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Genetic transformation studies
298 BBA
BIOCHIMICA ET BIOPHYSICA ACTA
8263 GENETIC TRANSFORMATION STUDIES III. EFFECT OF ULTRAVIOLET LIGHT ON THE MOLECULAR PROPERTIES OF NORMAL AND HALOGENATED DEOXYRIBONUCLEIC ACID ZOFIA O P A R A - K U B I N S K A , ZOFIA KURYLO-BOROWSKA AND WACLAW SZYBALSKI
McArdle Memorial Laboratory, University of Wisconsin, Madison, Wisc. (U.S.A.) (Received November 3oth, 1962)
SUMMARY
The transforming activity of 5-bromodeoxyuridine-labeled DNA is more sensitive to ultraviolet light than that of normal (non-labeled) DNA. The more rapid ultravioletirradiation inactivation of 5-bromodeoxyuridine labeled DNA, as compared with nonlabeled, is reflected also by (a) greatly increased affinity of ultraviolet-irradiated 5-bromodeoxyuridine-labeled DNA for the methylated albumin colunm, (b) decrease in density (dehalogenation) and very pronounced band spreading (degradation, increased heterogeneity), as revealed by CsC1 equilibrium-density centrifugation of ultraviolet-irradiated native 5-bromodeoxyuridine-labeled DNA's, and (c) marked cross-linking between complementary strands of 5-bromodeoxyuridine-labeled DNA, as determined with ultraviolet-irradiated and denatured material centrifuged in the Cs2SO4 density gradient. INTRODUCTION
It was demonstrated that BUdR-labeled transforming DNA is more sensitive to ultraviolet light (2537~) than BUdR-free (normal) DNA 1. Since this finding indicated that the ultraviolet sensitivity of intact cells is principally governed by the sensitivity of their DNA component 1, it became important to assess the molecular basis of ultraviolet-light effects on normal versus BUdR-labeled DNA. Several chemical events have been detected during ultraviolet irradiation of normal DNA or its components, including water addition across the 5-6 double bond of cytosine 2,3, and thymine dimerization*. Substitution of a 5-halouracil for thymine would obviously eliminate the latter reaction, which, however, could be replaced by 5-halouracil dimerization, a not generally accepted reaction s, and/or by photochemical dehalogenation6, 7. Whichever elementary photochemical reactions occur, the result Abbreviations: BUdR, 5-bromodeoxyuridine; FUdR, 5-fluorodeoxyuridine; NN DNA, normal native DNA; dN, normal denatured DNA; NB DNA, native unifilarly BUdR-labeled ("hybrid") DNA (thymidine replaced by B U d R in one strand only); BB DNA. native bifilarly BUdR-labeled DNA (thymidine in both strands replaced by BUdR); BU DNA, mixture of NB and BB; dB, denatured BUdR-labeled DNA components (strands); SSC, o.15 M NaC1 + O.Ol5 M Trisodium citrate; DSC, o.oi 5 M NaC1 + o.ool 5 M Trisodium citrate; sTA, specific transforming activity (number of transformant colonies per ~ug of transforming DNA).
Biochim. Biophys. Acta, 72 (1963) 298-3o9
IRRADIATION OF HALOGENATED TRANSFORMING
DNA
299
could be a multitude of changes in the polymeric structure of the DNA, including partial strand separation, single- and double-chain breaks, intra-strande, s, inter-strand 9 and protein-to-DNA crosslinking 1°. Many of these changes could be quantitatively evaluated by measuring the buoyant densities of native and denatured DNA or by assessing their chromatographic behaviour. CsCP 1 and Cs2S04 TM density-gradient centrifugation and chromatography on the methylated albumin column ~a-15 were employed as the primary methods for comparing the ultraviolet induced macromolecular changes in normal versus BUdR-labeled DNA. These physico-chemical studies were accompanied by parallel determinations of the biological activity of the DNA. EXPERIMENTAL
Strains
The prototrophic and indole-requiring strains of Bacillus subtilis were the same as previously used by SZYBALSKIet al. le. Media
Nutrient broth, 0.8% (Difco); VB minimal liquid m e d i u m - stock solution (MgSOi.7H20, IO g; citric acid.H,O, IOO g; K2HPO 4, 500 g; NaNH4HPO4"H,O, I75 g; water, I 1) diluted 1:50 and supplemented with 0.5% glucose17; S minimal solid medium (K,HPO a, 14 g; KH,PO,, 6 g; Trisodium citrate. 2 H20, i g; MgSO,. 7 H20, 0.2 g; (NH,),SO,, 2 g; glucose, 5 g; agar, I6 g; water, I 1). Isolation of D N A
DNA was isolated from cells lysed with lysozyme and sodium laurylsulfate, deproteinized by shaking with chloroform and butanol, and freed of RNA16, TM. The final purification steps included preparative CsCl-gradient fractionation 19, and column chromatography, as described in the following sections. To prepare the unifilarly BUdR-labeled DNA (NB) a nutrient broth culture was diluted 1:6 in VB minimal medium supplemented with IOO/zg BUdR and 4 Pg FUdR per ml and vigorously agitated for 2 h at 37 °. Any admixture of NN or BB was removed by preparative CsCl-gradient centrifugation. By prolonging the growth period in the presence of BUdR and FUdR to 4-5 h, a mixture of bifilarly and unifilarly BUdR-labeled DNA was obtainedX6, 20, the CsC1 banding pattern of which is presented in Fig. IA*. Transformation procedure
An overnight culture of indole-requiring cells was first diluted 1:3 in nutrient broth, and after 2.5 h diluted 1:3 in VB minimal medium. When the competence was reached (2.5 h), o.5-ml aliquots of this culture were mixed o.05-ml samples of transforming DNA and incubated for I h. All incubations
fresh peak with were
* Over 95 % of the thymidine was replaced by BUdR in the BUdR-labeled DNA strands synthesized under these conditions, since paper chromatographic analysis tt of acid-hydrolyzed BB DNA revealed less than 5 % of the normal thymine content; the NB DNA contained approximately equal amounts of 5-bromouracil and thymine. Biochim. Biophys. Acta, 72 (i963) 298-309
300
Z. OPARA-KUBINSKA, Z. KURYLO-BOROWSKA AND W. SZYBALSKI
carried out at 37 ° with vigorous shaking, o.o5-o.5-ml samples were plated on minimal agar and scored for prototrophic colonies after 24 h incubation at 37 °. Ultraviolet irradiation DNA dissolved in SSC at a concentration of 20 #g/ml was irradiated in quartz capillaries (inner diameter I ram) or in 6o-mm Petri dishes at a distance of 17 cm from a Westinghouse "Sterilamp" GI5TI8 emitting predominantly monochromatic ultraviolet light (2537A). The ultraviolet-light flux, which corresponded to 5" lO3 erg/mm2/min, was measured with a G.E. germicidal ultraviolet intensity meter (Haynes). No correction for ultraviolet self-absorption was necessary under these conditions of irradiation. Thermal denaturation of D N A DNA dissolved in DSC (pH = 7.8) at a concentration of 5 #g (normal DNA) or lO-15 #g (BUdR-labeled DNA) per ml was distributed in o.5-ml portions into I3-ml tubes and immersed in boiling water. Where indicated, 1% formaldehyde (HCHO) was present during denaturation. After 5 rain of heating, the samples were quenched by immersing in ice water. Density gradient centrifugation The general procedure for analytical density gradient centrifugation was similar to that described by MESELSON et al. 11. Approx. 0.2 ml (or 0.6 ml) of DNA solution (about I/~g per band) was added to 0.8 ml (or 0. 4 ml) of saturated CsC1 (or Cs,SO,) solution, and the final densities were adjusted to 1.7o (CsCI; normal DNA), 1.75 (CsC1; BUdR-labeled DNA), 1.42 (Cs•S04; normal DNA), or 1.45 (CszSO,; BUdRlabeled DNA), with the aid of refractometric measurements. The Cs,SO, density gradient was found very useful, since in this salt the buoyant density of the DNA is 6-times less dependent on its guanine + cytosine content, 3-times less dependent on the BUdR content, and shows a higher shift after denaturation than in the CsC1 gradient 12, Thus, when compared with the CsC1 gradient banding pattern, the bands in Cs2SO4 are relatively narrower, the effects of denaturation on the density are relatively more pronounced, and even at lower speed (31 41o rev./min) it is possible to band in the same cell both the native BUdR-free and the denatured BUdR-labeled DNA. Four I2-mm KeL-F cells were used for each run, and the photographs were taken first after 20 h at 47 44o rev./min and again after an additional 24-48 h at 31 41o rev./min. The details of the centrifugation procedure, which will be published elsewhere, are available on request in mimeographed form. The photographs were traced with a Spinco Analytrol Model RB equipped with a modified film microdensitometer attachment containing a gear-driven film plate. The synchronous motors in the Analytrol's paper drive and in the microdensitometer attachment were replaced by motors with 3-fold lower speed. The methods for preparative CsCI gradient centrifugation were described earlier xg. To obtain good separation not more than 30 #g DNA per band could be centrifuged Biochim. Biophys. Acta, 72 (1963) 298-309
IRRADIATION OF HALOGENATED TRANSFORMING D N A
3Ol
in one tube (Spinco S W - 3 9 L rotor; 3 ml CsCI solution + 2 ml paraffin oil; 35 ooo rev. per rain for 72 h at 20°).
Chromatography
The step-wise method of chromatography on the methylated albumin-kieselguhr (Hyflo Super Cel, Fisher Sci. Co.) column, according to SUEOKA A N D CHENG15, was employed throughout these studies. RESULTS
Sedimentation in density gradients
When centrifuged in the CsC1 or Cs,SO 4 gradient, native DNA extracted from B. subtilis sediments at the density of 1.7o 3 or 1.424 g/cm 3, respectively, forming a narrow band. Structural modifications of the DNA can often be easily detected by their effect on this banding pattern, e.g., a decrease in molecular weight or increase in density heterogeneity cause spreading of the band, while denaturation or BUdRfor-thymidine substitution increases the buoyant density of the DNA. The present study evaluates the effects of ultraviolet light and of subsequent denaturation on the sedimentation behaviour of BUdR-labeled and BUdR-free DNA. Effects of ultraviolet light on native D N A : Exposure of native DNA to ultraviolet light resulted in small increases in its buoyant density (Table I). Similar density changes, measured only in the CsC1 gradient ,were interpreted by MARMURet al. ~2 as indicating the denaturing effect of ultraviolet light. This simple interpretation would be difficult to reconcile with the present results, since the density increments observed in the Cs,S04 gradient were smaller than in the CsCI gradient, a result opposite to that observed for thermally denatured DNA (Table I). The band profile (band width) was not affected by ultraviolet light doses up to 25' lO4 erg/mm 2, although at still higher doses (125" lO4 erg/mm 2) pronounced band broadening was observed. The banding behaviour of native BUdR-labeled DNA was drastically altered even at the lower ultraviolet doses: the bands spread and the mean buoyant density TABLE
I
BUOYANT DENSITY OF NATIVE D N A AS AFFECTED BY ULTRAVIOLET LIGHT OR BY THERMAL DENATURATION N a t i v e D N A f r o m B. subtilis ( N N ) w a s e i t h e r i r r a d i a t e d w i t h i n d i c a t e d d o s e s o f u l t r a v i o l e t l i g h t o r d e n a t u r e d ( f o r i o m i n , a t lOO% i n D S C , q u e n c h e d a t o ° ) , a n d c e n t r i f u g e d i n CsC1 o r C s t S O 4 g r a d i e n t s , a s d e s c r i b e d i n MATERIALS AND METHODS. C o l i p h a g e T 6 D N A (CstSO4), Clostridium perfringens D N A (CsC1) o r n a t i v e B. subtilis D N A s e r v e d a s r e f e r e n c e d e n s i t y m a r k e r s .
Treatment
Thermal denaturation Irradiation with ultraviolet 5 " lO4 light 25 • i o 4 ( e r g / m m *) 125 • i o 4
Dem'ity ire'tease (g/cmt) ChSO,
CsCl
0.022
O.OI 5
o.ooi 0.004 o.oi6
o.oo 4 o.oio 0.036
Biochim. Biophys. Aaa, 72 (1963) 298-309
302
Z. OPARA-KUBINSKA, Z, KURYLO-BOROWSKA AND W. SZYBALSKI
].
BB
~^_~ i ,.8o=
NB
NN
i!i Cuntrol
/ !\ _Z. I\ ,,y==
ii! ov it:. ,.?=
,
Buoyant clensity in CsCI (g/crn 3) Fig. I. Microdensitometer tracings of photographs taken after 2o h of CsC1 equilibrium-densitygradient centrifugation (44 77° rev./min at 25°) of DNA extracted from B. subtilis cells grown for 4 h in the presence of ioo pg BUdR and 4 Pg FUd~ per ml, before (A, Control) and after ultraviolet irradiation with 5° kerg/mmz of germicidal ultraviolet light (B). The BB and NB peaks, represent the bifilarly and unifilarly (hybrid) BUdR-labeled DNA, respectively (solid lines), The NN peaks (dotted lines) were obtained by repeating the centrffugation after addition, directly to the cell, of 3 #1 (I #g) of nonlabeled B. subtilis DNA, serving as a reference density marker.
decreased (Fig. I). Both the spreading and the density decrease were more pronounced for bifilarly than for unifilarly BUdR-labeled DNA. Conditions oj denaturation: To facilitate the interpretation of the sedimentation patterns of the denatured DNA, the conditions for maximal and reproducible band shift upon denaturation were determined. It was known from earlier studies 23 that formaldehyde facilitates strand separation and prevents the renaturation of DNA during and subsequent to the cooling process. DNA was denatured by heating for 5 min at 96-9 80 at a concentration of 5/zg/ml in DSC, rapidly quenched at o °, and diluted 5-fold in CsC1 or Cs,S04 solutions of appropriate concentration. The effects of HCHO during both denaturation and centrifugation were evaluated, yielding four variants of the procedure. On the basis of the results presented in Fig. 2, variants A and C were selected for subsequent study, i.e. denaturation in the presence and absence of 1% HCHO and centrifugation without HCHO. It can be seen in Fig. 2 (B and D) that addi{ion of HCHO during centrifugation results in spreading of the band and decrease in the density differential between the native and denatured DNA. Although systematic studies on the effect of HCHO on the buoyant densities of native versus denatured DNA will be published elsewhere, it could be stated here that this apparent decrease in density of denatured DNA at increasing concentrations of HCHO in Cs2SO4 solution, measured versus a native DNA density marker, provides another simple experimental tool for the detection of the denatured state of DNA. Effects of ultraviolet light on the denaturation of D N A : Samples of NN, NB, and BB DNA were irradiated with ultraviolet light at three dose levels: I . lO4, 5" lO4, and 25" lO4 erg/mm 2. The DNA samples were then denatured in the presence and in the absence of 1% HCHO. The results of centrifugation of these samples in the Cs2SO4 gradient are presented in Figs. 3-5. Nonlabeled, nonirradiated DNA, which in the native state bands at the density Biochim. Biophys. Acta, 72 (1963) 298-309
IRRADIATION OF HALOGENATED TRANSFORMING
fii
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DNA
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density~ in Cs2SC)4 (g/crr?) Buoyant
f
Fig. 2. Microdensitometer tracings representing the buoyant density distribution of B. subtilis DlgA denatured (Den.) in the presence (A,B) or absence (C,D) of I % HCHO (for 5 min at 9 6 4 8 °, in DSC) and centrifuged in the CssSO 4 equilibrium-density gradient (31 41o rev./min at 28 h, for 25 °) containing o (C), o.2% (A), or 1.2% (B,D) HCHO. The denatured DNA is represented by the solid lines, while the NN peaks (dotted Lines) were obtained by repeating the centrifugation after addition of native B. subtilis DNA (I/~g) directly to the cells. The density increments correspond to o.o29 (A), O.Ol5 (B), o.o22 (C), and O.Ol4 g/cm s (D).
I
J
I
14120
i
Buoyant density in C%SO4 (9/cm3)
Fig. 3. Microdensitometer tracings representing the buoyant density distribution of B. subtilis DNA nonirradiated (A,B) or ultraviolet irradiated with 5° (C.D) or 250 {E,F) kerg/mm = and subsequently denatured in the presence (A,C,E) or in the absence (B,D,F) of 1% HCHO. The conditions of denaturation and centrifugation are the same as in Fig. 2. The dotted lines represent the position of the native DNA marker (NN). The shaded areas indicate schematically the position occupied by the "cross-linked" DNA.
of 1.424 g/cm s, becomes heavier by 0.029 or 0.022 g/cm s when heat-denatured in the presence (Fig. 3A) or in the absence (Fig. 3 B) of 1% HCHO, respectively. When ultraviolet-light-irradiated DNA is denatured, approx. 60% follows roughly the same pattern, while the behaviour of the remaining 40% depends on whether HCHO was present or absent during denaturation. In the absence of HCHO, this 40% fraction of the DNA bands at a density very close to that characteristic of the native state (Fig. 3D), even when cooling was very rapid so that thermal renaturation would not be operative. This behaviour of ultraviolet-light-irradiated DNA was interpreted as evidence for inter-strand cross-linkingg, ~, since covalent links between the complementary strands would not permit complete strand separation upon heating, with close to complete restoration of original structure even upon rapid quenching. This renaturation-like phenomenon was less efficient when HCHO was present during Biochim. Biophys. Aaa, 72 (!963) 298-309
304
Z. OPARA-KUBINSKA, Z. KURYLO-BOROWSKA AND W. SZYBALSKI
denaturation and thermal quenching, with the density" of the "cross-linked" DNA fraction approaching that of fully denatured DNA (Fig. 3C). After a 5-times higher ultraviolet dose, this cross-linked DNA increased from approx. 4 ° to 7o%, while the general banding behaviour was essentially the same (Figs. 3E and F). NB ("hybrid") DNA, purified by preparative CsCl-gradient fractionation sedimented at a density of approx. 1.452 g/cm 3 (band NB) in the Cs~SO4 gradient (Fig. 4A). Upon denaturation this DNA dissociated into dN and dB, of densities 1.446 and 1.525 g/cm 3, respectively (Fig. 4B). The denaturation process was carried out in the absence of HCHO, since the presence of HCHO caused an increase in the density of dN (cf. Fig. 2A versus Fig. 2C) and thus an overlapping of the dN and the hybrid (NB) DNA bands.
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1.420
Buoyont density in Cs2SO4 (g/era 3} Fig. 4. Microdensitometer tracings representing t h e b u o y a n t d e n s i t y distribution of t h e unifilarly B U d R - l a b e l e d ( " h y b r i d " ) D N A (NB), native (n) nonirradiated (A), and d e n a t u r e d (d) w i t h o u t (B) or after previous exposure to i o (C) a n d 5 ° (B) k e r g / m m 2 of ultraviolet light. The shaded areas indicate t h e absorbance of t h e " h y b r i d " D N A a n d o f its d e n a t u r a t i o n products. The peaks of t h e n a t i v e NN a n d o f t h e d e n a t u r e d d N nonlabeled B. subtilis D N A are indicated b y t h e d o t t e d (A-E) and broken (E) lines, respectively. The conditions of d e n a t u r a t i o n and centrifugation are t h e same as in Fig. 2C.
No dN or dB bands were detectable upon denaturation of NB previously irradiated with 5' lO4 erg] ram2 of germicidal ultraviolet light (Fig. 4D). Even at one fifth the ultraviolet dose (Fig. 4C) no dN band was visible and there was only a suggestion of the presence of a small quantity of a highly heterogeneous or degraded
" Centrifuged in CstSO 4 solution containing 0.2 % HCHO. I n t h e CsC1 gradient "'cross-linked" D N A b a n d s at a d e n s i t y very close to t h a t of n a t i v e DNA, i n d e p e n d e n t o f w h e t h e r H C H O was present during d e n a t u r a t i o n (1% H C H O during denaturation, 0.2% H C H 0 during centrifugation).
Biochim. Biophys. •aa, 72 (1963) 298-309
IRRADIATION OF HALOGENATED TRANSFORMING
DNA
305
dB-like material. Upon denaturation, this ultraviolet "cross-linked"* hybrid DNA spread wider and became heavier than the original NB band (Figs. 4 C and 4D), which indicates partial collapse of the native structure of NB in the absence of true strand separation, characteristic of nonirradiated NB material (Fig. 4B). BB DNA was also included in this study, but it was not separated from the NB of an ultraviolet-light-irradlated mixture of NB and BB DNA (Fig. 5D) fails to produce bands corresponding to the denatured dB and dN DNA's (Fig. 5B), suggesting that ultraviolet light causes cross-links not only in NN but also in NB and BB molecules. The shift toward higher density and the band spreading is more pronounced in the bifilarly labeled (BB) DNA than in the NB material irradiated and denatured under identical conditions (Fig. 5D).
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Buoyant density in Cs2SO 4 (g/cm 3) Fig. 5. Microdensitometer tracings representing the buoyant density distribution of a mixture of unifilarly (NB) and bifilarly (BB) BUdR-labeled DNA, native (n) nonirradiated (A), denatured (d) nonirradiated (B), native irradiated (C), and denatured after irradiation with 5° kerg/mm 2 of ultraviolet light. The peaks of the nonlabeled B. subtilis DNA marker (NN), are indicated by the dotted lines. The conditions of denaturation and centrifugation are the same as in Fig. 2C.
It also is notable that the increment in the density of the dB v e r s u s the BB band is almost twice as high as the density increment observed upon denaturation of the nonlabeled native DNA (dN v e r s u s NN). " An alternative interpretation of Fig. 4C (and Fig. 4 D) could be as follows : NB DNA is not cross-linked by ultraviolet irradiation, but the two shaded peaks correspond to the separated dB (wide-spread peak on the left) and dN components. Peak dB is shifted to the right because of photochemical debromination and spreads widely as the combined result of degradation and the inhomogeneity of dehalogenation. Peak dN is heavier than in Fig. 4B, partially as the result of ultraviolet light irradiation and partially from acquiring the bromine label coming off the dB strand. This interpretation, although less likely than the cross-linking hypothesis, has as yet not been rigorously excluded by experiments employing 14C-labeled B U d R in conjunction with radioactivity determination in the separated bands. Biochim. Biophys. Acta, 72 (1963) 298-3o 9
300
Z. OPARA-KUBINSKA,Z. KURYLO-BOROWSKAAND W. SZYBALSKI
Chromatographic analysis Two types of D N A were employed in this experiment : NN and BU DNA's. The Cs~SO 4 gradient sedimentation pattern of the latter is presented in Fig. 5 A. These D N A ' s were extracted, deproteinized, and freed from R N A as described in EXPERIMENTAL, and subsequently subjected to a preliminary chromatographic purificationon a m e t h y l a t e d albumin-kieselguhr column 18,14 prepared according to SUEOKA AND CHENG 1S. The 5 × I-cm column was charged with 8 ml of D N A (A,e 0 = 2.6) dissolved in 0.075 M NaC1. The primary elution patterns of the NN D N A and the BU D N A were essentially the same, with the BU D N A giving a somewhat broader distribution. Only the fractions eluting with 0.6 and 0. 7 M NaC1 (peak of DNA) were saved and dialyzed against 0.075 M NaC1. These chromatographically purified D N A ' s were employed in subsequent experiments, illustrated in Fig. 6. A 4.0 × o.6-cm column was charged with 5 ml o f D N A (A2~0 = 0.3) dissolved in 0.075 M NaC1, and eluted with 3 ml portions varying from o.I to 1. 5 M NaC1 in o.I M steps. The D N A content of the eluted fractions was estimated from the Az80. Again it is apparent t h a t the chromatographically purified NN (Fig. 6A) and I00 60
NN-D
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Fig. 6. Chromatographic elution patterns of nonlabeled (N-DNA) and of BUdR-labeld (BU) DNA, nonirradiated (A,B) or irradiated (C,D) with 5° kerg/mm*. The methylated albuminHyflo Super Cel column was charged with DNA dissolved in 0.o75 M NaC1; E = % of A ,60 in the effluent not retained by the column during the charging process. Stepwise elution in o. i M increments from o.I to i.o M NaC1, and final elution with 1.5 M NaC1.
BU D N A ' s (Fig. 6B) exhibited essentially the same elution patterns, although the total recovery of NN (88%) was higher than of the BU (70%). Aliquots of chromatographically purified D N A ' s were irradiated with ultraviolet light (5 .IO* erg/mm ~) and subjected to chromatographic analysis. Irradiation of normal D N A did not seem to effect grossly its chromatographic properties, reducing, however, its total recovery to 70%. Samples of irradiated DNA, collected before c h r o m a t o g r a p h y and from the 0.6 M NaC1 eluate (Fig. 6C), were denatured and
Biochim. Biophys. Acta, 72 (I963) 298-309
IRRADIATION OF HALOGENATEDTRANSFORMINGD N A
307
centrifuged in the Cs2SO 4 gradient under conditions identical to those represented in Fig. 3 D. Essentially the same centrifugation pattern was obtained, indicating t h a t the chromatographic behaviour of the cross-linked D N A was similar to t h a t of the nonirradiated N N D N A . Irradiation of BU D N A with the same dose (5" IO4 erg/mm 2) resulted in its rather firm a t t a c h m e n t to the column; only 12 % was eluted and only at molarities as high as 1.o-1.5 M (Fig. 6D). F u r t h e r elution with neutral 2 M NaC1 did not displace any D N A and with acidified 2 M NaC1 (pH 4) only an additional 6%. During charging of the column 4 % of the irradiated BU D N A was not retained (Fig. 6D), in contrast to the complete retention of the nonirradiated BU D N A (Fig. 6B). The specific transforming activity (sTA) of all the nonirradiated and irradiated samples was determined, before c h r o m a t o g r a p h y and after chromatography. The TABLE II T R A N S F O R M I N G A C T I V I T Y OF B U d R - F R E E
AND B U d R - L A B E L E D
DNA
A S A F F E C T E D B Y U L T R A V I O L E T L I G H T AND CHROMATOGRAPHIC FRA CT I O N A T I O N
Native DNA's from B. subtilis (ind+), BUdR-free (NN) or BUdR-labeled (BU = NB + BB, cf. Fig. i), were either irradiated with 5° kerg/mm 2 of ultraviolet light or nonirradiated and chromatographed on a methylated albumin column 15. Transforming activity (indole marker) was determined for the pre-chromatographed DNA (starting material), for the material Hot absorbed by the column during the charging process (E, o.075 M NaC1), and for the 0.6 M and i.o M NaC1 eluates (cf. Fig. 6). Transformants per I~g D N A Nonirradiated NN
Starting material
i.o. lOs
0.8. lO8
o
o
E Column eluates
0.6 M I.O M
BU
i.o. -
-
10 6
0.8" 10 6
-
-
Ultraviolet-irradiated NN
BU
250
20
o
o
400
--
-
-
o
results are summarized in Table II. The small difference in sTA between the NN and the B U D N A ' s used in this particular experiment cannot be considered significant, since the sTA of different batches of B U D N A equaled the sTA of normal D N A samples. Irradiated normal D N A retained 0.025% of its sTA; 7 0 % of this D N A eluting with 0.6-0. 7 M NaCI seemed to exhibit a 6 0 % higher sTA. Irradiated BU D N A retained only 0.0025% of its sTA and none of it could be found in the column eluate. In all the chromatographic experiments, the material eluting at molarities below 0.5 had no transforming activity. DISCUSSION AND CONCLUSIONS The change in the radiobiological properties of transforming D N A as a result of B U d R incorporation is paralleled b y changes in its radiochemical properties. A drastic difference in the chromatographic behaviour of the ultraviolet-irradiated B U D N A Biochim. Biophys. Acta, 72 (1963) 298-3o9
308
Z. OPARA-KUBINSKA, Z. KURYLO-BOROWSKA AND W. SZYBALSKI
(Fig. 6D), as compared with the NN DNA (Fig. 6C), indicates a high affinity of the former for methylated albumin. It would not be surprising if a similar reaction between BU DNA and basic nuclear proteins (cf. SMITH1°) were partially responsible for the dramatic ultraviolet sensitization by BUdR. Although no pronounced differences were observed in the chromatographic behaviour of the bulk of the NN, BU, and ultraviolet irradiated (5" lO4 erg/mm 2) NN DNA's, the step-wise elution employed was not designed to detect more subtle differences. Irradiation of NN DNA with higher ultraviolet doses results also in increased affinity for the column 24. The changes in the equilibrium-density-gradient centrifugation pattern as a result of ultraviolet irradiation could be divided into two operational classes: (a) those observed in native DNA and (b) those revealed after denaturation. I. The effect of moderate ultraviolet doses on the buoyant density of native BUdR-free DNA is rather small, with the density increases higher when measured in the CsC1 gradient than in the Cs2SO4 gradient. This indicates that ultraviolet lightaffected density changes are different than those produced by thermal denaturation, since the latter causes higher density increases in Cs2SO4 than in CsC1. This result points to the necessity of applying more than one density gradient medium when interpreting the effects of various agents on the basis of the buoyant density changes. In contrast with NN DNA, moderate ultraviolet doses result in pronounced widening of tile NB and BB bands and their shift to lower density. One interpretation of these changes would be the photochemical dehalogenation of the BU DNA 7, which would both decrease the buoyant density (this change probably partially compensated for by the ultraviolet-light-effected density increase similar to that observed with NN DNA) and magnify the heterogeneity with respect to density. The latter phenomenon and/or ultraviolet-light caused degradation of the BU DNA would result in spreading of the bands. 2. The ultraviolet elicited changes in the banding pattern of denatured DNA could be best interpreted as formation of cross-linkedg, 22 or "reversible ''z5 DNA. BU DNA is again much more sensitive to this modification than NN DNA (Figs. 3, 4,5 ). Since BB DNA used in the present study was practically free of thymine it would be difficult to interpret this cross-linking as caused solely by thymine dimerization across the strands. The dimerization of DNA-incorporated BUdR, the role of thymine dimerization in the cross-linking of complementary DNA strands, and the biological consequences.of the cross-linking are still open questions. ACKNOWLEDGEMENTS
This work was supported by Grant G-I8165 from the National Science Foundation. We are indebted to Dr. E. H. SZYBALSKAfor her editorial help and to Miss M. KING and to Mr. L. FENTON for the technical assistance. REFERENCES 1 Z. OPARA-KUBINSKA, Z. LORKIEWICZ AND W. SZYBALSKI, Biochem. Biophys. Res. Commun., 4 (1961) 288. 2 R. L. SINSHEIMER, Radiation Res., 6 (1957) i z i . s D. SHUGAR, in E. CHARGAFF AND J. N. DAVlDSON, The Nucleic Acids, Vol. 3, Academic Press, New York, i96o, p. 39.
Biochim. Biophys. Acta, 72 (1963) 298-309
IRRADIATION OF HALOGENATED TRANSFORMING
DNA
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4 R. BEUKERS AND W. BERENDS, Biochim. Biophys. Acta, 49 (I96I) 181. 5 D. WEINBLUM, Thesis, Technical University, Berlin, 196L 6 A. WACKER, J. CMm. Phys., 58 (1961) lO41. ? A. WACKER, H. D. MENNIGMANN AND W. SZYBALSKI, Nature. 196 (1962) 685. s W. ,BERENDS, J. Chim. Phys., 58 (1961) IO34. t j . MARMUR AND L. GROSSMAN, Proc. Natl. Acad. Sci. U.S., 47 (1961) 778. x0 K. C. SMITH, Biochem. Biophys. Res. Commun., 8 (1962) 157. 11 M. MESELSON, V. W. STAHL AND J. VINOGRAD, Prvc. Natl. Acad. Sci. U.S., 43 (1957) 581. x~ R. L. ERIKSON AND W. SZYBALSKI, Phage Information Service, (1962) 2, Cancer Research 23 (1963) 122; Abstracts Biophys. Soc. 7th Ann. Meeling (1963) W A 8. la L. S. LERMAN, Biochim. Biophys. Acta, 18 (1955) 132x* j . D. MANDELL AND A. D. HERSHEY, Anat. Bioehem., I (196o) 66. 15 N. SUEOKA AND T.-Y. CHENG, J. Mol. Biol., 4 (1962) 161. 15 W . SZYBALSKI, Z. OPARA-KUBINSKA, Z. LORKIEWICZ, E. EPHRATI-ELIZUR AND S. ZAMENHOF, Nature, 188 (196o) 743. aT H. J. VOGEL AND D. BONNER, J. Biol. Chem., 218 (1956) 97. 18 W. SZYBALSKI AND H. D. MENNIGMANN, Anal. Biochem., 3 (1962) 267. t9 W. SZYBALSKI, Experientia, 16 (i96o) 164. io W. SZYBALSKI, The Molecular Basis of Neoplasia, U n i v . o f T e x a s Press, A u s t i n , 1962, p. 147. tl B. DJORDJEVIC AND W. SZYBA~LSKI,J. Exptl. Med., 112 (196o) 509. ss j . MARMUR, W. F. ANDERSON, L. MATTHEWS, K. BERNS, E. GAJEWSKA, D. LANE AND P. DOTV, J. Cell, Comp. Physiol., Suppl. I, 58 (1961) p. 33u C. A. THOMAS~ JR. AND K. I. BERN-S, J. Mol. Biol,, 4 (1962) 309 • $4 A. D. HERSHEY, personal c o m m u n i c a t i o n . s5 E. P. GEIDUSCHEK, Prvc. Natl. Acad. Sci. U.S., 47 (1961) 95 °.
Biochim. Biophys. Acta, 72 (I963) 298-309
BIOCHIMICA ET BIOPHYSICA ACTA
8263 GENETIC TRANSFORMATION STUDIES III. EFFECT OF ULTRAVIOLET LIGHT ON THE MOLECULAR PROPERTIES OF NORMAL AND HALOGENATED DEOXYRIBONUCLEIC ACID ZOFIA O P A R A - K U B I N S K A , ZOFIA KURYLO-BOROWSKA AND WACLAW SZYBALSKI
McArdle Memorial Laboratory, University of Wisconsin, Madison, Wisc. (U.S.A.) (Received November 3oth, 1962)
SUMMARY
The transforming activity of 5-bromodeoxyuridine-labeled DNA is more sensitive to ultraviolet light than that of normal (non-labeled) DNA. The more rapid ultravioletirradiation inactivation of 5-bromodeoxyuridine labeled DNA, as compared with nonlabeled, is reflected also by (a) greatly increased affinity of ultraviolet-irradiated 5-bromodeoxyuridine-labeled DNA for the methylated albumin colunm, (b) decrease in density (dehalogenation) and very pronounced band spreading (degradation, increased heterogeneity), as revealed by CsC1 equilibrium-density centrifugation of ultraviolet-irradiated native 5-bromodeoxyuridine-labeled DNA's, and (c) marked cross-linking between complementary strands of 5-bromodeoxyuridine-labeled DNA, as determined with ultraviolet-irradiated and denatured material centrifuged in the Cs2SO4 density gradient. INTRODUCTION
It was demonstrated that BUdR-labeled transforming DNA is more sensitive to ultraviolet light (2537~) than BUdR-free (normal) DNA 1. Since this finding indicated that the ultraviolet sensitivity of intact cells is principally governed by the sensitivity of their DNA component 1, it became important to assess the molecular basis of ultraviolet-light effects on normal versus BUdR-labeled DNA. Several chemical events have been detected during ultraviolet irradiation of normal DNA or its components, including water addition across the 5-6 double bond of cytosine 2,3, and thymine dimerization*. Substitution of a 5-halouracil for thymine would obviously eliminate the latter reaction, which, however, could be replaced by 5-halouracil dimerization, a not generally accepted reaction s, and/or by photochemical dehalogenation6, 7. Whichever elementary photochemical reactions occur, the result Abbreviations: BUdR, 5-bromodeoxyuridine; FUdR, 5-fluorodeoxyuridine; NN DNA, normal native DNA; dN, normal denatured DNA; NB DNA, native unifilarly BUdR-labeled ("hybrid") DNA (thymidine replaced by B U d R in one strand only); BB DNA. native bifilarly BUdR-labeled DNA (thymidine in both strands replaced by BUdR); BU DNA, mixture of NB and BB; dB, denatured BUdR-labeled DNA components (strands); SSC, o.15 M NaC1 + O.Ol5 M Trisodium citrate; DSC, o.oi 5 M NaC1 + o.ool 5 M Trisodium citrate; sTA, specific transforming activity (number of transformant colonies per ~ug of transforming DNA).
Biochim. Biophys. Acta, 72 (1963) 298-3o9
IRRADIATION OF HALOGENATED TRANSFORMING
DNA
299
could be a multitude of changes in the polymeric structure of the DNA, including partial strand separation, single- and double-chain breaks, intra-strande, s, inter-strand 9 and protein-to-DNA crosslinking 1°. Many of these changes could be quantitatively evaluated by measuring the buoyant densities of native and denatured DNA or by assessing their chromatographic behaviour. CsCP 1 and Cs2S04 TM density-gradient centrifugation and chromatography on the methylated albumin column ~a-15 were employed as the primary methods for comparing the ultraviolet induced macromolecular changes in normal versus BUdR-labeled DNA. These physico-chemical studies were accompanied by parallel determinations of the biological activity of the DNA. EXPERIMENTAL
Strains
The prototrophic and indole-requiring strains of Bacillus subtilis were the same as previously used by SZYBALSKIet al. le. Media
Nutrient broth, 0.8% (Difco); VB minimal liquid m e d i u m - stock solution (MgSOi.7H20, IO g; citric acid.H,O, IOO g; K2HPO 4, 500 g; NaNH4HPO4"H,O, I75 g; water, I 1) diluted 1:50 and supplemented with 0.5% glucose17; S minimal solid medium (K,HPO a, 14 g; KH,PO,, 6 g; Trisodium citrate. 2 H20, i g; MgSO,. 7 H20, 0.2 g; (NH,),SO,, 2 g; glucose, 5 g; agar, I6 g; water, I 1). Isolation of D N A
DNA was isolated from cells lysed with lysozyme and sodium laurylsulfate, deproteinized by shaking with chloroform and butanol, and freed of RNA16, TM. The final purification steps included preparative CsCl-gradient fractionation 19, and column chromatography, as described in the following sections. To prepare the unifilarly BUdR-labeled DNA (NB) a nutrient broth culture was diluted 1:6 in VB minimal medium supplemented with IOO/zg BUdR and 4 Pg FUdR per ml and vigorously agitated for 2 h at 37 °. Any admixture of NN or BB was removed by preparative CsCl-gradient centrifugation. By prolonging the growth period in the presence of BUdR and FUdR to 4-5 h, a mixture of bifilarly and unifilarly BUdR-labeled DNA was obtainedX6, 20, the CsC1 banding pattern of which is presented in Fig. IA*. Transformation procedure
An overnight culture of indole-requiring cells was first diluted 1:3 in nutrient broth, and after 2.5 h diluted 1:3 in VB minimal medium. When the competence was reached (2.5 h), o.5-ml aliquots of this culture were mixed o.05-ml samples of transforming DNA and incubated for I h. All incubations
fresh peak with were
* Over 95 % of the thymidine was replaced by BUdR in the BUdR-labeled DNA strands synthesized under these conditions, since paper chromatographic analysis tt of acid-hydrolyzed BB DNA revealed less than 5 % of the normal thymine content; the NB DNA contained approximately equal amounts of 5-bromouracil and thymine. Biochim. Biophys. Acta, 72 (i963) 298-309
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Z. OPARA-KUBINSKA, Z. KURYLO-BOROWSKA AND W. SZYBALSKI
carried out at 37 ° with vigorous shaking, o.o5-o.5-ml samples were plated on minimal agar and scored for prototrophic colonies after 24 h incubation at 37 °. Ultraviolet irradiation DNA dissolved in SSC at a concentration of 20 #g/ml was irradiated in quartz capillaries (inner diameter I ram) or in 6o-mm Petri dishes at a distance of 17 cm from a Westinghouse "Sterilamp" GI5TI8 emitting predominantly monochromatic ultraviolet light (2537A). The ultraviolet-light flux, which corresponded to 5" lO3 erg/mm2/min, was measured with a G.E. germicidal ultraviolet intensity meter (Haynes). No correction for ultraviolet self-absorption was necessary under these conditions of irradiation. Thermal denaturation of D N A DNA dissolved in DSC (pH = 7.8) at a concentration of 5 #g (normal DNA) or lO-15 #g (BUdR-labeled DNA) per ml was distributed in o.5-ml portions into I3-ml tubes and immersed in boiling water. Where indicated, 1% formaldehyde (HCHO) was present during denaturation. After 5 rain of heating, the samples were quenched by immersing in ice water. Density gradient centrifugation The general procedure for analytical density gradient centrifugation was similar to that described by MESELSON et al. 11. Approx. 0.2 ml (or 0.6 ml) of DNA solution (about I/~g per band) was added to 0.8 ml (or 0. 4 ml) of saturated CsC1 (or Cs,SO,) solution, and the final densities were adjusted to 1.7o (CsCI; normal DNA), 1.75 (CsC1; BUdR-labeled DNA), 1.42 (Cs•S04; normal DNA), or 1.45 (CszSO,; BUdRlabeled DNA), with the aid of refractometric measurements. The Cs,SO, density gradient was found very useful, since in this salt the buoyant density of the DNA is 6-times less dependent on its guanine + cytosine content, 3-times less dependent on the BUdR content, and shows a higher shift after denaturation than in the CsC1 gradient 12, Thus, when compared with the CsC1 gradient banding pattern, the bands in Cs2SO4 are relatively narrower, the effects of denaturation on the density are relatively more pronounced, and even at lower speed (31 41o rev./min) it is possible to band in the same cell both the native BUdR-free and the denatured BUdR-labeled DNA. Four I2-mm KeL-F cells were used for each run, and the photographs were taken first after 20 h at 47 44o rev./min and again after an additional 24-48 h at 31 41o rev./min. The details of the centrifugation procedure, which will be published elsewhere, are available on request in mimeographed form. The photographs were traced with a Spinco Analytrol Model RB equipped with a modified film microdensitometer attachment containing a gear-driven film plate. The synchronous motors in the Analytrol's paper drive and in the microdensitometer attachment were replaced by motors with 3-fold lower speed. The methods for preparative CsCI gradient centrifugation were described earlier xg. To obtain good separation not more than 30 #g DNA per band could be centrifuged Biochim. Biophys. Acta, 72 (1963) 298-309
IRRADIATION OF HALOGENATED TRANSFORMING D N A
3Ol
in one tube (Spinco S W - 3 9 L rotor; 3 ml CsCI solution + 2 ml paraffin oil; 35 ooo rev. per rain for 72 h at 20°).
Chromatography
The step-wise method of chromatography on the methylated albumin-kieselguhr (Hyflo Super Cel, Fisher Sci. Co.) column, according to SUEOKA A N D CHENG15, was employed throughout these studies. RESULTS
Sedimentation in density gradients
When centrifuged in the CsC1 or Cs,SO 4 gradient, native DNA extracted from B. subtilis sediments at the density of 1.7o 3 or 1.424 g/cm 3, respectively, forming a narrow band. Structural modifications of the DNA can often be easily detected by their effect on this banding pattern, e.g., a decrease in molecular weight or increase in density heterogeneity cause spreading of the band, while denaturation or BUdRfor-thymidine substitution increases the buoyant density of the DNA. The present study evaluates the effects of ultraviolet light and of subsequent denaturation on the sedimentation behaviour of BUdR-labeled and BUdR-free DNA. Effects of ultraviolet light on native D N A : Exposure of native DNA to ultraviolet light resulted in small increases in its buoyant density (Table I). Similar density changes, measured only in the CsC1 gradient ,were interpreted by MARMURet al. ~2 as indicating the denaturing effect of ultraviolet light. This simple interpretation would be difficult to reconcile with the present results, since the density increments observed in the Cs,S04 gradient were smaller than in the CsCI gradient, a result opposite to that observed for thermally denatured DNA (Table I). The band profile (band width) was not affected by ultraviolet light doses up to 25' lO4 erg/mm 2, although at still higher doses (125" lO4 erg/mm 2) pronounced band broadening was observed. The banding behaviour of native BUdR-labeled DNA was drastically altered even at the lower ultraviolet doses: the bands spread and the mean buoyant density TABLE
I
BUOYANT DENSITY OF NATIVE D N A AS AFFECTED BY ULTRAVIOLET LIGHT OR BY THERMAL DENATURATION N a t i v e D N A f r o m B. subtilis ( N N ) w a s e i t h e r i r r a d i a t e d w i t h i n d i c a t e d d o s e s o f u l t r a v i o l e t l i g h t o r d e n a t u r e d ( f o r i o m i n , a t lOO% i n D S C , q u e n c h e d a t o ° ) , a n d c e n t r i f u g e d i n CsC1 o r C s t S O 4 g r a d i e n t s , a s d e s c r i b e d i n MATERIALS AND METHODS. C o l i p h a g e T 6 D N A (CstSO4), Clostridium perfringens D N A (CsC1) o r n a t i v e B. subtilis D N A s e r v e d a s r e f e r e n c e d e n s i t y m a r k e r s .
Treatment
Thermal denaturation Irradiation with ultraviolet 5 " lO4 light 25 • i o 4 ( e r g / m m *) 125 • i o 4
Dem'ity ire'tease (g/cmt) ChSO,
CsCl
0.022
O.OI 5
o.ooi 0.004 o.oi6
o.oo 4 o.oio 0.036
Biochim. Biophys. Aaa, 72 (1963) 298-309
302
Z. OPARA-KUBINSKA, Z, KURYLO-BOROWSKA AND W. SZYBALSKI
].
BB
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Buoyant clensity in CsCI (g/crn 3) Fig. I. Microdensitometer tracings of photographs taken after 2o h of CsC1 equilibrium-densitygradient centrifugation (44 77° rev./min at 25°) of DNA extracted from B. subtilis cells grown for 4 h in the presence of ioo pg BUdR and 4 Pg FUd~ per ml, before (A, Control) and after ultraviolet irradiation with 5° kerg/mmz of germicidal ultraviolet light (B). The BB and NB peaks, represent the bifilarly and unifilarly (hybrid) BUdR-labeled DNA, respectively (solid lines), The NN peaks (dotted lines) were obtained by repeating the centrffugation after addition, directly to the cell, of 3 #1 (I #g) of nonlabeled B. subtilis DNA, serving as a reference density marker.
decreased (Fig. I). Both the spreading and the density decrease were more pronounced for bifilarly than for unifilarly BUdR-labeled DNA. Conditions oj denaturation: To facilitate the interpretation of the sedimentation patterns of the denatured DNA, the conditions for maximal and reproducible band shift upon denaturation were determined. It was known from earlier studies 23 that formaldehyde facilitates strand separation and prevents the renaturation of DNA during and subsequent to the cooling process. DNA was denatured by heating for 5 min at 96-9 80 at a concentration of 5/zg/ml in DSC, rapidly quenched at o °, and diluted 5-fold in CsC1 or Cs,S04 solutions of appropriate concentration. The effects of HCHO during both denaturation and centrifugation were evaluated, yielding four variants of the procedure. On the basis of the results presented in Fig. 2, variants A and C were selected for subsequent study, i.e. denaturation in the presence and absence of 1% HCHO and centrifugation without HCHO. It can be seen in Fig. 2 (B and D) that addi{ion of HCHO during centrifugation results in spreading of the band and decrease in the density differential between the native and denatured DNA. Although systematic studies on the effect of HCHO on the buoyant densities of native versus denatured DNA will be published elsewhere, it could be stated here that this apparent decrease in density of denatured DNA at increasing concentrations of HCHO in Cs2SO4 solution, measured versus a native DNA density marker, provides another simple experimental tool for the detection of the denatured state of DNA. Effects of ultraviolet light on the denaturation of D N A : Samples of NN, NB, and BB DNA were irradiated with ultraviolet light at three dose levels: I . lO4, 5" lO4, and 25" lO4 erg/mm 2. The DNA samples were then denatured in the presence and in the absence of 1% HCHO. The results of centrifugation of these samples in the Cs2SO4 gradient are presented in Figs. 3-5. Nonlabeled, nonirradiated DNA, which in the native state bands at the density Biochim. Biophys. Acta, 72 (1963) 298-309
IRRADIATION OF HALOGENATED TRANSFORMING
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Fig. 3. Microdensitometer tracings representing the buoyant density distribution of B. subtilis DNA nonirradiated (A,B) or ultraviolet irradiated with 5° (C.D) or 250 {E,F) kerg/mm = and subsequently denatured in the presence (A,C,E) or in the absence (B,D,F) of 1% HCHO. The conditions of denaturation and centrifugation are the same as in Fig. 2. The dotted lines represent the position of the native DNA marker (NN). The shaded areas indicate schematically the position occupied by the "cross-linked" DNA.
of 1.424 g/cm s, becomes heavier by 0.029 or 0.022 g/cm s when heat-denatured in the presence (Fig. 3A) or in the absence (Fig. 3 B) of 1% HCHO, respectively. When ultraviolet-light-irradiated DNA is denatured, approx. 60% follows roughly the same pattern, while the behaviour of the remaining 40% depends on whether HCHO was present or absent during denaturation. In the absence of HCHO, this 40% fraction of the DNA bands at a density very close to that characteristic of the native state (Fig. 3D), even when cooling was very rapid so that thermal renaturation would not be operative. This behaviour of ultraviolet-light-irradiated DNA was interpreted as evidence for inter-strand cross-linkingg, ~, since covalent links between the complementary strands would not permit complete strand separation upon heating, with close to complete restoration of original structure even upon rapid quenching. This renaturation-like phenomenon was less efficient when HCHO was present during Biochim. Biophys. Aaa, 72 (!963) 298-309
304
Z. OPARA-KUBINSKA, Z. KURYLO-BOROWSKA AND W. SZYBALSKI
denaturation and thermal quenching, with the density" of the "cross-linked" DNA fraction approaching that of fully denatured DNA (Fig. 3C). After a 5-times higher ultraviolet dose, this cross-linked DNA increased from approx. 4 ° to 7o%, while the general banding behaviour was essentially the same (Figs. 3E and F). NB ("hybrid") DNA, purified by preparative CsCl-gradient fractionation sedimented at a density of approx. 1.452 g/cm 3 (band NB) in the Cs~SO4 gradient (Fig. 4A). Upon denaturation this DNA dissociated into dN and dB, of densities 1.446 and 1.525 g/cm 3, respectively (Fig. 4B). The denaturation process was carried out in the absence of HCHO, since the presence of HCHO caused an increase in the density of dN (cf. Fig. 2A versus Fig. 2C) and thus an overlapping of the dN and the hybrid (NB) DNA bands.
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Buoyont density in Cs2SO4 (g/era 3} Fig. 4. Microdensitometer tracings representing t h e b u o y a n t d e n s i t y distribution of t h e unifilarly B U d R - l a b e l e d ( " h y b r i d " ) D N A (NB), native (n) nonirradiated (A), and d e n a t u r e d (d) w i t h o u t (B) or after previous exposure to i o (C) a n d 5 ° (B) k e r g / m m 2 of ultraviolet light. The shaded areas indicate t h e absorbance of t h e " h y b r i d " D N A a n d o f its d e n a t u r a t i o n products. The peaks of t h e n a t i v e NN a n d o f t h e d e n a t u r e d d N nonlabeled B. subtilis D N A are indicated b y t h e d o t t e d (A-E) and broken (E) lines, respectively. The conditions of d e n a t u r a t i o n and centrifugation are t h e same as in Fig. 2C.
No dN or dB bands were detectable upon denaturation of NB previously irradiated with 5' lO4 erg] ram2 of germicidal ultraviolet light (Fig. 4D). Even at one fifth the ultraviolet dose (Fig. 4C) no dN band was visible and there was only a suggestion of the presence of a small quantity of a highly heterogeneous or degraded
" Centrifuged in CstSO 4 solution containing 0.2 % HCHO. I n t h e CsC1 gradient "'cross-linked" D N A b a n d s at a d e n s i t y very close to t h a t of n a t i v e DNA, i n d e p e n d e n t o f w h e t h e r H C H O was present during d e n a t u r a t i o n (1% H C H O during denaturation, 0.2% H C H 0 during centrifugation).
Biochim. Biophys. •aa, 72 (1963) 298-309
IRRADIATION OF HALOGENATED TRANSFORMING
DNA
305
dB-like material. Upon denaturation, this ultraviolet "cross-linked"* hybrid DNA spread wider and became heavier than the original NB band (Figs. 4 C and 4D), which indicates partial collapse of the native structure of NB in the absence of true strand separation, characteristic of nonirradiated NB material (Fig. 4B). BB DNA was also included in this study, but it was not separated from the NB of an ultraviolet-light-irradlated mixture of NB and BB DNA (Fig. 5D) fails to produce bands corresponding to the denatured dB and dN DNA's (Fig. 5B), suggesting that ultraviolet light causes cross-links not only in NN but also in NB and BB molecules. The shift toward higher density and the band spreading is more pronounced in the bifilarly labeled (BB) DNA than in the NB material irradiated and denatured under identical conditions (Fig. 5D).
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Buoyant density in Cs2SO 4 (g/cm 3) Fig. 5. Microdensitometer tracings representing the buoyant density distribution of a mixture of unifilarly (NB) and bifilarly (BB) BUdR-labeled DNA, native (n) nonirradiated (A), denatured (d) nonirradiated (B), native irradiated (C), and denatured after irradiation with 5° kerg/mm 2 of ultraviolet light. The peaks of the nonlabeled B. subtilis DNA marker (NN), are indicated by the dotted lines. The conditions of denaturation and centrifugation are the same as in Fig. 2C.
It also is notable that the increment in the density of the dB v e r s u s the BB band is almost twice as high as the density increment observed upon denaturation of the nonlabeled native DNA (dN v e r s u s NN). " An alternative interpretation of Fig. 4C (and Fig. 4 D) could be as follows : NB DNA is not cross-linked by ultraviolet irradiation, but the two shaded peaks correspond to the separated dB (wide-spread peak on the left) and dN components. Peak dB is shifted to the right because of photochemical debromination and spreads widely as the combined result of degradation and the inhomogeneity of dehalogenation. Peak dN is heavier than in Fig. 4B, partially as the result of ultraviolet light irradiation and partially from acquiring the bromine label coming off the dB strand. This interpretation, although less likely than the cross-linking hypothesis, has as yet not been rigorously excluded by experiments employing 14C-labeled B U d R in conjunction with radioactivity determination in the separated bands. Biochim. Biophys. Acta, 72 (1963) 298-3o 9
300
Z. OPARA-KUBINSKA,Z. KURYLO-BOROWSKAAND W. SZYBALSKI
Chromatographic analysis Two types of D N A were employed in this experiment : NN and BU DNA's. The Cs~SO 4 gradient sedimentation pattern of the latter is presented in Fig. 5 A. These D N A ' s were extracted, deproteinized, and freed from R N A as described in EXPERIMENTAL, and subsequently subjected to a preliminary chromatographic purificationon a m e t h y l a t e d albumin-kieselguhr column 18,14 prepared according to SUEOKA AND CHENG 1S. The 5 × I-cm column was charged with 8 ml of D N A (A,e 0 = 2.6) dissolved in 0.075 M NaC1. The primary elution patterns of the NN D N A and the BU D N A were essentially the same, with the BU D N A giving a somewhat broader distribution. Only the fractions eluting with 0.6 and 0. 7 M NaC1 (peak of DNA) were saved and dialyzed against 0.075 M NaC1. These chromatographically purified D N A ' s were employed in subsequent experiments, illustrated in Fig. 6. A 4.0 × o.6-cm column was charged with 5 ml o f D N A (A2~0 = 0.3) dissolved in 0.075 M NaC1, and eluted with 3 ml portions varying from o.I to 1. 5 M NaC1 in o.I M steps. The D N A content of the eluted fractions was estimated from the Az80. Again it is apparent t h a t the chromatographically purified NN (Fig. 6A) and I00 60
NN-D
20
IB,,,
I00 _®
12j
~. 6 0 ~" 2 0
6O
NN-DNA
2O I00 BU-DNA
60
UV
20
T No CI
~
t
~
CONCENTRATION (M)
Fig. 6. Chromatographic elution patterns of nonlabeled (N-DNA) and of BUdR-labeld (BU) DNA, nonirradiated (A,B) or irradiated (C,D) with 5° kerg/mm*. The methylated albuminHyflo Super Cel column was charged with DNA dissolved in 0.o75 M NaC1; E = % of A ,60 in the effluent not retained by the column during the charging process. Stepwise elution in o. i M increments from o.I to i.o M NaC1, and final elution with 1.5 M NaC1.
BU D N A ' s (Fig. 6B) exhibited essentially the same elution patterns, although the total recovery of NN (88%) was higher than of the BU (70%). Aliquots of chromatographically purified D N A ' s were irradiated with ultraviolet light (5 .IO* erg/mm ~) and subjected to chromatographic analysis. Irradiation of normal D N A did not seem to effect grossly its chromatographic properties, reducing, however, its total recovery to 70%. Samples of irradiated DNA, collected before c h r o m a t o g r a p h y and from the 0.6 M NaC1 eluate (Fig. 6C), were denatured and
Biochim. Biophys. Acta, 72 (I963) 298-309
IRRADIATION OF HALOGENATEDTRANSFORMINGD N A
307
centrifuged in the Cs2SO 4 gradient under conditions identical to those represented in Fig. 3 D. Essentially the same centrifugation pattern was obtained, indicating t h a t the chromatographic behaviour of the cross-linked D N A was similar to t h a t of the nonirradiated N N D N A . Irradiation of BU D N A with the same dose (5" IO4 erg/mm 2) resulted in its rather firm a t t a c h m e n t to the column; only 12 % was eluted and only at molarities as high as 1.o-1.5 M (Fig. 6D). F u r t h e r elution with neutral 2 M NaC1 did not displace any D N A and with acidified 2 M NaC1 (pH 4) only an additional 6%. During charging of the column 4 % of the irradiated BU D N A was not retained (Fig. 6D), in contrast to the complete retention of the nonirradiated BU D N A (Fig. 6B). The specific transforming activity (sTA) of all the nonirradiated and irradiated samples was determined, before c h r o m a t o g r a p h y and after chromatography. The TABLE II T R A N S F O R M I N G A C T I V I T Y OF B U d R - F R E E
AND B U d R - L A B E L E D
DNA
A S A F F E C T E D B Y U L T R A V I O L E T L I G H T AND CHROMATOGRAPHIC FRA CT I O N A T I O N
Native DNA's from B. subtilis (ind+), BUdR-free (NN) or BUdR-labeled (BU = NB + BB, cf. Fig. i), were either irradiated with 5° kerg/mm 2 of ultraviolet light or nonirradiated and chromatographed on a methylated albumin column 15. Transforming activity (indole marker) was determined for the pre-chromatographed DNA (starting material), for the material Hot absorbed by the column during the charging process (E, o.075 M NaC1), and for the 0.6 M and i.o M NaC1 eluates (cf. Fig. 6). Transformants per I~g D N A Nonirradiated NN
Starting material
i.o. lOs
0.8. lO8
o
o
E Column eluates
0.6 M I.O M
BU
i.o. -
-
10 6
0.8" 10 6
-
-
Ultraviolet-irradiated NN
BU
250
20
o
o
400
--
-
-
o
results are summarized in Table II. The small difference in sTA between the NN and the B U D N A ' s used in this particular experiment cannot be considered significant, since the sTA of different batches of B U D N A equaled the sTA of normal D N A samples. Irradiated normal D N A retained 0.025% of its sTA; 7 0 % of this D N A eluting with 0.6-0. 7 M NaCI seemed to exhibit a 6 0 % higher sTA. Irradiated BU D N A retained only 0.0025% of its sTA and none of it could be found in the column eluate. In all the chromatographic experiments, the material eluting at molarities below 0.5 had no transforming activity. DISCUSSION AND CONCLUSIONS The change in the radiobiological properties of transforming D N A as a result of B U d R incorporation is paralleled b y changes in its radiochemical properties. A drastic difference in the chromatographic behaviour of the ultraviolet-irradiated B U D N A Biochim. Biophys. Acta, 72 (1963) 298-3o9
308
Z. OPARA-KUBINSKA, Z. KURYLO-BOROWSKA AND W. SZYBALSKI
(Fig. 6D), as compared with the NN DNA (Fig. 6C), indicates a high affinity of the former for methylated albumin. It would not be surprising if a similar reaction between BU DNA and basic nuclear proteins (cf. SMITH1°) were partially responsible for the dramatic ultraviolet sensitization by BUdR. Although no pronounced differences were observed in the chromatographic behaviour of the bulk of the NN, BU, and ultraviolet irradiated (5" lO4 erg/mm 2) NN DNA's, the step-wise elution employed was not designed to detect more subtle differences. Irradiation of NN DNA with higher ultraviolet doses results also in increased affinity for the column 24. The changes in the equilibrium-density-gradient centrifugation pattern as a result of ultraviolet irradiation could be divided into two operational classes: (a) those observed in native DNA and (b) those revealed after denaturation. I. The effect of moderate ultraviolet doses on the buoyant density of native BUdR-free DNA is rather small, with the density increases higher when measured in the CsC1 gradient than in the Cs2SO4 gradient. This indicates that ultraviolet lightaffected density changes are different than those produced by thermal denaturation, since the latter causes higher density increases in Cs2SO4 than in CsC1. This result points to the necessity of applying more than one density gradient medium when interpreting the effects of various agents on the basis of the buoyant density changes. In contrast with NN DNA, moderate ultraviolet doses result in pronounced widening of tile NB and BB bands and their shift to lower density. One interpretation of these changes would be the photochemical dehalogenation of the BU DNA 7, which would both decrease the buoyant density (this change probably partially compensated for by the ultraviolet-light-effected density increase similar to that observed with NN DNA) and magnify the heterogeneity with respect to density. The latter phenomenon and/or ultraviolet-light caused degradation of the BU DNA would result in spreading of the bands. 2. The ultraviolet elicited changes in the banding pattern of denatured DNA could be best interpreted as formation of cross-linkedg, 22 or "reversible ''z5 DNA. BU DNA is again much more sensitive to this modification than NN DNA (Figs. 3, 4,5 ). Since BB DNA used in the present study was practically free of thymine it would be difficult to interpret this cross-linking as caused solely by thymine dimerization across the strands. The dimerization of DNA-incorporated BUdR, the role of thymine dimerization in the cross-linking of complementary DNA strands, and the biological consequences.of the cross-linking are still open questions. ACKNOWLEDGEMENTS
This work was supported by Grant G-I8165 from the National Science Foundation. We are indebted to Dr. E. H. SZYBALSKAfor her editorial help and to Miss M. KING and to Mr. L. FENTON for the technical assistance. REFERENCES 1 Z. OPARA-KUBINSKA, Z. LORKIEWICZ AND W. SZYBALSKI, Biochem. Biophys. Res. Commun., 4 (1961) 288. 2 R. L. SINSHEIMER, Radiation Res., 6 (1957) i z i . s D. SHUGAR, in E. CHARGAFF AND J. N. DAVlDSON, The Nucleic Acids, Vol. 3, Academic Press, New York, i96o, p. 39.
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DNA
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4 R. BEUKERS AND W. BERENDS, Biochim. Biophys. Acta, 49 (I96I) 181. 5 D. WEINBLUM, Thesis, Technical University, Berlin, 196L 6 A. WACKER, J. CMm. Phys., 58 (1961) lO41. ? A. WACKER, H. D. MENNIGMANN AND W. SZYBALSKI, Nature. 196 (1962) 685. s W. ,BERENDS, J. Chim. Phys., 58 (1961) IO34. t j . MARMUR AND L. GROSSMAN, Proc. Natl. Acad. Sci. U.S., 47 (1961) 778. x0 K. C. SMITH, Biochem. Biophys. Res. Commun., 8 (1962) 157. 11 M. MESELSON, V. W. STAHL AND J. VINOGRAD, Prvc. Natl. Acad. Sci. U.S., 43 (1957) 581. x~ R. L. ERIKSON AND W. SZYBALSKI, Phage Information Service, (1962) 2, Cancer Research 23 (1963) 122; Abstracts Biophys. Soc. 7th Ann. Meeling (1963) W A 8. la L. S. LERMAN, Biochim. Biophys. Acta, 18 (1955) 132x* j . D. MANDELL AND A. D. HERSHEY, Anat. Bioehem., I (196o) 66. 15 N. SUEOKA AND T.-Y. CHENG, J. Mol. Biol., 4 (1962) 161. 15 W . SZYBALSKI, Z. OPARA-KUBINSKA, Z. LORKIEWICZ, E. EPHRATI-ELIZUR AND S. ZAMENHOF, Nature, 188 (196o) 743. aT H. J. VOGEL AND D. BONNER, J. Biol. Chem., 218 (1956) 97. 18 W. SZYBALSKI AND H. D. MENNIGMANN, Anal. Biochem., 3 (1962) 267. t9 W. SZYBALSKI, Experientia, 16 (i96o) 164. io W. SZYBALSKI, The Molecular Basis of Neoplasia, U n i v . o f T e x a s Press, A u s t i n , 1962, p. 147. tl B. DJORDJEVIC AND W. SZYBA~LSKI,J. Exptl. Med., 112 (196o) 509. ss j . MARMUR, W. F. ANDERSON, L. MATTHEWS, K. BERNS, E. GAJEWSKA, D. LANE AND P. DOTV, J. Cell, Comp. Physiol., Suppl. I, 58 (1961) p. 33u C. A. THOMAS~ JR. AND K. I. BERN-S, J. Mol. Biol,, 4 (1962) 309 • $4 A. D. HERSHEY, personal c o m m u n i c a t i o n . s5 E. P. GEIDUSCHEK, Prvc. Natl. Acad. Sci. U.S., 47 (1961) 95 °.
Biochim. Biophys. Acta, 72 (I963) 298-309