312

Biochimica et Biophysica Acta, 561 ( 1 9 7 9 ) 3 1 2 - - 3 2 3 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 99374

GENOMIC INTEGRITY OF T1 DNA A F T E R 7- AND U L T R A V I O L E T IRRADIATION

HEIDI MARTIN-BERTRAM and ULRICH HAGEN

Kernforschungszentrum Karlsruhe, Institut fiir Genetik und fi~r Toxikologie yon Spaltstoffen, Postfach 3640, D-7500 Karlsruhe 1 (F.R.G.) (Received July 13th, 1978)

Key words: Phage DNA; Irradiation; Genome integrity; (T1 phage)

Summary T1 DNA, 7-irradiated in the phage particle or irradiated with ultraviolet light was checked for structural integrity by kinetics of melting and reannealing. 7-Irradiated DNA differed in all thermokinetic properties by a factor of 3--4 from DNA degraded by mechanical or enzymatical treatments. Ultraviolet irradiation caused much smaller effects than v-irradiation. Considering the frequency of pyrimidine dimers in relation to the v-ray induced lesions, strong evidence can be derived, that in addition to single base damages, local denatured regions are produced by v-irradiation. Such regions, formed possibly by direct absorption of radiation energy in DNA, i.e. by primary ionizations, are associated with base lesions and are passed over during reannealing.

Introduction Ionizing radiation of the living organism induces in the nucleic acids strand breaks of the nucleotide chain as well as a variety of damages in all four nucleotide bases and in the sugar residues. Most progress in radiobiology was achieved in the studies of the formation and repair of strand breaks and in the chemical identification of the major lesions [1--6]. However, radiation-induced changes in the molecular structure of DNA and their significance for survival and mutagenesis have been neglected [7--8]. The action of even small lesions on the structural integrity of the genome may well interfere with repair and cause mutations. It is expected that chemically or physically modified bases lead to a significant helix-distortion and to non-complementarity. Both would reduce thermal stability and cause mismatch during reannealing of the denatured DNA Abbreviations: Thy-Thy, thymine-thymine; Cyt-Thy, cytosine-thymine; Ura-Thy, uracil-thymine.

313 strands. The amount of mismatch or nonhomology can be correlated with the a m o u n t of damaged bases, as has been clearly demonstrated for single types of lesions [ 9--11 ]. Hanson and Zhivotovsky [ 12 ] interpreted their slower reassociation rates of the single copy fraction from DNA of ascites hepatoma cells as indicating base changes caused by X-irradiation. This suggests the possibility of correlating altered thermokinetics the multiple lesions induced by ionizing radiation. This paper will show the reassociation behavior and the thermal stability of a single copy genome, carrying radiation-induced lesions in the nucleotide bases. Since the genomic parameters are well known, DNA from phage T1 was chosen. The lesions in the T1 DNA were induced either by 7-irradiation of the phage, leading to a variety of unknown damages or, for comparison, by ultraviolet light, producing a definable number of pyrimidine dimers. We present our contribution as a model study of the general influence of base damage on the structural integrity of the genome. Materials and Methods Strains. Coliphage T1 wildtype, the wildtype of Escherichia coli B and the m u t a n t E. coli CR 34/C 416 T h y - were originally obtained from the Institut fiir Genetik, Cologne, and from the California Institute of Technology. Preparation of radioactive phages. [2-14C]Thymine (spec. act. 5.9 Ci/mol) was obtained from Dr. Schweer, Institut fiir Radiochemie, Kernforschungszentrum, Karlsruhe. E. coli CR 34/C 416 T h y - were grown in minimal medium I plus 20 pg/ml cold thymine [13]. After collection, bacteria were suspended in minimal medium II [13] plus 20 ~g/ml [2-14C]thymine, infected with phages at a multiplicity of 0.1 and incubated for 2--3 h. The phages were precipitated by saturation with 50% ammonium sulphate and purified from bacterial debris by a digestion with DNAase (deoxyribonucleate 5'-oligonucleotidohydrolase, EC 3.1.4.5, l pg/ml), RNAase (ribonucleate 5'-oligonucleotidohydrolase, EC 3.1.4.22, 1 gg/ml) and trypsin (2 pg/ml). After differential centrifugation, the •resuspended phage pellet was highly concentrated by banding in CsC1 (titre about 1013 phages/ml). Finally the phages were dialysed against absorption buffer [14]. DNA isolation. Phages were diluted in absorption buffer plus 0.8 M NaC1 to a titre of 1012/ml. After 4% sodium dodecyl sulphate was added, the mixture was incubated for 30 min at 37 ° C. The lysate was gently mixed with an equal volume of redistilled, buffer saturated phenol which was removed by centrifugation. After the treatment was repeated twice, the aqueous phase was finally dialysed against various buffers as indicated below. Irradiation procedure. 3,-Irradiation of T1 phages was performed according to Coquerelle and Hagen [15] in a [6°Co]7-source (Gammacell 220, Atomic Energy of Canada Ltd., dose rate 0.35 Mrad/h). Phages with a titre of 1012 were diluted in 0.165 M NaC1 plus 0.1 M L-histidine, saturated with nitrogen and irradiated at 0°C. Ultraviolet-irradiation of DNA solutions at 0°C and a concentration of 50 ~g/ml was achieved with a specially designed low-pressure mercury lamp

314

(Vycor glass, Gr~intzel, Karlsruhe). The dose rate at a distance of 22 cm was 3 J/m 2 per s (cf. ref. 8). Analysis o f thymine-containing dimers. Ultraviolet-irradiated [2-14C]DNA was hydrolysed in 98% formic acid at 176°C for 30 min. After evaporation to dryness, the hydrolysate was dissolved in a small volume of water and submitted to two-dimensional thin-layer chromatography (Cel MN 300 polygram, Macherey and Nagel) according to Goldman and Friedberg [17]. Thyminecontaining dimers and monomers were analysed by their radioactivity in a liquid scintillation counter after removal from the cellulose layer. Degradation procedures. To produce strand breaks, the DNA was sheared by ultrasound (Branson sonifier S 125) or by pressing the DNA solution through a no. 22 needle at 0°C. Single-strand breaks were introduced during incubation of DNA solution with pancreatic DNAase. 200 pg/ml DNA in 50 mM Tris-HC1, pH 7.5, and 5 mM MgC12 were digested with 1 ~g/ml DNAase for 30--60 min at 37°C. After a desired degradation, followed in a viscosimeter, the enzyme was inactivated and removed by chloroform/isopentanol treatment. Finally, the DNA solution was extensively dialysed against the buffers indicated below. Molecular weight determinations. Molecular weights of DNA samples were measured by sedimentation in the analytical ultracentrifuge (Beckman Instruments, model E). Sedimentation coefficients and molecular weights were calculated according to standard procedures [15,18,19] under neutral conditions and after denaturation in alkali and by heating [20]. The frequency of strand breaks (B) were calculated after formula 1 or 2:

Bss = m/Mn (single-strand breaks)

(1)

Bds = 2m/Mn (double-strand breaks)

(2)

where m is the mean molecular weight of a nucleotide and M n the number average molecular weight. The frequency of breaks refers here directly to one nucleotide (single chain breaks) or to one pair of nucleotides (double chain breaks). T m analysis and reassociation kinetics. Melting and reassociation experiments were performed in 0.06 M sodium phosphate buffer, pH 6.8, in a Gilford spectrophotometer with a thermoprogrammer and an electrically thermostated cuvette-block for four samples. After denaturation at a heat rate of 0.5°C/min up to 92°C, the cuvette block was cooled down to 70°C within 1--2 min. The change of absorbance during the time this temperature-jump was omitted in the calculation of reassociation kinetics. Reassociation was followed up to 70% reannealed molecules. To control the specificity of the reaction, the reassociates were remelted and the profiles of this second melting were compared with those of the first melting. The reassociation rate constant k: was calculated according to Wetmur and Davidson [21]: Ass

-- Ads _ kaPw

t +1

(3)

A t --Ads

where A t is the absorbance of the reassociate, Ads the absorbance of the

315 double-strand DNA, As~ of the single-strand DNA, t is the relaxation time and PT the total DNA nucleotide concentration: PT = 1 . 4 7 . 10 -4 Ads mol • 1-1

(4)

The dimension of h2 is 1 • mo1-1 • s -1. Mean ks-values were calculated by polynominal regression analysis of multiple A t data, taken at the particular relaxation time from the absorbance decline plot registrated by the spectrophotometer at 260 nm. Results C o n d i t i o n s o f reassociation and the e f f e c t o f degradation

Low ionic strength (0.06 M phosphate buffer, pH 6.8) and a relatively high reassociation temperature of Tm --15°C were chosen to detect small changes in the reassociation kinetics. It was necessary, however, to test whether under these conditions a second-order reaction also takes place. Generally, when single-stranded DNA reanneals, we observe a biomolecular reaction, where the rate constant k2 is proportional to the concentrations of single-stranded reactants. This second-order reaction can be described by the following Eqn.: - - d C A / d t = -- d C n / d t = h2CAC B

(5)

where CA, CB is the concentration of both single strands and t the relaxation time. During reannealing CA changes to cA =

-

ct

(6)

where C ° is the initial concentration and Ct the concentration of the reassociate at the time t. ks is here the second-order rate constant for helix formation. Since the concentrations of the single strands are the same (CA = CB), we can write: d C t / d t = k2(C°A - - Ct )2

(7a)

or after integration: ks - 1 t

Ct 0 ( C 0A _ Ct ) CA

(7b)

Using different initial DNA concentrations, h2 remains constant, which is a critical test for the second-order reaction of the reassociation. Such an independence of the DNA concentration was indeed found in our experiments; a few examples are shown in Table I, indicating second-order specificity of our reassociation conditions. Wetmur and Davidson [21] have demonstrated the dependence of k2 on the genomic complexity N and the molecular weight L of the single strand by the equation: ks = fl3kN(L/N)

(8)

where fl is the density of nucleation sites and kN the elementary rate constant, responsible for the initiation of the reaction. Since N remains constant within a defined, although degraded genome, k2 is expected to be proportional to L,

316 TABLE

I

REASSOCIATION

RATE

CONSTANT

Treatment -

-

--

AS A FUNCTION

OF INITIAL

DNA CONCENTRATION

Mw(ss ) X 10 -6

Ads

k 2 (1 " m o l e -1 " s -1 )

15.0 15.0

0.979 0.468

6.131 ± 0.190 6 . 6 3 5 -+ 0 . 2 9 0

Sheared

7.60 7.60

0.950 0.433

5 . 0 1 1 _+ 0 . 1 6 0 5 . 3 4 0 +- 0 . 2 3 0

DNAase degraded

1.90 1.90

1.050 0.450

6.821 + 0.080 6.569 + 0.110

100 krad

8.90 8.90

1.035 0.498

6 . 4 4 6 +- 0 . 1 8 0 6 . 1 9 1 +- 0 . 1 1 0

~'-Irradiated: 200 krad

6.41 6.41

1.030 0.540

4 . 8 3 8 +- 0 . 1 1 4 5.453 + 0.129

7-Irradiated:

Ultraviolet irradiated:

720 J/m 2

15.0 15.0

1.008 0.474

4 . 5 0 0 +- 0 . 1 1 0 4.070 + 0.120

Ultraviolet irradiated:

1800 J/m 2

15.0 15.0

1.020 0.530

2.089 + 0.170 2 . 6 9 0 -+ 0 . 2 1 0

according to Eqn. 8. This was, however, not observed in the experiment. Under the reassociation conditions of Wetmur and Davidson (1.0 M Na+), k2 was found to be proportional to L °' 50, which was attributed to an excluded volume or steric hindrance effect. The effect of a defined type of chain scission under our reassociation conditions was studied. Strand breaks were introduced with shearing forces, ultrasound or DNAase digestion. The relation between the extent of degradation and the respective k2 value forms a straight line in the double log plot (Fig. 1). The

°//

? 7

c3

/(.

x

6E 54 62 50

o

J

6

r

10

o

T/~o

/o j

0!5

/.

o'6

0'7

toglo kz

0'8

0'9

L

I

~

100

1

,

ZOO

Dose [krod]

F i g . 1. C o r r e l a t i o n b e t w e e n r e a s s o c i a t i o n r a t e c o n s t a n t k 2 a n d s i n g l e - s t r a n d e d m o l e c u l a r w e i g h t ( M w ( s s ) ) f r o m T 1 D N A , d e g r a d e d in t w o w a y s : ( o ) s h e a r i n g o r u l t r a s o u n d ; ( o ) d i g e s t i o n w i t h D N A a s e . M o l e c u l a r w e i g h t s a r e g i v e n in d a l t o n . R e a s s o c i a t i o n conditions: 0.06 M phosphate buffer, pH 6.8, and 70°C (Tm --15° C). F i g . 2. I n d u c e d s t r a n d b r e a k s i n T 1 D N A a f t e r ' ) ' - i r r a d i a t i o n o f t h e p h a g e p a r t i c l e p e r 1 0 5 n u c l e o t i d e s , Single- and double-strand breaks were analysed by sedimentation analysis.

317 molecular weight of DNA with double-strand breaks resulting from the shearing forces or ultrasound was related to the rate constant k2 ~ L °'253 +- 0.020, and the molecular weight of DNA bearing single-strand breaks induced by DNAase digestion, followed the relation k2 ~ L °'~79 -+ 0.013. The error of the exponent was calculated by regression analysis from the data in Fig. 1. With doublestrand breaks, the observed correlation is independent of the m e t h o d of degradation (ultrasound or shearing) but the slope differs by a factor of 3/2 because of the random cuts by DNAase digestion.

Reassociation kinetics and Tm analysis of DNA from 7-irradiated phage By irradiating phage with 7-rays, a number of strand breaks were introduced into the DNA as shown in Fig. 2. When the rate-constant k2 was plotted against induced strand breaks, we obtained the straight lines of Fig. 3. The slope for 7-irradiated DNA is steeper by a factor of 3.5 than the slope obtained with the DNA samples degraded by either DNAase or shearing forces and ultrasound (not shown). This means that in the 7-irradiated DNA in addition to the induced strand breaks other lesions are present which lower the reassociation rate constant k2. Before and after reassociation the DNA samples were denatured at a heat rate of 0.5°C/min until no further hyperchromic rise was observed. From the sigrnoid melting curves, the mean-point (T=) was determined graphically and the derivatives were calculated (not shown). The Tm depression of 7-irradiated DNA and of mechanically or enzymatically degraded DNA were related to the respective rate constant (percent k2). With DNAase-treated DNA samples there

0.9~

9O 100 90 80

0°2

\

E r..4

70 60 50

I

Induced brenks per 105nucteotides

05

10

Aim [°~]

Fig. 3. D e c r e a s e o f the r e a s s o c i a t i o n r a t e c o n s t a n t k 2 w i t h the i n d u c t i o n of b r e a k s and o t h e r lesions i n t o D N A . e, D N A a s e d i g e s t e d D N A ; o, D N A f r o m 7 - i r r a d i a t e d p h a g e particles. T h e slope w a s d r a w n a f t e r p o l y n o m i n a l r e g r e s s i o n analysis o f all e x p e r i m e n t a l d a t a . % k 2 m a x = % k 2 o f t h e u n m o d i f i e d c o n t r o l , h 2 o f 1 0 0 ~ c o r r e s p o n d s t o an e x p e r i m e n t a l v a l u e of 6 . 6 3 5 + 0 . 2 9 0 (1 • m o l e -1 • s - 1 ) as c a l c u l a t e d f r o m t h e a b s o t b a n c e decline p l o t (see M e t h o d s and T a b l e I). Fig. 4. C o r r e l a t i o n b e t w e e n r a t e c o n s t a n t - d e v i a t i o n (% k 2 m a x , see Fig. 3) a n d T m d e p r e s s l u n ( d T m ) in D N A f r o m 7 - i r r a d i a t e d p h a g e , o, T m e s t i m a t i o n b e f o r e r e a n n e a l i n g (first m e l t i n g ) ; e, T m e s t i m a t i o n a f t e r r e a s s o c i a t i o n ( s e c o n d m e l t i n g ) ; +, D N A a s e d i g e s t e d D N A s a m p l e s , first and s e c o n d m e l t i n g , d T m c o r r e s p o n d s t o a m e l t i n g p o i n t o f 8 4 . 6 ° C , d T m at 1 ° C t o 8 3 . 6 ° C .

318 was only a sight depression of the melting point (Fig. 4, data from sheared DNA not shown). A distinct Tm depression, however, was observed with v-irradiated DNA during the first melting, indicating again a further radiation damage in addition to the strand breaks. This effect, however, disappeared at the second melting of the irradiated DNA. Then, the same Tm was measured with v-irradiated and with mechanically or enzymatically degraded DNA. The nonirradiated samples showed also no difference between the first and the second melting, because of the complementary reannealed strands.

Reassociation kinetics and Tm analysis of DNA irradiated with ultraviolet light T1 DNA was irradiated with ultraviolet light and the amount of thyminecontaining dimers was estimated by two-dimensional TLC of the acid DNA hydrolysate (Fig. 5). Before submitting to melting and reassociation the ultraviolet.irradiated DNA samples were examined for strand breaks by alkaline sedimentation. There was no indication of a degradation of DNA molecules or depurination due to exposure to ultraviolet light. Related to the percent of dimers per total thymine content in the DNA, there is a distinct decrease of the reassociation rate constant k2 (Fig. 6). Also a depression of the Tm value was observed becoming more pronounced with higher amounts of dimers. To compare these data with the experiments on DNA samples, subjected to other treatments, calculations were done considering a thymine containing dimer as one lesion in the DNA double helix per total number of nucleotides in the molecule, e.g. 2% dimers per total thymine means one lesion per 400 nucleotides. Thus, 1% dimers corresponds to 1.25 lesions in the DNA per 1000 nucleotides and 10 -3 dimers per nucleotide, produced by ultraviolet light, cause a k2 depression of 8.4% and a Tm decrease of 0.48°C.

g0 o~ f

~o

o

c_n o

o i

/

o/ 61 ,of

m m ~

l

I m m m m i I00~ 2000

Dose []/m -2]

% (]/Ttotot

F i g . 5. A n a l y s i s o f p y r i m i d i n e d i m e r s i n T 1 D N A a f t e r u l t r a v i o l e t - i r r a d i a t i o n b y t w o d i m e n s i o n a l T L C . E s t i m a t i o n o f d i m e r s f r o m t h e t o t a l [ 2 - 1 4 C ] t h y m i n e c o n t e n t ( % T T / T t o t a l ) . A l l v a l u e s are m e a n s f r o m a t least three estimations. F i g . 6. T h e e f f e c t o f p y z i m i d i n e d i m e r s o n t h e r e a s s o c i a t i o n r a t e c o n s t a n t k 2 ( o ) a n d t e m p e r a t u r e d e p r e s s i o n ( d T m ; o), F o r i n i t i a l . k 2 a n d T m v a l u e s see F i g s . 3 a n d 4.

on the melting

319 10( gC 8(

,o

\

],,÷

40 30

~,Tm [°C] Fig. 7. E f f e c t of T m d e p r e s s i o n ( d T m ) o n the r a t e c o n s t a n t d e v i a t i o n ( % k 2 m a x , see Fig. 3) f o r the first (o) a n d s e c o n d m e l t i n g ( e ) o f u l t r a v i o l e t i r r a d i a t e d T1 D N A .

Since about four times as many Thy-Thy dimers, counting twice in radioactivity, are formed as Cyt-Thy dimers [22], which are discovered in the same area from TLC profiles (Cyt-Thy dimers convert to Ura-Thy dimers during hydrolysis [22]), a underestimation of about 15% in our data can be assumed [23]. Ultraviolet-irradiated DNA reacted differently from 7-irradiated DNA during the second melting, the Tm depression is even higher. A linear correlation to the rate constant is observed for both melting procedures (Fig. 7). Discussion

General conclusions and the effect o f chain breaks The method of melting and reannealing was applied to identify universal structural changes in 7- and ultraviolet-irradiated DNA. Because these techniques detect the influence of one single base damage on genome structure [9,10] they should be sensitive means to investigate DNA after 7-irradiation which induces a variety of lesions. The detection of mismatch depends on criteria and one of the criteria to detect even small lesions kinetically was to select the temperature of reassociation (Tr) close to Tin. As T~ approaches Tin, a decrease in the stability of base-pairing is observed [25]. The same effect is observed by lowering the ionic strength of the DNA solution. If Tr and ionic strength are carefully chosen, complementary covalent reanneaiing is just maintained b u t non-homologous base-pairs will fail to match. The specificity of the second-order reaction under these conditions was ensured in all modified DNA samples used. To study the effect o f chain scissions on the genome structure, we examined T1 DNA degraded by various techniques: ultrasound, shearing forces and DNAase digestion. Compared to a corresponding plot by Wetmur and Davidson [21], the slopes in Fig. 1 were about half as steep, i.e. ks was less dependent on the length of the molecule than under the conditions of Wetmur and Davidson

320 (1 M Na ÷, Tr = Tm -- 25°C). This may be attributed to the lower ionic strength in our conditions, where large molecules are less folded [25] and possibly more susceptible for nucleation than in high ionic strength. The lower slope in the log Mr/log k2 plot of DNAase digested samples (random cut DNA) to that of sheared DNA by a factor of 2/3 is in agreement with respective considerations of Wetmur and Davidson [21]. As the Tm depression is negligible after mechanical or enzymatical degradation and the kinetics are specific, we conclude that in all these cases the complementary strands matched perfectly•

Melting and reannealing of ultraviolet irradiated DNA After ultraviolet irradiation the d o m i n a n t lesion in the DNA is the pyrimidine dimer. Dimer formation increased linearly with ultraviolet dose; the slope became slower in the range of 1600 J • m -2 (Fig. 5) which may be attributed to the small genome size of T1, limiting the number of adjacent t h y m i n e moieties Similar observations were made by Wulff on E. coli DNA [26]. With higher amounts of dimers we observed a linear depression of the melting point and a linear decrease of the reassociation rate constant k2. The Tm depression in our experiments was larger than that reported by Kahn [11] (see below, Table II), which may be due to the different ionic strength used. Also our delay of k2 was more pronounced. The k2 response after ultraviolet irradiation, however, depends on the temperature of reannealing (Kahn [11]). The decrease of k2 was linearly related to the Tm depression (Fig. 7 and ref. 11). In both plots, there was no indication of a shoulder, as reported by H u t t o n and Wetmur [9] for deaminated DNA or glyoxal-modified DNA where a significant decrease of k2 could be observed only at a T m depression of 10--15°C. This indicates that the pyrimidine dimer affects the structural integrity of DNA in a way other than the chemical modification of a single

T A B L E II EFFECT OF DIFFERENT TREATMENTS ON THE MELTING THE REASSOCIATION RATE CONSTANT k 2 OF DNA DNA treatment respectively

d T m p e r 1 0 -4

induced lesions

DNA modification

POINT DEPRESSION

(dTm)

AND ON

k 2 d e p r e s s i o n per 1 0 -.4 i n d u c e d l e s i o n s per n u c l e o t i d e (%)

Reference

per nucleotide (o C)

Strand breaks by shearing

forces

0.2

1 2 . 0 -+ 2

this work

Strand breaks b y D N A a s e treatment

0.2

11.5 + 2.5

this work

Strand b r e a k s b y T - i r r a d i a t i o n o f the phage particle

0.7

33

this work

Thymine dimers by ultraviolet i~adiation

0.048 *

0.84

Thymine dimers (ultraviolet)

0.025 *

--

11

Deamination b y H N O

0.032 *

--

24

2

Average value for m o d i f i e d bases * Extrapolated

0•022 *

from dT m per % modified bases

-+ 5

k 2 depression not linear

this work

21

321 base. Furthermore, the small difference between the first and the second melting point indicates that the dimer remains stable during thermic treatment and mismatch comprises more bases around the pyrimidine dimer.

Thermokinetic properties of ~,-irradiated DNA To get some information about the radiation effect on DNA in living organism, T1 DNA was 7-irradiated in the phage particle [27]. The frequency of the radiation-induced single-strand and double-strand breaks has been measured in phages by various authors. The yield found for DNA in T1 phage by Bohne et al. [28] (Pl = 3.6 • 10 -1° single-strand breaks per rad per nucleotide) corresponds to that found in our experiments (Pl = 3.8 • 10-1°). From analysis of base damages in aqueous solutions [29] or in irradiated mammalian cells [30], it can be deduced that also in 7-irradiated phages about three times more base lesions are produced than chain scissions. 7-Irradiated DNA denatured differently than ultraviolet-irradiated DNA. A depression of Tm is only measurable at the first melting. Furthermore, the derivatives of the melting profile revealed a random distribution of the bases in the genome; there is only a slight shift towards lower melting temperatures, e.g. an earlier opening of H bonds by two centigrades (data n o t shown}. The different melting kinetics of 7- and ultraviolet-irradiated DNA demonstrate that 7-ray induced lesions in DNA may be structural more diverse than those induced by ultraviolet light. The action of 7-irradiation on the decline of Tm and k2 was compared with the influence of other treatments like ultraviolet irradiation and chemical base modifications. Such a comparison is summarized in Table II, where the alterations of Tm and k2 are related to a frequency of 10 -4 lesions per nucleotide. It becomes evident that the decay of Tm and k2 in 7-irradiated DNA is about three times higher than in DNA degraded mechanically or enzymatically. The effect in addition to the strand breaks, i.e. the Tm depression of 0.5°C per 10 -4 induced strand breaks or the k2 decrease of about 21%, may be attributed to base damage as well as other structural or conformational changes in the DNA. This conclusion is supported assuming similar efficiencies of all base lesions in our experiments and comparing the action of pyrimidine dimers to 7-ray induced Tm and k2 alterations (Table II). We propose that ionizing radiation produces in the double helix local denatured regions, not accompanied by a chain scission. Evidence for such sites after ~-irradiation of DNA in auqeous solutions has been obtained previously by other authors [31,32]. DNA molecules carrying single stranded sites were characterized by their retention on methylated serum albumin kieselgur columns [31]. The radiation induced denatured regions were not able to reanneal under otherwise appropriate conditions. Local conformational changes of the DNA were described also by Vorli~kova and Pale~ek [32], from pulse-polarographic measurements and circular dichroism spectra. The formation of denatured regions can be caused indirectly by the radiolysis of water to H30 ÷. The pH in the direct environment of HaO ÷ will then temporarily be lowered [33]. If radiolysis of water occurs at DNA molecules, the acidic pH may disturb or weaken H bonds, leading to local singlestranded sites.

322 Yet, 7-irradiation of the phage leads to a direct absorption of radiation energy within the DNA and the effect can be attributed also to primary ionizations in the molecules, where a mean energy of about 60 eV is deposited [34]. Several bases are presumably damaged next to each other, leading, together with the indirect action of radiolysis, to a denatured region with one or more base lesions. Furthermore, primary ionization gives rise to polarization waves; the sudden appearance of an electrostatic charge within a macromolecule may cause an opening of hydrogen bonds with limited extension [ 35]. The Tm of the first melting plot reflects directly the influence of base damages and labile H bonds on the genome structure. The thermokinetic alterations on 7-irradiated DNA observed could be explained by the formation of unpaired regions. The first step in reassociation is the formation of a few base pairs in correct register, called nucleation. This reaction is reversible and will become slower, if nonhomologous bases or conformational changes of the nucleotide chain are present. If the radiation-induced base lesions combined with conformational changes of the helix are unable to reanneal and are passed over, k2 will be reduced considerably. Otherwise, a mismatched structure would result altering the melting behavior of the reassociate [36]. This, however, was not observed. The reannealed base pairs are considered to be unaltered. Thus, the perfectly matched reassociates behave like molecules which are structural intact with respect to initially reacting strands but reduced in size. The second Tm of 7-irradiated DNA is therefore unchanged as compared to sheared DNA, which indicates the specificity and homology of the complementary strands. Denatured regions with damaged bases, unable to reassociate, may considerably perturb genetic recombination in living organisms, since this seems to be the biological process requiring recognition between complementary parts of DNA molecules.

Acknowledgment H.M.B. whishes to thank Dr. I. Essigmann-Capesius, Botanisches Institut der Universit~it Heidelberg, for her initial help with the Gilford spectrophotometer.

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