Alteration of DNA reassociation kinetics due to base mismatch caused by thymine dimerisation

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Biochlmica et Biophyszca Acta, 374 (1974) 324--331 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98156

ALTERATION OF DNA REASSOCIATION KINETICS DUE TO BASE MISMATCH CAUSED BY THYMINE DIMERISATION

D. W E I N B L U M , H.J. B R E T E R , R.K. Z A H N and J. B E R G E R Physiologisch-chernisches Institut and lnstitut fiir medizinische Statistik und Dokumentation der Universitiit Mainz, 65 Mainz (G.F.R.) (Received May 20th, 1974)

Summary Base mismatch was introduced into Escherichia coli DNA by photodimerisation between contiguous thymine molecules. With increasing dimer content both the melting temperature and the reassociation velocity of the DNA decrease. The reassociation rate is reduced by a factor of approx. 2 when the melting temperature is lowered by 5° C. Since the dimers are distributed statistically over the DNA molecules, in a DNA sample, molecules with different dimer content and hence with different reassociation rates will occur. Consequently, molecules with a dimer content below the average will reassociate preferentially early during the course of reassociation while molecules with high dimer content will reassociate later. During the progress of the reaction the average dimer content will, therefore, increase in the single-stranded fraction. Base mismatch does not only reduce the rate of reassociation but also causes a deviation from second-order kinetics.

Introduction Repetitive sequences, occurring in eukaryotic DNA, are generally not identical but only similar. Due to random mutational events over many generations, the originally identical base sequences have been altered differently. Upon reassociation such DNA will form imperfect helices with varying amounts of mismatched base pairs. Britten [1] was the first to suspect that this mismatch may reduce the reassociation velocity of repetitive DNA. Such an effect would help to explain the reduced reassociation velocities of particular repetitive DNA [2,3]. Recently several attempts have been made to determine the exact magnitude of this effect [4--6]. Since the reduced thermal stability of reassociated DNA is roughly proportional to the amount of base mismatch all investigators have tried to correlate the amount of reduction in the reassociation rate to the

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a m o u n t of reduction of thermal stability. However, the results obtained so far have been different. These differences can only be partially explained by the different models of base mismatch which have been used in the investigations. Another aspect of the influence of base mismatch on the reassociation kinetics of DNA also has to be considered. As we have pointed out [3], the course of reassociation of repetitive DNA cannot be described exactly by second-order kinetics, if base mismatch causes a decrease in the rate and if the base mismatch is distributed statistically over the DNA molecules. In order to demonstrate this effect, we have used the thymine dimer-containing D N A as a model to investigate the influence of base mismatch on the kinetics of reassociation, since it is easy to follow the distribution of the mismatching dimers during the course of the reaction in the double- and singlestranded DNA. Materials and Methods D N A isolation Escherichia coli, Strain CR 34/C 416, was cultivated in a synthetic medium, containing [3 H] thymine. The bacteria were digested with lysozyme, from the lysate the D N A was extracted b y the m e t h o d of HSnig et al. [7]. The specific activity of the DNA was 5.8 #Ci/mg. D N A irradiation A 1.5 • 10 -4 M DNA solution in 0.12 M phosphate buffer of pH 6.8 was irradiated in the presence of 0.02 M acetophenone in a water-cooled pyrex vessel with a high-pressure mercury lamp for various time intervals. After irradiation the~ acetophenone was removed by extraction with chloroform and subsequent dialysis against 0.12 M phosphate buffer overnight. The thymine dimer c o n t e n t of the irradiated DNA was determined after formic acid hydrolysis by high-pressure liquid cation-exchange chromatography according to Breter et al. [8]. Reassociation kinetics Reassociation kinetics of the DNA samples, containing various amounts of thymine dimers, were measured by hydroxyapatite column chromatography as described elsewhere [3]. The denatured DNA was incubated at 60°C in 0.12 M phosphate buffer. Aliquots from the single- and double-stranded fraction of each h y d r o x y a p a t i t e separation were analysed for their dimer content as described above. Separation of larger amounts of DNA into single- and doublestranded fractions was carried o u t on a hydroxyapatite column with a 3-cm diameter. Melting curves were taken with an especially equipped Zeiss spectrophotometer. Sedimentation coefficients of the DNA were determined in a Beckman model E analytical ultracentrifuge b y velocity sedimentation. Results

Irradiation o f D N A According to Ben-Ishai et al. [9] irradiation of DNA with ultraviolet light

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above 313 nm wavelength in the presence of acetophenone yields practically only thymine dimers of the cyclobutane type (syn, head to head) as the sole photoproduct. Only approx. 4% of the total pyrimidine dimers are thymine-cytosine dimers. Since we used thymine-labelled DNA, the specific activity of the thymine--cytosine dimers is only half of the activity of the thymine-thymine dimers, therefore, the true amount of pyrimidine dimers may be slightly higher as reported. It appears also, that the irradiation introduces a small amount of interstrand crosslinks into the DNA, since after the irradiation a very fast reassociating fraction is observed. For the DNA samples containing 6.46, 13.72 and 23.81% of dimer, respectively, the amount of crosslinked DNA was estimated to be 1, 2 and 4%. Reassociation curves were corrected according to these values. As Zierenberg et al. [10], have shown, thymine dimerisation does not cause chain breaks to an appreciable extent. S values of the sheared double- and single-stranded DNA before and after irradiation were 7.0 and 5.2, respectively, corresponding to a length of approx. 400 nucleotides [ 11].

Base mismatch reduced by thymine dimerisation Upon photodimerisation, the distance between two neighbouring thymine rings in one DNA strand is shortened, so that base pairing of these thymine molecules with the complementary two adenine molecules becomes impossible. Hayes et al. [12] have shown that in an oligothymidylate--polydeoxyadenylate complex a total of four base pairs per thymine dimer is broken, e.g. not only the directly affected base pairs but also hydrogen bonds in the adjacent two base pairs are ruptured. This must not be the same for thymine dimers in DNA, since firstly even the sheared DNA is considerably longer than the oligonucleotides used by Hayes and coworkers and secondly since the hydrogen bonds between adjacent guanine--cytosine pairs may not be affected due to their higher strength. Considering that in E. coli DNA thymine--adenine accounts for 50% of the base pairs, we find an average reduction of the DNA melting temperature (ATm) of 0.65°C per 1% dimerised base. ATm values for thymine-adenine base pairs have been determined from deaminated poly(deoxyadenosine--thymidine) complexes to 0.7°C per 1% altered base [13]. For 1% mismatched average base ATm values from 1.2 to 1.5 have been reported recently [14,15]. We, therefore, conclude that in DNA only two hydrogen bonds per dimer are broken. Influence of thymine dimers on D N A reassociation Reassociation kinetics of E. coli D N A , containing various amounts of thymine dimers, were determined by hydroxyapatite chromatography. As can be seen from Fig. 1, with increasing dimer content the reassociationvelocity of the D N A decreases. It is also evident from the figure, that the slope of the reassociation curve flattens with increasing dimer content. This indicates deviation from ideal second-order kinetics. In Table I the dimer content, melting temperature and Co t~/2 values are listed. The c0tl/2 values were calculated assuming second-order kinetics according to Britten and Kohne [16], although we think that at a higher amount of mismatch deviation from second-order

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kinetics is considerable. Assuming the Co tl/2 values to be a function of ATm, it appears that the Co tl/2 values are growing faster than linearly with growing ATm. In order to prove this, higher ATm values should have been included in this investigation. Unfortunately the maximal amount of dimers which can be introduced into E. coli DNA is limited since only 37% of the total thymine is in dimerisable positions [17]. To achieve maximal dimerisation, prolonged irradiation would he necessary which would also produce unwanted side products to an intolerable extent.

TABLE I E F F E C T OF DIMER CONTENT ON T m AND APPARENT Cotl/2 VALUES Dimer content

T m (reassociated

AT m

(%)

DNA) (°C)

(°C)

0 6.46 13.72 23.81

90.0 88.4 87.2 83.7

0 2.5 3.7 7.2

Cotli2

Factor of increase

7.2 9.2 12.2 17.2

1 1.3 1.7 2.4

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Distribution o f dimers m early and late reassociating DNA If thymine dimers are introduced into D N A by ultraviolet irradiation, the amount of dimer per D N A molecule is not constant but the dimers will be distributed statistically over the D N A molecules, e.g. a considerable percentage of molecules will contain more respectively less dimers than the average. Since the D N A molecules with a low dimer content have a higher reassociation rate than those with an average or high dimer content as we have shown above, one would expect that in a given sample of uniformly irradiated, denatured D N A the early reassociating D N A molecules will have a dimer content below the average of the sample. In the remaining singie-stranded fraction on the other hand an enrichment of dimers should occur. We have, therefore, analysed the dimer content of the double- and singiestranded fraction which we obtained from determining the reassociation kinetics by hydroxyapatite chromatography during the course of reassociation. The results for the D N A samples, containing 6.46 and 13.72% dimer respectively, are listed in Table II. As it was expected, the dimer content of the double-stranded fraction at the beginning of the reassociation is indeed significantly below the average dimer content and rises as the reassociation proceeds while the dimer content of the single-stranded fraction towards the end of reassociation reaches values considerably above the average sample content. In Fig. 2 the average dimer content for the double-stranded fraction of the D N A samples containing 6.46 and 13.72% dimer respectively, is plotted as a function of the progress of reassociation. For comparison integrated Poisson distribu-

T A B L E II DISTRIBUTION OF DIMERS BETWEEN THE DOUBLE- AND SINGLE-STRANDED FRACTIONS AT DIFFERENT STAGES OF REASSOCIATION

V a l u e s o f G r o u p (a) w e r e d e t e r m i n e d from D N A c o n t a i n i n g 6.46% dimer, values o f Group (b) f r o m D N A containing 13.72% dimer. T h e values o f the last c o l u m n (average d i m e r c o n t e n t total) w e r e calculated f r o m the values o f c o l u m n s 2--5. Group

(a)

(b)

Reassociation time (h)

0.1 0.5 1 3 6 10 24 48 0.1 0.5 1 3 6 I0 24 48

Single-stranded D N A

Double-stranded D N A

% of total

Average dimer c o n t e n t

% of total

Average (timer c o n t e n t

Average dimer content total

93.8 87.4 82.6 68.5 56.0 45.8 33.8 24.7 93.1 86.9 84.6 72.2 59.0 50.0 36.8 31.1

6.48 6.49 6.54 6,61 6.65 6,76 7.30 7.41 13.90 14.03 14.09 14,33 14.35 14.66 15.18 16.28

6.2 12.6 17.4 31.5 44.0 54,2 66.2 75.3 6.9 13.1 15.4 27.8 41.0 50.0 63.2 68.9

6.01 5.94 6.08 6.13 6.23 6,24 6.41 6.27 11.25 11.58 11.81 12.13 12.52 12.78 12,80 12.63

6.45 6.42 6,46 6.46 6.47 6.48 6.71" 6.55 13.72 13.71 13.74 13.72 13.60 13.72 13.68 13.76

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Fig. 2. A v e r a g e dLmer c o n t e n t o f t h e r e a s s o c m t e d D N A f r a c t i o n s as a f u n c t m n o f t h e p r o g r e s s o f t h e r e a s s o c i a t l o n r e a c t i o n . C u r v e s a a n d c: i n t e g r a t e d Poxssion d i s t r i b u t i o n s for 6 . 4 6 a n d 1 3 . 7 2 % dLmer respectively. C u r v e s b a n d d: e x p e r i m e n t a l l y o b s e r v e d a v e r a g e d i m e r c o n t e n t m t h e r e a s s o c l a t e d f r a c t i o n contalrLing 6 . 4 6 a n d 1 3 . 7 2 % d i m e r , r e s p e c t i v e l y .

tions for these dimer contents are also plotted. Such curves one would obtain, if the dimers were Poisson distributed and if the DNA molecules would reassociate exactly in the order of increasing dimer content. Since the change in rate due to different dimer c o n t e n t for these two samples does n o t exceed the factor of two and since the concentration of DNA molecules with extreme dimer c o n t e n t is rather low, the experimentally observed curve must be considerably more shallow. In order to demonstrate more convincingly the magnitude of the uneven distribution o f dimers in early and late reassociating DNA the following experim e n t was undertaken: denatured DNA with an average dimer content of 13.72% was incubated under reassociating conditions until approx. 30% of the DNA were reassociated. The double-stranded DNA was completely separated

TABLE III D I M E R C O N T E N T O F D N A F R A C T I O N S , O B T A I N E D BY F R A C T I O N A T E D R E A S S O C I A T I O N Fraction

Percentage of total DNA

Average dimer c o n t e n t (%)

0 - - 29 29-- 44 4 4 - - 68 6 8 - - 77 77--100

29 15 24 9 23

11.67 13.24 14.09 15.52 17.02

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off by hydroxyapatite chromatography and the remaining singie-stranded DNA was incubated again, until another part was reassociated, which again was separated from the single-stranded DNA. This procedure was twice repeated. The size and the dimer content of the fractions are listed in Table III. As it can be seen from the table, by this procedure the dimer content increases from the first to the last fraction by almost 50%.

Discussion It is generally assumed, that the rate
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difficult to explain the conflicting results. Again we suspect that the production of interstrand crosslinks has led to an underestimation of the effect of base mismatch. From our data we conclude, that the reduction in rate is observable already at rather small ATm values, although the reduction is not as high as given by Sutton and McCallum. A reduction in rate due to dimer-induced mismatch is further supported by our findings, that the dimer content of early reassociating D N A molecules is below the sample average. The only rational explanation for this result is a decrease in the reassociation rate with increasing dimer content. Since it is reasonable to assume that mismatched bases are also distributed statistically over the repetitive D N A sequences it follows, that in such D N A populations molecules exist, which will reassociate with different rates. Increasing the amount of mismatch, therefore, will not only reduce the overall reassociation velocity but also will cause increasing deviation from "ideal" reassociation kinetics as it is found for prokaryotic DNA. The exact mathematical description of the reassociation reaction of repetitive eukaryotic D N A is obviously very complicated. Presently attempts are made to simulate the course of such a reaction in a computer. Acknowledgement We thank Mrs U. Gtingerich and Mrs H. Steffens for excellent assistance. Professor Dr B. Heicke helped us to cultivate the bacteria, Dr M. Geisert determined the sedimentation coefficients. The investigation was supported by a grant from the Deutsche Forschungsgemeinschaft. References 1 Brltten, R J . (1970) Carnegie Inst. Wash. Yearb. 69, 503---506 2 Southern, E.M. (1970) Nature 227, 794--798 3 Weinblum, D., Giingerich, U., Geisert, M. and Zahn, RJ~. (1973) Biochim. Biophys. Acta 299, 231--240 4 Sutton, W.D. and McCaUum, M. (1971) Nat. New Biol. 232, 83--85 5 McCarthy, B J . and Farquhar, N.N. (1972) Evolut4on o f Genetic Systems, Brookhaven S y m p o s i u m No. 23, p. I , Gordon and Breach, N e w Y o r k 6 Hutton, J,R. and Wetmur, J.G. (1973) Biochemistry 12, 558---563 7 H~nig, W.. Zahn, R.K. and Heitz, W. (1973) Anal. Biochem. 55, 34--50 8 Breter, H J . . Weinblum, D. and Zahn, R.K. (1974) Anal. Biochem., in the press 9 Ben-lshai, R., Ben-Hut, E. and Hornfeld, Y. (1968) Isr, J. Chem. 6,769--775 10 Zierenberg, B.E., Kr~rner, D.M., Gemert, M.G. and Kirste, R.G. (1971) P h o t o c h e m . Photoblol. 14, 515--520 11 Prunell, A. and Bernardi, G. (1973) J. Biol. Chem. 248, 3433--3440 12 Hayes, F.N., Williams, D.L., Ratliff, R.L., Varghess, A~/. and Rupert, C.S. (1971) J. Am. Chem. Soe. 93, 4940--4942 13 Kotaka, T. and Baldwin, R.L. (1964) J. Mol. Biol. 9, 323--339 14 Ullman, J.S. and McCarthy, B~/. (1973) Biochim. Biophys. A e t a 294, 416--424 15 Gralla, J. and Crnther, D.M. (1973) J, Mol. Biol. 78, 301--319 16 Brltten, R ~ . and K o h n e , D.E. (1968) Carnegie Inst. Wash. Yearb. 65, 78--106 17 Lamola, A.A. and Yamane, T, (1967) Proc. Natl. Acad. Sci. U.S. 58, 443--449 18 Southern, E.M. (1971) Nat. N e w Biol. 232, 82---83 19 Hutton0 J.R. and Wetmur, J.G. (1973) Biochim. Biophys. Res. C o m m u n , 52, 1148--1155 20 Geiduschek, E.P. (1961) Proc. Natl. Acad. Sin. U,S, 47,950---955 21 Kahn, M. (1974) Biopolymers 13, 669--675