 h , Fig, 2. S e d i m e n t a t i o n a n a l y s i s o f a l k a l i - d e n a t u r e d D N A a f t e r p r o n a s e t r e a t m e n t o f cell l y s a t e s : ( A ) Cell l y s a t e s w e r e p r e p a r e d i n t h e p r e s e n c e o f 1 m g / m l (o o), 3 m g / m l (B o) a n d 6 m g / m I o f a u t o digested pronase (l o). ( B ) C e l l l y s a t e s w e r e p r e p a r e d f r o m [ 1 4 C ] t h y m i d i n e - l a b e H e d cells a n d t z e a t e d w i t h 6 m g / r n l proxtase (B D). S e p a r a t e l y cell l y s a t e s w e r e p r e p s , r e d f r o m [ 3 H ] t h y m i d i n e l a b e l l e d cells a n d t r e a t e d w i t h 1 2 m g / m l p r o n a s e (o -'). F o l l o w i n g t h e p r o n a s e t r e a t m e n t t h e cell lysates were carefully mixed, alkali denatured and analysed on alkaline 5--20% sucrose gradients, 5°C, 20000 rev./min, 13 h.

248 concentration used during the lysis procedure (Fig. 2A). Higher pronase concentrations result in both shorter chains and reduced polydispersity. However, a limit size exists which is reached at pronase concentrations of a b o u t 6 mg/ml. At higher pronase concentrations there is no further reduction in average size. DNA treated with 6 mg/ml or 12 mg/ml of autodigested pronase and analyzed on the same gradient sediments to a b o u t the same position (Fig. 2B). If DNA were being degraded continuously by a site-unspecific nuclease activity present in the pronase the DNA digested with 12 mg/ml of pronase should be smaller than that digested by 6 mg/ml pronase. These results indicate that the change in size of single-stranded DNA induced by pronase is limited and results in DNA strands with an average size of a b o u t 8.5 • 106 daltons. Fig. 3 shows that even a 2400-fold dilution of the [3H]DNA with unlabelled but pronasepretreated DNA shows no effect on the size change of [3H]DNA strands induced by pronase. This indicates that the activity which changes the size of denatured DNA during the treatment with pronase is not competitively inhibited by large amounts of pretreated DNA. Therefore, the average phosphodiester bond of DNA is not the substrate which is cleaved during the protease treatment of DNA. The somewhat broader size distributions at high concentrations of unlabelled DNA are probably caused by boundary anomalities due to the high DNA concentrations [10].

Size o f denatured DNA after cell lysis in the presence o f other proteases After incubation of cell lysates in the presence of other proteases which are active in the presence of SDS and EDTA, such as proteinase K, subtilisin, trypsin and chymotrypsin the denatured DNA released by alkaline sucrose gradient centrifugation is also smaller than the denatured DNA which is released by cell lysis in the absence of proteases. However, while 6 mg/ml of pronase is sufficient to release DNA of the limit size more drastic conditions (37°C, higher concentrations than 6 mg/ml) are needed to release denatured DNA close the limit size by the other proteases mentioned above (Fig. 4). In contrast to these enzymes, which are active in the presence of EDTA, thermo16> u ru o

14-

0~

10-

12-

8 6 *E

42-

2 4

8

Fractions

12 from

16

20

24

Top

Fig. 3. Sedimentation ~nalysis o f D N A from [3H]thymidine-labelled cells r e p r e s e n t i n g 0.5/~g o f l a b e l l e d D N A a f t e r lysls in t h e p r e s e n c e o f 6 mg/ml o f p r o n u e (e e); 6 m g / m l o f p r o n a s e and 300 ~g (o o), 6 0 0 ~g (~ D), 1200/~g (• ~) of unlabelled but pronase-predigested D N A ; Alkaline 5--20% sucrose gradients, 5 ° C, 20 0 0 0 rev./min, 13 h.

249 lysin needs Ca 2+ for full activity. D N A digested by this enzyme exhibits less size change than D N A digested by proteases which do not need activation by divalent ions. Thus, there is a correlation between proteolytic activity and the change in size of denatured DNA. The p h e n o m e n o n of protease-induced alkali lability of D N A is a general p h e n o m e n o n which is not restricted to pronase or to contaminating enzymatic activities only present in pronase. However, under identical conditions pronase is somewhat more effective in making D N A alkali labile than the other proteases under investigation. This may be due to different sensitivities of the various enzymes against denaturation by SDS.

Size of denatured DNA after cell lysis in the presence of two proteases The number of alkali-labile sites which are introduced into D N A by the treatment with two 'proteases (pronase and proteinase K) are non-additive. This indicates that identical and specific sites are made alkali labile by different proteases. If the two enzymes would introduce alkali-labile sites randomly and independently from each other one would expect shorter D N A strands after cell lysis in the presence of both enzymes than after cell lysis in the presence of

A

12-

12 ¸

C

16-

D

4

o r~

B 12-

12-

u £_ n

8

8-

J 4

4-

8

12

16

20

Fractions

4

24 from

8

12

16

20

24

Top

Fig. 4 . S e d i m e n t a t i o n analysis o f d e n a t u r e d D N A r e l e a s e d a f t e r cell lysis in t h e p r e s e n c e o f various p r o t e ases. Cell l y s a t e s c o n t a i n e d 1 0 m g / m l o f p r o t e a s e s . A f t e r i n c u b a t i o n at 3 7 ° C for 1 3 h D N A w a s alkali d e n a t u r e d and s u b m i t t e d t o alkaline 5 - - 2 0 % s u c r o s e gradients, 5 ° C, 2 0 0 0 0 r e v . / m i n , 1 3 h. ( A ) • •, pronase; • "-, p r o t e i n a s e K. ( B ) • a~ p r o n a s e plus p r o t e i n a s e K; • -', Pronase a l o n e . (C) • ~, t r y p s i n ; • - "-, c h y m o t r y p s i n . ( D ) • ~', subtillsin; • , •, thermolysin.

250 only one enzyme. Fig. 4B shows that the DNA strands released after digestion with pronase alone are of the same size as the DNA strands isolated after simultaneous treatment with pronase and proteinase K.

Inhibition of the protease-inducible alkali lability of DNA by specific protease inhibitors The protease inhibitor phenylmethylsulfonyl fluoride (PMSF) inhibits reactions catalyzed by serine and thiol proteases [11,12]. Under the conditions of cell lysis, inhibition of the proteolytic activity (release of acid-soluble radioactivity from 14C-labelled protein) of pronase by 1.5 mM PMSF is approx. 30% during the first 5--6 h (not shown). At longer incubation periods, inhibition decreases, which is probably due to the instability of the inhibitor [11]. The inhibition of the pronase-inducible alkali lability of DNA by the protease 16-

A

/-25

°/o

A

12-

8-

+d o O2

> .u u - 6 0 O/o 12~

~d

o2 +~

o

© 0_

-~-r 4

,

12

16

20

24

,

i

4

,

L ,

8

Fractions Fractions

from

l

12

,

i

16

,

i

20

,

l

24

,

f r o m Top

Top

Fig. 5. I n h i b i t i o n o f t h e p r o n a s e - i n d u c i b l e alkali lability o f d e n a t u r e d D N A b y t h e p r o t e a s e i n h i b i t o r P M S F . ( A ) Cell lysis b y m e a n s o f 1 0 m g / m l o f p r o n a s e f o r 5 h in t h e p r e s e n c e o f 1 . 5 m M o f P M S F (A A) a n d w i t h o u t P M S F ( e -'). ( B ) Cell lysis b y m e a n s o f I 0 m g / m l o f p r o n a s e for 1 6 h in t h e p r e s e n c e o f 1 . 5 m M o f P M S F (-" -') and w i t h o u t P M S F (A A). A l k a l i n e 5---20% s u c r o s e gradients, 5 ° C , 2 0 0 0 0 r e v . l m i n , 1 3 h. Fig. 6 . I n h i b i t i o n o f t h e t r y p s i n - i n d u c i b l e alkali lability o f d e n a t u r e d D N A b y p - a m i n o b e n z a m i d i n e and t h e t r y p s i n i n a c t i v a t i o n T L C K . ( A ) Cell lysls b y m e a n s o f for 5 h . ( B ) Cell lysls b y m e a n s o f 1 0 m g / m l o f T L C K - i n a c t l v a t e d t r y p s i n ( e 1 0 m g / m l o f t r y p s i n in t h e p r e s e n c e o f 1 . 5 m M o f p - a m i n o b e n z a m i d i n e f o r 5 h 5 - - 2 0 % s u c r o s e gradients, 5 ° C , 2 0 0 0 0 r e v . / m i n , 1 3 h.

t h e trypsin-inhibitOr I0 mgfml of trypsin ~) a n d b y m e a n s o f (" A). Alkaline

251 inhibitor PMSF parallels the inhibition of the proteolytic activity of pronase (Fig. 5A and B). Although 5 h of digestion in the presence of pronase is not sufficient to result in limit size {8.5.106 daltons) DNA, the size change induced by pronase under these conditions is obvious. However, after cell lysis for 5 h in the presence of pronase and 1.5 mM PMSF the denatured DNA is significantly larger. This indicates that the change in size of denatured DNA is inhibited by PMSF (Fig. 5A). After longer cell lysis periods the difference in size between the DNA released from PMSF-inhibited lysates and controls without PMSF is smaller, but still observable (Fig. 5B). This indicates that the change in the size of denatured DNA observed after pronase treatment is due to a reaction catalyzed by the protease. The trypsin-induced alkali lability of DNA is similarly inhibited by PMSF (not shown) and additionally by other inhibitors which are more specific for the tryptic activity such as p-aminobenzamidine [13] and L-l-chloro-3-ptosylamido-7-amino-2-heptanon [14,15]. At 1.5 mM p-aminobenzamidine the trypsin-induced change in size of denatured DNA is almost completely inhibited (Fig. 6). More than 60% of the denatured DNA released from p-aminobenzamidine-inhibited lysates sediments close to the bottom of the centrifuge tube whereas most of the denatured DNA released from cell lysates which were treated with trypsin in the absence of p-aminobenzamidine is released as much shorter strands. Although inhibition of trypsin by TLCK is less pronounced when compared with the effect of p-aminobenzamidine, TLCK is of special interest because this compound is highly specific for the inactivation of the tryptic activity. Even closely related enzymes such as chymotrypsin are not inhibited by this compound [16,17]. These experiments indicate that it is the protease activity which is responsible for the protease-induced change in the size of denatured DNA. Naturally occurring protease inhibitors such as the inhibitor from soy beans are not suitable to inhibit the proteolytic activity of proteases under the conditions of cell lysis because the protease inhibitor complex is highly sensitive to denaturing conditions [18]. Treatment o f isolated native and denatured DNA with high protease concentrations An effective method used to isolate high molecular weight native DNA is based on neutral cell lysis in the presence of SDS and proteinase K followed by digestions with both pancreatic and T1 ribonucleases [8]. As described above, the concentrations of proteinase K which are used to release DNA by this procedure (50 #g/ml) are too small to make considerable numbers of the protease-sensitive alkali-labile sites in DNA. Therefore, DNA prepared by this method contains relatively long alkali-stable strands. The number average molecular weight of Ehrlich ascites cell DNA isolated by this procedure is 450 • 106 for native DNA and 50 • 106 for denatured DNA indicating that each of the antiparallel strands of an isolated DNA molecule contains not more than 3--4 additional alkali-sensitive sites (Fig. 7). Treatment of this DNA with pronase followed by alkali denaturation results in shorter single strands compared to similarly incubated but not pronase-treated DNA (Fig. 8A). This indicates that

252

in D N A isolated in the presence of low concentrations of proteinase K only a fraction of the protease-sensitive sites are made alkali-labile during the isolation procedure. Additional treatment with higher protease concentrations makes more sites alkali-labile. The broader size distribution observed after the additional pronase treatment of isolated D N A compared to direct digestion by pronase during cell lysis is probably caused by the difficulty of mixing long D N A strands with the enzyme in such a way that shear forces are avoided as far as possible. The size of isolated alkali-denatured DNA, subsequently neutralized by dialysis and treated with high levels of pronase is also changed to shorter size whereas similarly treated D N A not pronase digested sediments much faster (Fig. 8B). This indicates that the pronase-sensitive sites do not depend on diges4 6 °/o

12

u

/

4

/

I,A-I

\

/ .J" \-, i l

+-,~2 4 _0 P,"

i

0_

16-

"6

i

,

i

,

r

,

i

,

?

20-

~,

,

12

T4

-

,

-

/

f\,

\

8-

~D 4-

4

8

12 Fractions

16

20 from

24 Top

'

4

'

~ '1'~' 1'6' 2'o' J4' Fractions

from

T op

Fig. 7. S e d i m e n t a t i o n analysis o f high m o l e c u l a r weight D N A isolated in the presence of 50 ~ g / m l o f Proteinase K. A l k a l i n e 5--20% s u c r o s e gradient (e --), neutral 5--20% sucrose gradient (e --); p o s i t i o n o f T 4 ,marker in b o t h , neutral a n d alkaline gradients ( ). Centrlfugation was at 5 ° C, 1 5 0 0 0 r e v . / m i n , 13 h. Fig. 8. S e d i m e n t a t i o n analysis o f i s o l a t e d D N A treated w i t h pronase. ( A ) D N A isolated in the presence o f 50 ~ g / m l o f p r o t e i n a s e K and a d d i t i o n a l l y i n c u b a t e d w i t h 6 rag/m1 o f pronase ( i m). Similarly treated D N A b u t n o t pronase i n c u b a t e d (e "-). (B) D N A isolated in the presence o f 50 , g / m l o f proteinase K, alkali d e n a t u r e d , dialysed against lysis b u f f e r and a d d i t i o n a l l y i n c u b a t e d w i t h 6 m g / m l o f pronase (m i ) . Similarly treated D N A b u t n o t pronase i n c u b a t e d (e ~). Alkaline 5--20% sucrose gradients, 5 ° C, 20 0 0 0 r e v . / m i n , 13 h.

253 tion in crude lysates, b u t can be seen following digestions of isolated native or denatured DNA. Moreover, the latter indicates that double-stranded DNA is n o t necessary for recognition of sites by pronase. Furthermore, these experiments show that the material which is made alkali labile during the protease treatment is unlikely to consist of RNA. This R N A had to be resistant to alkali and to the ribonucleases used during the isolation procedure b u t sensitive to a ribonuclease contamination present in the protease preparations.

Nuclease $1 digestion o f pronase-treated DNA The specific degradation of single-stranded DNA by nuclease S l [19] was used to demonstrate .'nicks' or 'gaps' and their relative positions in pronasetreated DNA. Pronase-treated DNA is rapidly cleaved by nuclease $1 resulting in native DNA pieces which migrate in 0.6% agarose gels (Fig. 9C). This indicates that the protease-treated native DNA is a 'nicked' or 'gapped' duplex molecule. The number average molecular weight of the double-stranded DNA pieces formed during digestion of pronase-treated DNA by nuclease S1 is a b o u t 9.0 • 106 which is roughly half the length of the single-stranded DNA released after optimal pronase treatment of DNA followed by alkali denaturation. This indicates that the 'nicks' or 'gaps' which are introduced by proteases are arranged alternatively on the opposite strands. In contrast, DNA isolated in the presence of low concentrations of protease and thereby containing fewer alkali labile sites results in DNA pieces which, after comparable incubation periods in the presence of nuclease $1, are t o o large to migrate in 0.6% agarose gels (Fig. 9A). DNA duplex molecules which are nicked or gapped by the treatment with high concentrations of pronase cannot serve as substrate for nick translation by E. coli polymerase I (Fig. 10). These experiments were designed in such a way that the number of polymerase molecules was in the same order as the number o f the protease-induced nicks. Under the assumption that the prGtease-induced nicks are distanced 8--9 • 10 6 daltons (Fig. 9) the number of nicks/A260 unit (1 ml) is in the order of 3.5 • 101:. The aliquots analysed (50 pl) represented DNA containing 1.75" 1011 nicks. If only one [3H]dTTP molecule would have been added per nick in the polymerase reaction one had to expect an incorporation of a b o u t 0.3 pmol of [3H]dTTP per aliquot. Since the specific activity of the [3H]dTTP was 44 Ci/mmol this is equivalent to 2.5- 104 dpm or at least to 1--1.5 • 104 cpm. This shows that the a m o u n t of label which could be introduced per protease,induced nick is sufficient to be detected. However, the rate of incorporation of [3H]dTTP was lower than 5% of this expected value indicating that the protease-induced nicks could n o t serve as substrates for nick translation. In contrast, after addition of a trace a m o u n t of deoxyribonuclease I, inducing 3'-OH/5'-P ends, nick translation started immediately. This indicates that the 3'-ends which are liberated by pronase are blocked, either by phosphate or, alternatively, by incompletely removed linker material. DNA containing protease-induced nicks which was additionally treated with alkaline phosphatase under conditions designed to remove internal phosphate groups [9] still could n o t serve as substrate for nick translation. The rate of incorporation of [3H]dTTP was lower than 20% of the expected incorporation for nick

254

0 X E

o_

60-

o_

40-

/

240-

T

20'4~_\ r~,~,.~.==.~.=.=...=.=.=., l

~

4

40-

cY

30-

~

20-

~ lO-

,

~_

,

8

i

12

,

~ ~ ~ i

16

,

q

20

=.=.=

,

( Slices)

~

12@

,

24

-

c

~/~

B

%

t,

t

.p-/ ,

\ ,

4

,

8

~

,

-

12

~

,o.~

16

20

(Slices)

24

E D_ u ck F--

16~ i m

12-

~'~ 8-

4.73 3.6

!

I

2.13x 106 D~ltons

!

?

4-

. . . . . . -, 4 8 12 16 Distance from Top

20

24

I

,

,

/

x 100-

, ~

@

i

6c

• .j!-/

k) C

1

20 30 40 50 incubation (rain)

0

Fig. 9. D i s t r i b u t i o n o f radioactivity after agarose gel e l e c t r o p h o r e s i s o f D N A i n c u b a t e d w i t h nuclease S I . ( A ) D N A isolated in t h e presence o f 50 # g / m l o f p r o t e i n a s e K and i n c u b a t e d with nuclease $1 for zero rain (e "-), o r 20 vain (u u). (B) D N A isolated in the presence o f 6 m g / m l o f pronase and i n c u b a t e d for z e r o vain (e -') w i t h nuclease $1. (C) D N A isolated in the presence o f 6 m g / m l o f pronase and i n c u b a t e d w i t h nuclease $1 for 10 vain (a e ) , or 20 m i n (u ~). A r r o w s indicate p o s i t i o n s o f ~ p h a g e / E c o R I digestion p r o d u c t s . Fig. i0. Pzotease-induced 'nicks'or 'gaps'in D N A are not attacked by E. coli polymerase I. After addition of trace amounts of deoxyribonucleue I (a~ows) nick txanslationstartsafter about 5 vain. CA) D N A (1 A 2 6 0 unit) isolated by means of 6 vag/ml of pronase. ( B ) D N A 41,5 A 2 6 0 units)isolatedb y means of 6 mg/val of pronase but additionally treated with alkaline phosphatase at 65°C (three times 2 units) and deproteinized by C H C I 3/pentan-2-ol.

translation. The somewhat higher background incorporation before addition of deoxyribonuclease I in this case is most probably due to some breaks introduced in the DNA during heating with alkaline phosphatase. Thus, it must be assumed that one or both ends which are liberated by pronase are blocked by a material other than phosphate. Discussion Double-stranded subunits of chromosomal DNA have been postulated by several authors [20--23]. However, since native DNA is an extremely long and apparently continuous molecule [1--3] and since it is possible to isolate linear native DNA with a molecular weight of more than 500 • 106 [8] it is probable

255 that the smaller native DNA subunits demonstrated earlier were either the result of shear degradation [20], chemical degradation [21] or other artefacts due to special isolation procedures [22,23]. Other authors [24,25] have interpreted changes in the sedimentation rates of denatured DNA induced by prolonged alkali treatment and/or irradiation as indicative of DNA subunits. However, these changes in the sedimentation rates may be due to alkali or irradiation-induced conformational changes such as unfolding of supercoiled loops present in histone-depleted chromatin [26]. In contrast, the changes of the sedimentation rates demonstrated in this paper are true changes in DNA size. Native DNA which is released from mammalian cells by protease concentrations as low as 50 ~g/ml is completely unfolded and linear [8]. Thus, the DNA described in this paper, which is released by cell lysis in the presence of 1 mg/ml of pronase followed by alkali denaturation, is unfolded and the changes in sedimentation seen here represents true changes in size. Furthermore, the change in size induced by protease treatm e n t cannot be explained by R N A or by any enzyme present in the cells or activated b y the cell lysis procedure since DNA isolated in the presence of ribonucleases is also sensitive to additional protease treatment. Therefore, there are only two remaining mechanisms by which the protease-inducible alkali lability o f denatured DNA could be explained: DNA is either nicked b y a very unusual deoxyribonuclease present in all proteases tested except thermolysin or, alternatively, a peptide/protein linker joining single-stranded subunits is cleaved by protease-catalyzed reactions. If any nuclease contamination were involved in the induction of the alkali labile sites it could n o t be a site-unspecific nuclease. We demonstrated earlier that phage DNA (~X174, T7) is n o t changed in size b y pronase [5]. This paper contains additional evidence ruling o u t such an unspecific nuclease. If a site-unspecific nuclease caused the observed change in size of denatured DNA one would expect continued degradation of DNA by higher enzyme concentrations. This is not observed. Similarly, an unspecific nuclease is incompatible with the results of the competition experiment. If 6 mg/ml of pronase contained just enough nuclease to introduce the number of nicks into a small a m o u n t of labelled DNA such that the limit size of denatured DNA is observed, one would expect many fewer nicks after competition by a 2400-fold excess of unlabelled DNA. Since the size of the labelled DNA strands in both cases are identical, it is clear that the activity which makes DNA alkali labile during the protease treatment is site specific. Finally, another evidence for this site specificity comes from the result indicating that the number of alkali-sensitive sites introduced into DNA molecules by optimal doses of t w o different proteases (pronase and proteinase K) are non-additive. If the typical phosphodiester bonds were sensitive to the treatment with proteases, one should expect additional effects after treatment with pronase and proteinase K. This is n o t observed. Thus, the change in size of denatured DNA is clearly n o t due to a site-unspecific nuclease activity present in the protease preparations. Furthermore, it is highly unlikely that a site-specific nuclease is working during the incubation of DNA with proteases. Such a site-specific nuclease would have to be present in proteases of various origin including mammalian cells (trypsin, chymotrypsin), fungi (proteinase K) and bacterial cells (subtilisin, pronase). Furthermore, this site-specific nuclease would have to be heat stable (10 min,

256 80°C) and resistant to the autodigestion procedure (30 mg/ml of pronase, 37°C, 1 h}, active under the conditions of cell lysis (SDS, EDTA), able to recognize a site in both native or denatured DNA. It would recognize sites present in eukaryotic DNA but n o t present in ¢X174 and T7 DNA. Such a strange nuclease is highly unlikely but it cannot be ruled o u t unequivocally. However, since we are able to present direct evidence for the involvement of a proteasecatalyzed reaction in the phenomenon of protease-induced alkali lability of DNA, we feel that the best explanation of this p h e n o m e n o n is n o t cleavage by nuclease contamination but by the protease itself. The release of short singlestranded DNA by the protease treatment of cell lysates followed by alkali denaturation is significantly inhibited by specific protease inhibitors. Since these inhibitors are highly specific for reactions catalyzed by the proteases we consider this result as direct evidence for the involvement of protease activity in the p h e n o m e n o n of protease-induced alkali lability of denatured DNA. Treatment of DNA with high concentrations of proteases causes adjacent strands to become disconnected and thus alkali labile. Since the sites which can be changed by proteases are also nuclease $1 sensitive after the protease treatment, we propose that the protease-treated DNA is a nicked or gapped duplex molecule. This result is also incompatible with a protein-protected R N A linker joining single-stranded DNA. Removal of the protective material by high concentrations of proteases from such a D N A / R N A hybrid region would render it alkali labile but not $1 sensitive. Thus, the non-deoxynucleotide material joining single-stranded DNA subunits almost certainly consists of peptide/ protein material joining adjacent strands by bridging nicks or gaps in DNA. This internucleotide linker material is somewhat protease resistant (high concentrations of protease are necessary for optimal cleavage) and alkali stable b o u n d to DNA. As a consequence, it should be copurified during the isolation of DNA. The presence of a small a m o u n t of peptide material in isolated native DNA is well known [27--32]. Furthermore, we have demonstrated that distinct proteins which are between 54 000 and 68 000 daltons in size are not removed from DNA despite treatment with proteases, phenol and alkali [33]. Since for example, nucleotide-(P-N)-peptides (phosphoamides) and nucleotide-(P-O)seryl (or threonyl) peptides (phosphoesters) are alkali stable [34] the alkali-stable joining of single-stranded DNA subunits by a protein/peptide material can be well explained on a molecular basis. Although the internucleotide protein material is not completely removed by high concentrations of proteases [33] it could have some protease-susceptible site{s), resulting in adjacent DNA strand gaps with some of the material remaining associated with one or both DNA ends. This view is supported by the results indicating that at least one of the ends liberated by proteases is blocked by a material other than a phosphate group because the nicks or gaps induced b y proteases are not utilized b y E. coli DNA polymerase I. A similar observation was made earlier when the ¢ X 1 7 4 RFII (A) complex was investigated [35]. The non-susceptibility of this complex to utilization by E. coli DNA-polymerase I was considered to reveal a blocked 5'-end.

257

The results demonstrated in this paper are compatible with the scheme shown below -

v

~

I

i

~

NaOH

Pronase

~'~8 5.106d~ltons

E~

...........

,:- . . . . . . . . . . . . . . . . .

: ..................

• ................

i~lmH[i,-ul

1Pr°nase ~,n,,,,ll"~ ,H,'.................... IUIIHH,Hj,,H!!E',!!,,,,,H,mh

l ~8.5.106daltons

.,~

......

1 ...........

~9.10 6daltons

It appears, that DNA contains special structural sites (e) which are arranged at relatively regular distances and alternatively on the opposite DNA strands. Alkali-denatured DNA which was n o t treated with proteases behaves as a long molecule. This indicates that the special structural sites are alkali stable. Treatment of alkali-denatured DNA with pronase results in much shorter singlestranded DNA which is due to the sensitivity of the special structural sites to pronase. ( B ) D u r i n g the treatment of DNA with proteases the special structural sites (e) are changed by a protease-catalyzed reaction, however, the resulting native DNA molecule behaves still as a long molecule because the sites which are changed by the protease treatment are n o t arranged opposite or close to each other. When this DNA is alkali denatured, single strands of an apparent molecular weight of 8 . 5 . 1 0 6 are released. By nuclease S1 double: stranded DNA pieces of similar molecular weight are released. The biological role of protein/peptide discontinuities in DNA is n o t known. However, covalently b o u n d proteins are also found in various prokaryotic systems (reviewed in Ref. 36). In some of these systems these proteins were shown to be attached to DNA in the vicinity of the origin of replication and it was assumed by the authors that these proteins covalently b o u n d to DNA are involved in nucleic acid synthesis [37]. The peptide/protein linkers demonstrated by us could play a similar role in eukaryotic DNA. According to Taylor and Hozier [38] an analysis of the spacing between origins of replications shows that such sites are available at intervals of a b o u t 4 #m along eukaryotic DNA. This is almost exactly the distance between two sequential discontinuities observed by us (8.5 • 106 daltons, 27 kilo bases) (Scheme I). Other possible roles of these proteins such as the involvement in the maintenance of chromatin structure were discussed earlier [33].

258

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Kavenoff, R. and Zimm, B.H. (1973) C h r o m o s o m a 41, 1--27 Kavenoff, R. and Zimm, B.H. (1973) Cold Spring Harbor Syrup. Quant. Mol. Biol. 38, 1--8 Sasaki, M.S. and Norman, A. (1966) Exp. Cell Res. 44, 6 42--645 Hays, J.B. and Zimm, B.H. (1970) J. Mol. Biol. 48, 297--317 Hershey, H.V. and Wemer, D. (1976) Nature 262, 148--150 Karzel, K. and Schmid, O.J. (1968) Arzneimittelforschu ng 18, 1500--1509 Shaw, E., Mares-Guia, M. and Cohen, W. (1965) Biochemistry 4, 2219--2224 Gross-Bellard, M., Oudet, P. and Chambon, P. (1973) Eur. J. Biochem. 36, 32--38 Weiss, B., Live, T.R. and Richardson, C.C. (1968) J. Biol. Chem. 243, 4530--4541 R o s e n b l o o m , J. and Schumaker, V.N. (1963) Biochemistry 2, 1206--1211 Fahrney, D.E. and Gold, A.M. (1963) J. Am. Chem. Soc. S5, 997--1009 Barter, A.J. (1977) in Proteinases in Mammalian Cells and Tissues (Dingle, J.T., ed.), p. 16, N ort h Holland Publishing Company, A m s t e r d a m Mares-Guia, M. and Shaw, E. (1965) J. Biol. Chem. 240, 1 5 7 9 - - 1 5 8 5 Schoellmann, G. and Shaw, E. (1962) Biochem. Biophys. Res. Commun. 7, 36---40 Petra, P.H., Cohen, W. and Shaw, E. (1965) Biochem. Biophys. Res. Commun. 2 1 , 6 1 2 - - 6 1 8 Smith, E.L., Markland, F.S., Kasper, C.B., DeLange, R.J., Landon, M. and Evans, W.H. (1966) J. Biol. Chem. 241, 5974--5976 Radcliffe, R.D. and Barton, P.G. (1972) J, Biol. Chem. 247, 7735--7742 Vogel, R., Trauschold, J. and Werle, E. (1966) in Natiirliche Proteasen und Inhibitoren, p. 8, Thieme Verlag, Stuttgart Shishido, K. and Ando, T. (1972) Biochim. Biophys. Acta 287, 477--484 Sadron, Ch., Pouyet, J. and Vendrely, R. (1957) Nature 179, 263--264 Bendich, A., Borenf~eund, E., Korngold, G.C., Krim, M. and Balls, M.E. (1964) Abstracts of the Meeting on Nucleic Acids and Their~Biological Roles, pp. 214--237, I n s t i t u t o Lomba rdo, Milano, Italy, 1963, Tipografia Successori Fusi, Pavia Hilgartner, C.A. (1964) Exp. Cell Res. 3 6 , 2 2 8 - - 2 4 1 Welsh, R.S. and Vyska, K. (1971) Arch. Biochem. Biopliys. 142, 132--143 Lett, J.T., Klucis, E.S. and Sun, C. (1970) Biophys. J. 10, 277--292 Elkind, M.M. (1971) Biophys. J. 11, 502--520 Nakane, M., Ide, T., Anzal, K., Ohara, S. and Andoh, T. (1978) J. Biochem. 84, 145--157 Kirby, K.S. (1957) Biochem. J. 6 6 , 4 9 5 - - 5 0 4 Kirby, K.S. (1958) Biochem. J. 70, 260--265 Kirby, K.S. (1959) Biochim. Biophys. Acta 3 6 , 1 1 7 - - 1 2 4 Borenfreund, E., Fitt, E. and Bendich, A. (1961) Nature 191, 1375--1377 Bianehi, P.A. and Shooter, K.V. (1961) Biochem. J. 78, 372--376 Salser, J.S. and Balls, M.E. (1967) Biochim. Biophys. Acta 149, 220--227 Krauth, W. and Werner, D. (1979) Biochim. Biophys. Acta 564, 390--401 Shabarova, Z.A. (1970) Progress in Nucleic Acid Research (Davidson, J.N. and Cohn, W.E., eds.), Vol. 10, pp. 145--182, Academic Press New York, Londo n Ikida, J., Yudelevich, A. and Hurwitz, J. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2669--2673 Salas, M., Mellado, P. and Vifiuela, E. (1978) J. Mol. Biol. 119, 269--291 Rek osh , D.M.K., Russel, W.C. and Bellet, A.J.D. (1977) Cell 1 1 , 2 8 3 - - 2 9 5 Taylor, J.H. and Hozier, C. (1976) C h r o m o s o m a 5 7 , 3 4 1 - - 3 5 0