Strand separation of DNA induced by ultraviolet irradiation in vitro

145

Biochirnica et Biophysica Acta, 374 ( 1 9 7 4 ) 1 4 5 - - 1 5 8 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 9 8 1 4 3

STRAND SEPARATION OF DNA INDUCED BY ULTRAVIOLET IRRADIATION IN VITRO

R A N J I T D A S G U P T A a ' * and S A N K A R M I T R A a ' b ' * *

a Bose Institute, Calcutta-9 (India), and b Biology Division, Oak Ridge National Laboratory, Oak Ridge, Ten~ 37830 (U.S.A.) (Received May 20th, 1 9 7 4 )

Summary 1. Ultraviolet irradiation of double stranded DNA in vitro caused strand separation, as judged by the susceptibility of the DNA to degradation by an endonuclease from Neurospora crassa that is specific for single strands, by its behavior on elution from hydroxylapatite and by its sedimentation properties. The extent o f degradation by the endonuclease was almost linearly proportional to the ultraviolet dose up to 2.8 • 10 s J / m 5, when 45% of the DNA was degraded. Increased DNA concentration and high concentrations of Na ÷ and Mg2÷ were inhibitory to degradation. The pH of the DNA solution had no significant effect. 2. The ultraviolet-irradiated DNA was resistant to single strand-specific exonuclease I from Escherichia coli even after removal of 5'-phosphate termini or limited endonucleolytic cleavage. Ultraviolet-induced denaturation was not reversible even under o p t i m u m conditions. 3. The elution profile of irradiated DNA from hydroxylapatite was gradually shifted, depending on the ultraviolet dose, from the position of double stranded DNA toward that of single stranded DNA, although there was no significant overlap with the profile of single stranded DNA. The elution profile also indicated that the denaturation was not an all-or-none p h e n o m e n o n and that all the DNA molecules were similarly affected. Unlike X-irradiation, ultraviolet light did n o t generate single stranded fragments. 4. The sedimentation coefficient of T7 phage DNA increased from 31 to 67 S after ultraviolet irradiation (1.4 • 10 s J / m 2 incident dose), presumably due to localized collapse of the double stranded structure and formation of interstrand cross-links. 50% of the total irradiated DNA was rapidly renaturable after denaturation by alkali. * Present address: B i o p h y s i c s L a b o r a t o r y , University o f Wisconsin, Madison, Wisc. 53706, U.S.A. ** Present address: B i o l o g y Division, O a k Ridge N a t i o n a l Laboratory, Oak Ridge, Tenn. 37830, U.S.A. T o w h o m reprint requests s h o u l d be addressed.

146 5. Localized strand separation in DNA after ultraviolet irradiation was related to pyrimidine dimer formation; proflavine reduced both. The denatured regions contained almost all of the pyrimidine dimers. When the irradiated DNA was treated with N. crassa endonuclease, thymine-containing dimers were released as acid-soluble products more readily than was thymine. 6. DNA inside T7 phage was also denatured after ultraviolet irradiation of the whole phage. Such DNA was not denatured b y alkali, and was covalently cross-linked with the phage coat.

Introduction Formation of pyrimidine dimers after ultraviolet irradiation is expected to produce denatured regions in double stranded DNA b y destruction of hydrogen bonds. Evidence for such denaturation has been d o c u m e n t e d [1--4]. Recently, Kato and Fraser [5], using a N e u r o s p o r a crassa endonuclease specific for single stranded DNA, showed the presence of denatured regions in ultravioletirradiated ~X replicative-form DNA; and Trifonov and his co-workers [6,7] used the kinetic formaldehyde method to determine the number of locally denatured regions in DNA after ultraviolet irradiation. Independently, we have attempted to estimate the extent of denaturation in DNA after ultraviolet irradiation, using the single strand-specific nucleases N crassa endonuclease [8] and Escherichia coli exonuclease I [9]. Efforts were also made to correlate the partial denaturation of DNA after ultraviolet irradiation with its elution behavior from hydroxylapatite and its sedimentation properties. Materials and Methods Strains E. coli B and BB strains were obtained from S. Champe (Rutgers University), and thymine-requiring E. coli 15T- was obtained from S.B. Bhattacharya (Calcutta University). E. coli C was obtained from R.K. Poddar (Calcutta Uni-

versity). Wild-type T7 phage was supplied b y R. Fujimura (Oak Ridge National Laboratory). N. crassa Y73A was obtained from S.K. C h a t t o p a d h y a y (Bose Institute). Reagents

Carrier-free H332 PO4 and [2-~4 C] thymidine were purchased from Bhabha Atomic Research Centre, India, and Radiochemical Centre, Amersham, respectively. The growth media were Difco products. Proflavine sulfate was a gift o f R.K. Poddar. Lysozyme, pancreatic ribonuclease, and most other biochemicals were Sigma products. N. crassa e n d o n u c l e a s e

The enzyme was purified from fungal mycelia by a slightly modified procedure of Linn [ 1 0 ] . A wild-type strain of N. crassa Y73A was grown in 10-1 flasks containing 8 1 of Fries' complete medium [11] under forced aeration for 2 days at 23 ° C. The mycelia were filtered and washed with distilled

147 water and then ground with 3 times their weight of Superbrite glass beads in a Sorvall Omnimixer in 0.05 M glycylglycine buffer (pH 7.0). After MgC12 treatment and (NH4)2SO4 fractionation [ 1 0 ] , the phosphocellulose chromatography step was omitted and Linn's procedure was followed in the subsequent steps. The e n z y m e was assayed on heat-denatured 3: P-labeled E. coil DNA as substrate. The assay mixture (0.3 ml) contained 20 nmoles of DNA (expressed as phosphorous equivalent), 3 g m o l e s MgC12, 0.3 pmole mercaptoethanol, and 30 ~zmoles Tris--HC1 buffer (pH 7.5) with an appropriate a m o u n t of enzyme. 1 unit of enzyme is defined as that converting 1 pmole of denatured DNA into trichloroacetic acid-soluble products in 30 min at 37 ° C. The final enzyme had a specific activity of 1600 units/mg protein, which is a b o u t one-sixth that of Linn's preparation [10]. The a m o u n t of enzyme that degraded 95% of the denatured E.coli DNA (20 nmoles) in an incubation mixture made only 1.5% of t h e native E. coli DNA and none of the T7 phage acid soluble. E. coli exonuclease I Exonuclease I was purified from E. coli B b y the procedure of Lehman [9]. The cells were grown to late log phase in glucose--yeast extract. The frozen cells were homogenized in 3 times their weight o f Superbrite glass beads in a Sorvall Omnimixer. The enzyme was purified through chromatography on hydroxylapatite and had a specific activity of 2000 units/mg protein. It had trace endonuclease activity. The assay procedure and definition of unit activity were according to Lehman [9]. The amount of enzyme required to degrade 85% of 20 nmoles of denatured E. coli DNA made 1.5% of the native E. coli DNA and none of the T7 phage DNA acid soluble. E. coli alkaline phosphatase The alkaline phosphatase from E. coli C spheroplast supernatant was purified according to Malamy and Horecker [ 1 2 ] . The enzyme was twice recrystallized. Hydroxylapatite chromatography Hydroxylapatite was prepared according to Miyazawa and Thomas [13]. A column of 1.2 cm × 3.5 cm was used. The hydroxylapatite was equilibrated with 0.05 M sodium phosphate buffer (pH 6.8). The DNA in the same buffer was loaded on the column, and a 200-ml linear gradient of 0.05 M--0.4 M sodium phosphate buffer (pH 6.8) was used to elute the DNA at 30 ° C. Denatured DNA was eluted at 0.19 M and native DNA at 0.26 M sodium phosphate. More than 95% of the total DNA was recovered. 2p-Labeled E. eoli D N A 32 P-Labeled DNA was isolated from E. eoli B grown to late log phase in TCG medium [14] containing 3--4 mCi 32 P/1. The cells were suspended in 20% sucrose in 0.033 M Tris--HC1 buffer (pH 8.0), and egg-white lysozyme and EDTA were added to 50 pg/ml and 10 -3 M, respectively. After incubation at r o o m temperature for 5 min, the spheroplasts were lysed with 2% Sarkosyl NL97 at 60°C for 15 rain. The lysate was deproteinized according to Marmur [15]~ and the DNA was purified b y alcolhol precipitation. R N A was removed

148

by incubation with 1 0 0 p g / m l pancreatic ribonuclease (preheated at 100°C for 10 min) for 30 min, followed by deproteinization and alcohol precipitation. The final DNA had less than 1% trichloroacetic acid-soluble counts and had a specific activity of a b o u t 106 cpm/pmole. The yield was 8--15 pmoles DNA (P) per 1 of culture. 14C-Labeled E. coli DNA was prepared in the same way as above from E. coli 15T- in a supplemented salt--glucose medium [16] containing 4 pg [14C] thymidine (0.15 pCi) per ml. 32P-Labeled T7 phage DNA E. coli BB was grown in TCG medium [14]. 90 min before infection (multiplicity of infection, 0.2) 32p was added, and 2--3 h after infection, after complete lysis, the phage was purified [14] b y high-speed centrifugation (78 000 × g, 1 h) after removal of cell debris b y low-speed centrifugation (10 000 × g, 15 min). The phage suspension in 1 M NaC1--0.01 M Tris--HCl buffer (pH 7.5), was subjected to another cycle of low- and high,speed centrifugation and finally banded in CsCI gradient in an SW-39 swinging-bucket tube and centrifuging for 2 h at 100 000 × g. The phage solution was dialyzed against 0.01 M Tris--HC1 buffer (pH 8.0). DNA was extracted from the phage usually b y gentle shaking with phenol [14] and exhaustively dialyzed against 0.01 M Tris--HC1 buffer (pH 8.0). Denatured DNA was prepared by heating the DNA solution in 0.01 M Tris--HCl buffer or 0.05 M sodium phosphate buffer (pH 6.8) at 100°C for 10 min and then rapidly immersing it in an ice bath. Radioactive counting 32p was counted in a G-M end-window counter and 14C in a Beckman liquid scintillation spectrometer, LS-100. The fluor solutions were either toluene containing 0.4% p-terphenyl and 0.005% POPOP or dioxane containing 10% naphthalene and 0.5% PPO. Analytical methods Thymine--thymine dimers and other pyrimidine dimers were estimated in irradiated DNA b y a slight modification of the procedure of Setlow and Carrier [17]. 14 C-Labeled DNA (irradiated or unirradiated) was mixed with 50 pg unlabeled DNA and hydrolyzed with 70% HC104 at 100°C for 1 h. The bases and pyrimidine dimers were separated by descending paper chromatography. The chromatogram was cut into strips and counted in a liquid scintillation counter. Protein was estimated according to Lowry et al. [18] with bovine serum albumin as standard. For very small quantities o f protein, Waddell's spectrophotometric method [19] was used. Double-stranded DNA was esimated spectrophotometrically based on 50 pg as one absorbance unit at 260 nm. UZtraviolet irradiation Ultraviolet irradiation was performed with a GE germicidal lamp emitting a b o u t 95% of its radiant energy at 254 nm. The D N A solution was made up in 0.01 M Tris--HC1 buffer (pH 8.0), or 0.005 M sodium phosphate buffer (pH

149 6.8), unless otherwise stated. 0.5 ml DNA solution (0.2--0.4 pmole/ml) in a shallow petri dish (2.5 cm diameter) was kept at a distance of about 9 cm, and the light intensity as measured by a dosimeter (kindly supplied by R. Laterjet) was adjusted to 40 J/rn-2 • s. The depth of the irradiated layer was 1.5--2 mm. Irradiation was carried out at room temperature in the dark with occasional stirring of the solution. After irradiation, DNA solutions were kept at 0°C when n o t immediately used. Results

Evidence for denaturation o f ultraviolet-irradiated D N A Susceptibility to N. crassa endonuclease. When 32 P-labeled double stranded DNAs of E. coli and T7 phage were exposed to ultraviolet light and then treated with single strand-specific endonuclease from N. crassa, they were partially degraded by the e n z y m e (Table I), indicating the presence of denatured regions in the DNAs. The a m o u n t of enzyme used was the minimum necessary to produce 85--90% degradation of an equivalent sample o f unirradiated, single stranded DNA in 30 min, since a 10-fold excess was found to cause almost complete degradation of ultraviolet-irradiated samples (7.2 • 104 J / m ~) in the same a m o u n t of time (see Discussion). The extent of denaturation indicated by this assay agrees closely w i t h the estimate by Salganik et al. [ 3 ] , who used a different m e t h o d to show that an ultraviolet dose of 1.5 • 10 s J / m 2 resulted in 21--25% denaturation of a DNA sample. Several factors were found to affect the extent of the denaturation induced by ultraviolet irradiation, as measured by the present endonuclease assay. First, denaturation increased in proportion to the ultraviolet dose up to a fluence of 2.9 " l 0 s J / m 2, which caused 45% of the total DNA sample to be susceptible to the e n z y m e (Fig. 1). It is possible that interstrand cross-linking, which is also induced by ultraviolet in proportion to dose [20], rendered the DNA resistant to further strand separation at high doses. After exposure to 5.8

TABLE I SUSCEPTIBILITY OF ULTRAVIOLET-IRRADIATED E. C O L I E X O N U C L E A S E I

D N A TO N. CRASSA

ENDONUCLEASE AND

3 2 p - L a b e l e d E. C o l i a n d T 7 D N A s ( 0 . 2 ~ m o l e / m l ) in 0.01 M T r i s - - H C l b u f f e r ( p H 8.0), w e r e i r r a d i a t e d and t h e n a l i q u o t s ( 2 0 n m o l e s ) w e r e d i g e s t e d w i t h 0 . 0 4 u n i t N . crassa e n d o n u c l e a s e and 4 u n i t s o f E. coli e x o n u c l e a s e I s e p a r a t e l y f o r 30 m i n at 3 7 ° C a c c o r d i n g t o t h e assay p r o c e d u r e d e s c r i b e d in M e t h o d s and in [ 9 ] , r e s p e c t i v e l y . T h e n 0.2 m l u n l a b e l e d E. c o l i D N A ( 2 . 5 m g / m l ) a n d 0.5 m l 7% HCIO 4 w e r e a d d e d at 0°C. A f t e r 1 0 m i n a t O°C, t h e p r e c i p i t a t e w a s c e n t r i f u g e d a t 10 0 0 0 X g f o r 1 0 m i n in the c o l d and a n aliquot of the s u p e r n a t a n t was c o u n t e d for radioactivity. DNA source

Ultraviolet dose (J/m2)

% DNA degraded by N . crassa endonuclease

E. c o i l exonuclease I

E. c o l i

7 . 2 . 104 1.4" 10 s

15 26

0 0

T7

7 . 2 " 104 1.4 • 1 0 s

14 25

0 0

150

50 40 ¢D o

30

~

o 10

/

o

0 0

4

8

12 16 20 24 UV-DOSE {J/rn 2 x I0 -4)

28

32

Fig. 1. Ultraviolet d o s e - d e p e n d e n c e o f D N A d e n a t u r a t i o n . A l i q u o t s (0.1 ml) of 32p-labeled E. c o l i (20 n m o l e s ) w e r e assayed for e x t e n t o f degradation w i t h 0 . 0 4 units o f N. crassa e n d o n u c l e a s e for 3 0 rain at 37 ° C.

• 10 s J/m 2 , only 65% of the DNA was susceptible to the endonuclease. Almost no degradation was found to result from irradiation alone, less than 0.5% of the DNA being acid-soluble before nuclease treatment after the highest dose tested. The concentration of DNA in the irradiated sample also had an effect on the results. When DNA at different concentrations was exposed to a given dose of ultraviolet, the susceptibility of the irradiated material to degradation by the enzyme was lower for the higher concentrations (Table II). The effect was most marked for solutions containing 0.01--0.05 /~moles of DNA per mL Although renaturation of denatured DNA shows a similar concentration effect [ 2 1 ] , it is possible that here interstrand cross-linking, which increases with increasing DNA concentration, was responsible for the reduced susceptibility to the nuclease. The effect of monovalent and divalent cations on the ultraviolet-induced denaturation o f DNA were also examined in the present experiments, since the melting point (Tin) of DNA in solution is known to increase with the logarithm of the ionic strength of the solvent [ 2 2 ] . Both Na ÷ and Mg 2÷ inhibited denaturation as detected by the endonuclease assay (Table III), although no obvious T A B L E II EFFECT OF DNA CONCENTRATION T O N. C R A S S A E N D O N U C L E A S E

ON SUSCEPTIBILITY OF ULTRAVIOLET-IRRADIATED

DNA

3 2 p - L a b e l e d E. c o i l D N A w a s irradiated w i t h 7 . 2 . 104 J / m 2 at different c o n c e n t r a t i o n s , and t h e n aliquots ( 2 0 n m o l e s ) f r o m each sample w e r e treated w i t h 0 . 0 4 unit N. crassa e n d o n u c l e u e as described in Table I. DNA concentration 0~mole/n'd)

N . crassa e n d o n u c l e a s e

% D N A degraded b y

0.01 0.05 0.10 0.20 0.50 1

36.5 19 14 12 9 9

151 TABLE III E F F E C T OF C A T I O N C O N C E N T R A T I O N IRRADIATION

ON D E N A T U R A T I O N

OF D N A A F T E R U L T R A V I O L E T

3 2 p - L a b e l e d E . c o l i D N A ( 0 . 4 ~ m o l e / m l ) in 0.01 M Tris-HCl b u f f e r ( p H 8.0) b u f f e r w a s m a d e u p t o 2+ d i f f e r e n t N a ÷ a n d Mg c o n c e n t r a t i o n s and t h e n irradiated w i t h u l t r a v i o l e t light ( 1 . 4 • 105 j / m 2 ) . A l i q u o t s ( 2 0 n m o l e s ) o f t h e D N A s o l u t i o n w e r e d i g e s t e d w i t h 0 . 0 4 u n i t of N . crassa e n d o n u c l e a s e s o l u t i o n at 3 7 ° C f o r 3 0 rain. C o n t r o l e x p e r i m e n t s s h o w e d that t h e e n z y m e a c t i v i t y w a s n o t a f f e c t e d b y the a m o u n t of NaCI a n d MgCI 2 p r e s e n t in t h e a l i q u o t s o f D N A u s e d f o r assay. Salt

None NaCI

MgCl 2

C o n c n (M)

0.2 0.5 1 0.001 0.01 0.05 0.1

% D N A d e g r a d e d b y N . crassa endonuclease 26 11 9.4 6.9 25 22 15 14

relation b e t w e e n the ionic strength of the medium and the extent of denaturation was observed. On the other hand, the pH of the medium had no significant effect on the extent of denaturation for DNA irradiated in 0.05 M sodium acetate, sodium succinate, Tris--HC1 and sodium glycinate buffer at pH 4.0--9.9. The activity of the endonuclease was n o t affected b y cation concentration or b y t h e presence o f different buffer ions. Resistance to E. coli exonuclease /. Ultraviolet-irradiated DNAs of E. coli and T7 phage were n o t at all susceptible to E. coli exonuclease I (Table I), which degrades single stranded DNA from the 3'-OH end [9]. We first attributed this p h e n o m e n o n to the possibility that the denatured regions produced b y ultraviolet might be inside the double helical backbone of the DNA; however, when irradiated DNA was partially degraded with N. crassa endonuclease; which produces 3'-OH ends within single strands, and then treated with the exonuclease, it still was n o t susceptible to the action of the second enzyme. Similarly, prior treatment with E. coli alkaline phosphatase to remove any monoesterified phosphates from the inside and ends of the DNA molecules (3'-phosphates at the ends of single strands would inhibit exonuclease action on the denatured regions) failed to affect the resistance of the irradiated samples to the exonuclease. These results suggested that the " b l o c k s " in the denatured regions of ultraviolet-irradiated DNAs that prevented the hydrolytic action of the exonuclease b u t not of the endonuclease were b o t h random and extensive. To examine the possibility that the " b l o c k s " might be ultraviolet-induced photoproducts, we irradiated single stranded E. coli DNA and subjected it to treatment with b o t h enzymes. Exonuclease I treatment of 10 nmoles of unirradiated single stranded DNA resulted in 80% degradation of the denatured material either in the presence or in the absence of 10 nmoles of ultraviolet-irradiated native E. coli DNA (results n o t shown), indicating that the irradiated DNA itself did not inhibit the activity o f the enzyme. However, ultraviolet irradiation did reduce the susceptibility of the single stranded DNA to E. coil exo-

152

T A B L E IV S U S C E P T I B I L I T Y OF U L T R A V I O L E T - I R R A D I A T E D E N D O N U C L E A S E A N D E. C O L I E X O N U C L E A S E I

S I N G L E - S T R A N D E D D N A T O N.

CRASSA

H e a t - d e n a t u r a t e d 3 2 p - l a b e l e d E, c o l i D N A w a s irradiated and a s s a y e d as d e s c r i b e d in Table I. Ultraviolet dose (J/m 2)

% D N A degraded by N. c r a s s a e n d o n u c l e a s e

4,8.103 1 . 2 . 104 2 . 4 . 104 4.8-104

E. e o l i e x o n u c l e a s e I

Before irradiation

After irradiation

Before irradiation

After irradiation

85 85 85 85

85 85 85 85

80 80 80 80

26 19 17 I0

nuclease I (but not to N. crassa endonuclease), and the extent of the reduction was dependent on the dose (Table IV). This it is possible that the resistance to exonuclease results from the combined effects of pyrimidine photoproducts and cross-links in the DNA. Hydroxylapatite chromatography. Denatured D N A can be separated from native D N A by elution from hydroxylapatite with a sodium phosphate gradient. When ultraviolet-irradiated D N A was chromatographed by the same method, its elution profile was between those of single and double stranded DNA; and as the ultraviolet dose was increased, the profile was closer to that of denatured D N A (Fig. 2). The peak for ultraviolet-irradiated D N A , even at the highest dose used (1.4 • 10 s J/m 2 ), did not significantly overlap that for single stranded DNA, indicating that ultraviolet, in contrast to X-irradiation [ 2 3 ] , does not produce single stranded D N A fragments. The peaks for D N A subjected to different doses of ultraviolet did overlap (Fig. 2), and their symmetri-

50~_40> -0,4 (~ 3 0 -

-0,3

(z: .j 200 ~-

10-

0

"~ o (3.

;_j-!;iJ-iI

...... 10 1'2

, 16

2'0 2'4 28 FRACTION NO.

52

-o,2 ~

if:

-0.1

;

56

Fig. 2. E l u t i o n p r o f i l e s o f ultraviolet-irradiated D N A f r o m h y d r o x y l a p a t i t e . A l i q u o t s o f 32p.Labele d E. D N A ( 2 0 n m o l e s ) after irradiation w e r e e h r o m a t o g r a p h e d o n h y d r o x y l a p a t i t e in i d e n t i c a l c o l u m n s and e l u t e d w i t h a gradient o f s o d i u m p h o s p h a t e s o l u t i o n as descn'bed in Materials and M e t h o d s . o o, unirradiated d o u b l e - s t r a n d e d D N A ; c c , untrradiated single-stranded D N A ; • •, doubles t r a n d e d D N A irradiated w i t h 1.1 • 104 J / m 2 u l t r a v i o l e t light; • o, 7.2 • 104 J / m 2 u l t r a v i o l e t light: ~, 1.4 • 10 S J / m 2 u l t r a v i o l e t light. coli

153

cal nature suggests, first, that ultraviolet-induced denaturation is not an all-ornone p h e n o m e n o n (i.e. partial denaturation of a DNA sample is due to the presence of denatured regions in all of the molecules rather than complete denaturation of some of the molecules} and, second, that all the DNA molecules in a given irradiated sample are unfolded to a smilax extent. Sedimentation properties. 32 P-Labled T7 phage DNA, which gives a symmetrical profile corresponding to 31 S upon band sedimentation in a sucrose gradient [24], was used for an examination of the effect of ultraviolet irradiation on sedimentation behavior. After exposure to 7.2 • 104 or 1.44 • 10 s J / m 2, the sedimentation coefficient of the samples was uniformly increased to approx. 67 S. The increased sedimentation coefficient of irradiated DNA could be due to denaturation of DNA with the collapse of its rigid structure [24] and/or to the aggregation of DNA molecules as a result of interstrand cross-linking. Interstrand cross-links are formed after ultraviolet irradiation [20], but denaturation must also be a major factor in the effect since the sedimentation coefficient o f the DNA at neutral pH is more than doubled.

Characteristics o f D N A denatured after ultraviolet irradiation Irreversibility o f denaturation and interstrand cross-linking. Ultravioletinduced photodimers between consecutive pyrimidine bases on the same strand of a DNA molecule cause local denaturation of the double helix, and interstrand cross-links, which are produced after ultraviolet irradiation [20], should stabilize the denatured regions. Such cross-links should also cause resistance to complete strand separation after heat- or alkali-induced denaturation; thus, the extent of cross-linking could be determined by the use of one or both of those methods to denature the irradiated DNA. When 32 P-labeled E. coli DNA was irradiated with 7.2 • 104 J/m 2 and then treated with alkali, only about half of the sample was denatured, as determined by its susceptibility to N. crassa e n d o n u c l e ~ e (Table V). When the irradiated, alkali-treated sample was chromatographed on hydroxylapatite (Fig. 3), approx. 40% of the DNA was eluted in the position of denatured DNA, and the remaining 60% was eluted in the position of DNA that had been subjected only to ultraviolet treatment (Fig. 2). It appeared, therefore, that approx. 60% of

TABLE V INTERSTRAND

C R O S S - L I N K I N G O F D N A I N D U C E D BY U L T R A V I O L E T

IRRADIATION

3 2 p - L a b e l e d E . c o l i D N A ( 0 . 4 ~ m o l c / m l ) w a s i r r a d i a t e d ( 7 . 2 • 1 0 4 J / m 2) i n 0 . 0 1 M Tris--HC1 b u f f e r ( p H 8 . 0 ) a n d t h e n m a d e u p t o 0 . 2 M in N a O H . A f t e r 1 5 r a i n a t 2 5 ° C , t h e s o l u t i o n s w e r e r a p i d l y a d j u s t e d t o p H 8 . 4 w i t h 1 M Tris--HC1 b u f f e r ( p H 7 . 2 ) , a t 0 ° C , a n d t h e n a l i q u o t s w e r e i n c u b a t e d w i t h 0 . 0 4 u n i t N. c r a s s a e n d o n u c l e a s e i n t h e s t a n d a r d i n c u b a t i o n m i x t u r e as d e s c r i b e d in T a b l e I. Treatment

% DNA degraded by N. crassa endonuclease

Unirradiated DNA (control)

U n i r r a d i a t e d D N A a f t e r alkali t r e a t m e n t Irradiated DNA I r r a d i a t e d D N A a f t e r alkali t r e a t m e n t

0 85 11 48

154 D-DNA

N-DNA

>- 4 0 L >

-0.4-~ 0

(J 30o

o -0,3 z

20-

-0,2~ o

10-

~

-0,1 ~ 010

1'2

I~

20

2'4

2's

~'2

36

F R A C T I O N NO,

F i ~ 3. H y d r o x y l a p a t i t e c h r o m a t o g r a p h y o f u l t r a v i o l e t - i r r a d i a t e d D N A ( 2 0 n m o l e s ) a f t e r a l k a l i d e n a t u r a t i o n . I r r a d i a t e d ( 7 . 2 • 1 0 4 J / m 2) 3 2 p - l a b e l e d E. coli D N A w a s t r e a t e d w i t h 0 . 2 N N a O H f o r 1 5 r a i n a t 3 0 ° C a n d t h e n q u i c k l y n e u t r a l i z e d w i t h 1 M T r i s - - H C l b u f f e r ( p H 7.2)° a t 0 ° C . A f t e r d i a l y s i s in 0 . 0 5 M s o d i u m p h o s p h a t e b u f f e r ( p H 6 . 8 ) , t h e D N A w a s c h r o m a t o g r a p h e d o n h y d r o x y i a p a t i t e as d e s c r i b e d in Materials and Methods. o o, i r r a d i a t e d c o n t r o l D N A ; -" -', i r r a d i a t e d D N A a f t e r a l k a l i tzeatm e n t . T h e po~dfions o f e l u t i o n o f n a t i v e (N) a n d d e n a t u r e d ( D ) D N A s a r e i n d i c a t e d b y a r r o w s .

the irradiated molecules contained one or more cross-links, which prevented strand separation in spite of the presence of ultravioletAnduced regions of denaturation in the molecules. Similarly, the irradiated DNA was found to be stable u p o n storage under various conditions. When irradiated samples were treated-with N. crassa endonuclease immediately after being exposed to ultraviolet light, 13% of the DNA was degraded, and the a m o u n t did n o t change significantly after incubation for 3 h at 0°C. Incubation at 65°C, which is the o p t i m u m condition for renaturation of denatured DNA [ 2 1 ] , made the sample slightly more susceptible (15% degradation) to the enzyme. Correlation o f denaturation and dimer formation. Thymine--thymine dimers (TT) and other pyrimidine-containing p h o t o p r o d u c t s are known to be induced in ultraviolet4rradiated DNA and can be separated from thymine residues b y paper chromatography of acid-hydrolyzed [2-14 C] thymine-labeled DNA [ 1 7 ] . Proflavine, which inhibits the formation of pyrimidine dimers [25, 2 6 ] , was used in the present study as a probe of the relationship between ultraviolet-induced denaturation and dimer formation. [2-14 C] Thymidinelabeled E. coli DNA was irradiated with 7,2 • 104 J/m 2 and then hydrolyzed, and thymine, TT and other thymine-containing p h o t o p r o d u c t s were isolated b y paper chromatography. TT and other p h o t o p r o d u c t s accounted for 12.0 and 6.3%, respectively, of the total label. For DNA irradiated in the presence of 2.5 10 -s M proflavine, however, the values were reduced to 3.6 and 0%, respectively. Such proflavine-treated, ultraviolet-irradiated DNA was found to be eluted from hydroxylapatite precisely in the position of native DNA (Fig. 4), as opposed to control irradiated DNA (Fig. 2), and was much less susceptible to N. crassa endonuclease (4% degradation) than was the control (10% degradation). In addition, single-stranded E. coli DNA, which was shown to be resistant to degradation b y E. coil exonuclease I after ultraviolet irradiation (Table IV), exhibited an increased susceptibility to the exonuclease when it was irradiated in the presence of proflavine (Table VI). Further evidence for the correlation of denatured regions with pyrimidine p h o t o p r o d u c t s was obtained from the kinetics of the release of labeled thymine •

155

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2ol

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~

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400

80-

-BO

60-

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40-

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20-

-20 ~

w o

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_ - I - - l / I-

//\?...

, ~ ~ o Z ~ , 24

28

52

___.__~ 36

40

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i

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15

GO

120

INCUBATION TIME (min)

FRACTION NO,

Fig. 4. H y d r o x y l a p a t i t e c h r o m a t o g r a p h y o f E. coil D N A i r r a d i a t e d w i t h u l t r a v i o l e t l i g h t in t h e p r e s e n c e of p r o f l a v i n e . 1 4 C - l a b e l e d D N A was i r r a d i a t e d ( 7 . 2 . 104 J / m 2) in t h e p r e s e n c e of 2.5 • 1 0 -s M p r o f l a v i n e s u l f a t e a n d t h e n , a f t e r dialysis, w a s c h r o m a t o g r a p h e d o n h y d r o x y l a p a t i t e as d e s c r i b e d in Materials a n d Methods. o o, c o n t r o l D N A i r r a d i a t e d in t h e a b s e n c e o f p r o f l a v i n e ; • --, D N A i r r a d i a t e d in t h e p r e s e n c e o f p r o f l a v i n e . T h e a r r o w s i n d i c a t e t h e p o s i t i o n s o f e l u t i o n of n a t i v e (N) a n d d e n a t u r e d (D) DNAs. Fig. 5. K i n e t i c s o f release of t h y m i n e p h o t o p r o d u c t s a n d t h y m i n e i n t o acid-soluble p r o d u c t s f r o m u l t r a v i o l e t - i r r a d i a t e d D N A b y N. crassa e n d o n u c l e a s e . A l i q u o t s o f [ 14C] t h y m i d i n e - l a b e l e d E. coil D N A ( 1 0 0 n m o l e s , 5 • 104 c p m ) a f t e r i r r a d i a t i o n (7.2 • 104 J / m 2) w e r e i n c u b a t e d w i t h 0 . 0 0 4 u n i t of N. crassa e n d o n u c l e a s e u n d e r o p t i m u m c o n d i t i o n s as d e s c r i b e d in Materials a n d M e t h o d s , f o r v a r i o u s t i m e s . T h y m i n e a n d t h y m i n e - c o n t a i n i n g d i m e r s w e r e s e p a r a t e d f r o m t h e a c i d - h y d r o l y s a t e o f D N A , as d e s c r i b e d in Materials a n d M e t h o d s . C o n t r o l i r r a d i a t e d D N A w i t h o u t e n z y m e d i g e s t i o n w a s u s e d to c a l c u l a t e t h e t o t a l a m o u n t o f t h y m i n e - c o n t a i n i n g d i m e r s . ~A thymine-containing dimers; o o thymine.

and its photoproducts from ultraviolet-irradiated E. coli DNA upon degradation by N. crassa endonuclease (Fig. 5). Almost all the thymine-containing photoproducts were released as acid-soluble fragments after 15 min of suboptimal enzyme treatment, when only 6% of the total thymine-containing regions had been degraded. Thymine continued to be released, however, even after all the thymine-containing photoproducts had been removed. This result indicates that there might be denatured regions present in the DNA that do not contain thymine photoproducts and are degraded only after the more denatured, dimer-containing areas have been removed. TABLE VI E F F E C T OF P R O F L A V I N E ON S U S C E P T I B I L I T Y S T R A N D E D D N A T O E. C O L I E X O N U C L E A S E I

OF

ULTRAVIOLET-IRRADIATED

SINGLE-

3 2 p - L a b e l e d , h e a t - d e n a t u r e d E coil D N A (0.2 # m o l e / m l ) in 0.01 M Tris--HC1 b u f f e r ( p H 8.0) was i r r a d i a t e d (3 • 1 0 3 J / m 2) in t h e p r e s e n c e or a b s e n c e of p r o f l a v i n e sulfate ( 2 . 5 • 105 M). 2 0 - n m o l e D N A a l i q u o t s w e r e t h e n t r e a t e d w i t h 4.0 u n i t E. coli e x o n u c l e a s e I as d e s c r i b e d in T a b l e I. DNA

% D N A d e g r a d e d b y E. coil exonuclease I

Before irradiation After irradiation A f t e r i r r a d i a t i o n in t h e p r e s e n c e o f p r o f l a v i n c

85 28 54

156

TABLE VII SUSCEPTIBILITY ENDONUCLEASE

OF

DNA

FROM

ULTRAVIOLET-IRRADIATED

T7

PHAGE

TO N.

CRASSA

3 2 p - L a b e l e d T7 p h a g e ( 1 2 A 2 6 0 n m u n i t s / m l ) in 0.01 M T r i s - - H C l b u f f e r ( p H 8.0) a f t e r i r r a d i a t i o n (7.2 • 104 J / m 2) was m a d e u p t o 0.4 M NaC1, 0.01 M s o d i u m p h o s p h a t e b u f f e r ( p H 7.8), a n d the D N A was r e l e a s e d b y h e a t i n g t h e s u s p e n s i o n at 7 0 ° C f o r 5 m i n f o l l o w e d b y q u i c k c o o l i n g t o 0°C. A l i q u o t s ( 2 0 n m o l e s of D N A ) of t h e m i x t u r e w e r e d i g e s t e d w i t h N . c r a s s a e n d o n u c l e a s e as d e s c r i b e d in T a b l e I. U n i r r a d i a t e d p h a g e p r o c e s s e d in t h e s a m e w a y w a s used as t h e c o n t r o l . T h e p r e s e n c e o f p h a g e p r o t e i n did n o t i n h i b i t t h e a c t i v i t y of t h e e n z y m e as i n d i c a t e d b y t h e s u s c e p t i b i l i t y of l a b e l e d d e n a t u r e d DI~A t o the e n z y m e in t h e p r e s e n c e of u n l a b e l e d h e a t - t r e a t e d p h a g e . Alkaline d e n a t u r a t i o n was as d e s c r i b e d in T a b l e V, T y p e of D N A

% DNA degraded by N. crassa endonuelease

DNA DNA DNA DNA

0 80 12.8 12

from after from from

unirradiated phage alkali t r e a t m e n t i r r a d i a t e d phage i r r a d i a t e d p h a g e a f t e r alkali t r e a t m e n t

Denaturation o f T7 phage D N A in situ after ultraviolet irradiation In all of the experiments described above, purified DNA was irradiated in vitro. T7 phage DNA in its natural organized state in association with proteins was then examined and found to be equally sensitive to ultraviolet-induced denaturation. When DNA that h a d been released from irradiated phage by heating at 70°C for 5 min i n the presence of 0.4 M NaC1, 0.01 M sodium phosphate (pH 7.8) [27] was treated with N. crassa endonuclease, it was partially susceptible to the enzyme (Table VII). The extent of degradation (12.8%) was comparable to that for purified T7 phage DNA (14%) irradiated with the same dose (Table I). Thus, ultraviolet irradiation either induced denaturation inside the phage head or so constrained the structure of the DNA inside the phage head that it was denatured as soon as it was released by heat treatment. No degraded DNA was released from the irradiated phage b y nuclease treatment before heat treatment. The D N A irradiated in situ was completely resistant to denaturation b y alkali (Table VII), possibly as a result of DNA--DNA and DNA--protein cross-links [ 2 8 ] . Further, sedimentation analysis in neutral and alkaline sucrose gradients showed that the irradiated DNA was not separated from the protein coat b y treatment with heat or alkali [ 2 9 ] .

Discussion The present studies confirm earlier observations that ultraviolet light induces localized strand separation of DNA [1--7]. In order to estimate quantitatively the extent of denaturation, we had to use ultraviolet doses far above the biological range, b u t the correlation of ultraviolet dose and the extent of denaturation in the range tested suggest that the relationship may hold even at a much lower level of irradiation. High doses of ultraviolet irradiation also induce other alterations in the DNA, such as cross-linking and single strand breakage, which may n o t be produced at the biological dose. The results of K a t o and Fraser [5] with a much lower level of irradiation support our contention that

157

denaturation may be more significant than cross-linking and strand breakage after ultraviolet irradiation. The extensive degradation of irradiated DNA, unlike unirradiated DNA, with excess N. crassa endonuclease is puzzling. It is possible that ultraviolet irradiation causes general destabilization of the DNA helix and that the endonuclease, after degrading the completely denatured regions, attacks partially damaged double helical regions. Alternatively, the endonuclease starting with the degradation of denatured regions may force open the undenatured regions of DNA molecules. It is worth noting that excess endonuclease does not show a similar effect on X-irradiated DNA, where single stranded regions are also generated [29]. In any case, considering the precautions taken (see Results) and the fact t h a t the values obtained with the nuclease assay in the present studies are consistent with those of Salganik e t a l . [3], it is safe to conclude that the quantitative values obtained here are not too far off. The ability of ultraviolet-irradiated DNA to resist degradation by E. coli exonuclease I, even when it is denatured before or after irradiation, suggests that the exonuclease cannot degrade regions containing pyrimidine dimer or other photoproducts formed after irradiation. At the same time, ultraviolet irradiation produced cross-links in DNA and thus made it rapidly renaturable after denaturation by alkali. Since pyrimidine dimers are formed within the same strand, the irreversibility of ultravioletAnduced denaturation is likely due to stabilization of the denatured regions by interstrand cross-linking of unk n o w n nature [20]. This was also clear from the hydroxylapatite chromatography data, which show t h a t single stranded fragments were never produced, even after a very high dose of ultraviolet light. The sedimentation coefficient of 67 S for irradiated T7 DNA is less than 86 S, the value for single stranded T7 DNA in high salt [24]. Completely collapsed DNA of the size of T7 would have a sedimentation coefficient of about 120 S [24]. Thus the increased sedimentation coefficient of T7 DNA after irradiation is probably due to a partial collapse of its structure as a result of localized denaturation. Intermolecular crosslinking may also contribute to the increase, by the extent cannot be determined. It is generally supposed that regions of DNA containing pyrimidine dimers are denatured. The concomitant reductions in the yield of pyrimidine dimers and in the extent of denaturation of proflavine-treated, ultraviolet-irradiated DNA strongly support this idea. It may be pointed out that proflavine has no effect on the denaturation of DNA after X-irradiation, where pyrimidine dimers are n o t produced [29]. It is n o t clear, however, whether pyrimidine dimers are the cause or the effect of ultraviolet-induced denaturation. We have shown (see Results) that all the pyrimidine dimers are located in the denatured regions. Whether there are other denaturated regions in the irradiated DNA t h a t do n o t contain dimers, but do contain other photoproducts, is n o t clear. Other workers [6,7] have concluded from kinetic formaldehyde experiments that pyrimidine dimers are clustered in ultraviolet-irradiated DNA. It appears likely from our results, particularly from hydroxylapatite chromatographic analysis, that denaturation occurs over extended regions of the DNA. Finally, it was interesting to observe that DNA of whole T7 phage was

158

denatured to an extent comparable to that of free DNA after ultraviolet irradiation. The denaturation may have occurred in situ inside the phage head, or after its release by heat treatment; however, no DNA was released from the intact phage after ultraviolet irradiation. At the same time, all the DNA was presumably linked with protein by covalent bonds and sedimented after heat treatment as a pellet during ultracentrifugation.

Acknowledgements This work was partly supported by research grants from the Council of Scientific and Industrial Research and Department of Atomic Energy, Government of India, and by the U.S. Atomic Energy Commission under contract with the Union Carbide Corp.

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