Mutation Research, 181 (1987) 57-60 57 Elsevier

MTR 04367

Appearance and repair of apurinic/apyrimidinic sites in D N A during early germination of Zea mays

Eric Dandoy a, Robert Schyns b, Roger Deltour a and Walter G. Verly b Laboratoires de Morphologie V~g~tale ae t de Biochimie b, Facultd des Sciences, Universit~ de Liege, Sart Tilman B22 a and B6 b,

4000 Liege I (Belgium)

(Received 5 January 1987) (Accepted 5 February 1987)

Keywords: Apurinic/apyrimidinic sites; DNA repair; Zea mays; Germination, early.

Summary

The number of AP (apurinic or apyrimidinic) sites found in DNA of radicle cells of Zea mays quiescent embryos after 2 years of storage is low; the low rate of spontaneous base loss is probably due to the low water content of the seed. But this number increases 4-fold during the first 20 h of germination, to decrease between 20 and 25 h, and increase again afterwards. These variations may well be due to competition between the formation of AP sites and their repair during early germination. Formation must be due to DNA glycosylases removing bases which have been damaged during the storage of the seeds. The first increase in the number of AP sites would be due to DNA glycosylases which have survived the storage period; ~e second increase might be the result of the synthesis of new DNA glycosylase molecules.

The nuclear DNA-repair synthesis monitored by autoradiography closely follows the number of AP sites during early germination, suggesting that the repair of most of the damage which has accumulated in DNA during storage has AP sites as an intermediary step.

The DNA of higher plant embryos undergoes damage which accumulates while the seeds are being stored (Osborne, 1980). Incorporation of thymidine into DNA during early germination suggests that part of these lesions might be re- paired before DNA replicates (Cheah and Osborne, 1978); successful repair might be a con- dition which has to be fulfilled before the embryo will grow into a plant.

Cheah and Osborne (1978) observed that al- kaline treatment of D N A from quiescent embryos

Correspondence: E. Dandoy, Laboratoire de Morphologie Vrg&ale, Facult~ des Sciences, Universit~ de Liege, Sart Til- man B22, 4000 Liege I (Belgium).

reveals numerous strand breaks. Such strand breaks have two origins: preexisting strand breaks (which may or may not be near AP sites), and alkali-labile sites (among them intact AP sites). Thinking that the spontaneous loss of bases might be an important cause of DNA degradation dur- ing seed storage, we directly assayed the AP sites in DNA of Zea mays embryos after storing the seeds for two years and during early germination before the first round of nuclear DNA synthesis. During this early germination, we also followed the DNA synthesis. The majority of embryo cells in Zea mays seeds are in G1 (Deltour, 1985) and, when rehydrated, it takes more than 36 h at 16 °C before they enter the S phase of the first mitotic

0027-5107/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

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cycle. Repair of nuclear DNA can thus be studied during this period of early germination in the absence of DNA replication.

Material and methods

The analyzed material After a 2-year storage period at about 20 ° C,

batches of 300 kernels were germinated in the darkness, at 16 ° C, in large petri dishes over cot- ton wool and filter paper saturated with distilled water. Every 6 h, 750 germinating kernels were dissected and the first 2 mm of the radicle of each embryo were cut off and immediately frozen in liquid nitrogen.

Assay of AP sites The excised root tips (750) were lyophilized,

then put in liquid nitrogen and ground. After addition of 7.7 ml 0.05 M Na borate, pH 7.0, 0.01 M EDTA, 1% sarcosyl, the suspension was treated in a Potter homogenizer. Pronase (1.5 mg in 0.75 ml 0.1 M Tris-HC1, 1 M NaC1, pH 7.0) was added and the mixture incubated at 40 °C for 2 h. After centrifugation, the supernatant was kept and the sediment was treated a second time in the same way. To the pooled supernatants, 1.5 ml 5 M NaC1 and 2 vol. of ethanol were added to precipi- tate the DNA. After centrifugation, the sediment was dissolved in 7 ml 1.5 mM Na citrate, 15 mM NaC1, 0.1 mM EDTA, pH 7.0 (SSC/10), contain- ing T1 (700 units) and T2 (140 units) RNAases, and the solution was incubated at 37 °C for 3 h. Proteinase K (0.7 mg in 700 /~1 10 mM NaC1, 5 mM CaCI2) was added and the incubation con- tinued at 37 °C for 2 h. NaC1 was added until the concentration was 2.5 M and the protein extracted twice with chloroform:isoamyl alcohol (24: 1, v:v) . DNA was again precipitated with ethanol. After centrifugation, the sediment was dissolved in SSC/10. All the steps (treatments with RNAases, proteinase K, protein extraction, ethanol precipitation) were repeated and the final DNA sediment dissolved in 1 ml 10 mM Na phosphate, 10 mM NaCI, pH 7.2. The DNA concentration was measured using the diaminobenzoic acid method of Thomas and Farquhar (1977).

To 114 /~1 DNA solution, 6 /zl 100 mM [14C]methoxyamine (NEN; 6.21 Ci /mole) in 10

mM Na phosphate, 10 mM NaCI, pH 7.2, were added and the mixture was incubated at 37 °C for 30 min (Talpaert-Borl6 and Liuzzi, 1983); three 35-btl aliquots were then placed on fiberglass filters which were immersed in 1 M ice-cold HC1 in a shaker; the HC1 solution was replaced after 1, 2 and 3 h. The filters were then rinsed 5 times with 1 M ice-cold HC1, 10 times with 25 mM metho- xyamine in 1 M ice-cold HC1, 7 times with ethanol, and finally twice with diethyl ether. The filters were dried for 30 min at 60 °C in counting vials and, after addition of the scintillation solution, assayed for radioactivity. The radioactivity of con- trois, carried out in the same way except that the initial solution did not contain DNA, was sub- tracted. Taking into account the [14C]methoxya- mine specific radioactivity and the amount of DNA in the sample, the AP site frequency in DNA (number of AP sites per 10 6 nucleotides) was calculated. The results given below are the averages of two independent [14C]methoxyamine assays using 6 filters for each germination time.

DNA-repair synthesis After 0, 7, 13, 19, 25 or 31 h of germination, the

excised whole embryos were immersed in a solu- tion of [3H]thymidine (Amersham; 5 Ci /mmole ; 1 /~Ci/ml) for 5 h, fixed at 4 ° C with 4 % glutaraldehyde in 0.1 M Na cacodylate buffer, pH 7.0, and washed with the same cold buffer. After a 1-h post-fixation in 2% OsO 4 at 4°C, they were washed with water, dehydrated in ethanol, and embedded in Epon. 1-/~m thick cross-sections were cut at 1 mm from the radicle tip. Autoradiography was carried out with a 4-fold dilution of Ilford L4 emulsion.

In order to check the specificity of [3H]thymi- dine incorporation, after the 5-h incubation with the radioactive deoxynucleoside, control embryos were fixed for 1 h in 1% formaldehyde, washed overnight with cold cacodylate buffer, exposed for 30 min to 10 mM HC1 at 20 °C to detach histones from DNA, and rinsed in water. Thin slices of radicle tissues were then incubated with DNAase I (Boehringer; 4 mg in 25 ml 3 mM MgSO4, pH 6.5) at 37°C for 11 h, then at 16°C for 13 h, and washed with cold cacodylate buffer. Postfixation in OSO4, rinsing, dehydration and embedding in Epon were carried out as above. 1-/tm thick sec-

tions were randomly cut and submitted to autora- diography.

After a 20-day exposure, the autoradiographs were developed and the sections stained with toluidine blue. Grains were counted above the cortical cells; for each germination time, 300 cells were examined, 50 in each of 6 different prepara- tions, and 150 cells for the DNAase-treated con- trois.

Results and discussion

The A P sites The D N A of embryos in seeds stored for 2

years at 2 0 ° C contains 38 AP sites per 106 nucleotides (Fig. 1); this means a rate of base loss of about 2 × 10- 5 per year. According to Lindahl and Nyberg (1972), the spontaneous depurination rate for naked D N A in water solution at 20 ° C is 2.5 × 10 -4 per year; since the depyrimidination rate is much slower (Lindahl and KarlstriSm, 1973), this means a rate of base loss of 12.5 × 10 -3 per year, i.e. 6 times faster than what is observed in

10 •

5 150 ~_

o 6 12 18 2/, 30 36 h

Fig. 1. AP sites and DNA-repair synthesis in radicle cells during early germination of Zea mays seeds. The seeds were placed on water-soaked cotton wool and filter paper in petri dishes in the dark at time 0. The AP sites in the isolated DNA were counted with [14C]methoxyamine; the results (A) are given in numbers of AP sites per 10 6 nucleotides (right scale). At different times of germination, the embryos were exposed for 5 h to [3H]thymidine and the radioactivity incorporated into DNA measured by autoradiography; the number of silver grains are given per cell (left scale), above the nucleus (O) and above the cytoplasm (El). Vertical bars represent the standard errors of the means used to draw the figure (an example of error calculation is given in Table 1).

59

dry seeds. It is unlikely that the difference is due to the repair of AP sites; it probably has two other causes: the D N A is in the chromatin structure; the dehydration of the seed must slow down the hy- drolytic loss of bases.

But the most surprising result is the 4-fold increase in AP sites during the first 20 h of germination (Fig. 1). This rapid increase is prob- ably due to D N A glycosylases which were inactive in the dry seed and which were reactivated by rehydration. These D N A glycosylases probably removed altered bases. Consequently, it seems that the lesions that accumulate in D N A during stor- age of the seeds are not so much AP sites, but rather damaged bases.

The number of AP sites decreases between 20 and 25 h of germination to increase again after- wards. The decrease indicates repair. Between 20 and 25 h of germination, the rate of AP site repair is greater than the rate of their formation. It is probable that the rate of their formation de- creases, either because most of the damaged bases have already been eliminated or because the activ- ity of the D N A glycosylases which had survived the storage of the seeds decreases. The second explanation is probably the more important since the number of AP sites increases again after 24 h. One hypothesis is that the embryo cells had time to synthesize new molecules of D N A glycosylases to completely clean the D N A before the first round of nuclear D N A replication.

DNA-repair synthesis Radioactivity incorporation during a 5-h ex-

posure of the embryos to [3H]thymidine at differ- ent times of germination was measured by autora- diography (Fig. 1, Table 1). The lack of strongly labelled nuclei in all the observed embryos (except one at 36 h) showed that no cell had entered the S phase during the studied period of germination. The mean number of silver grains per nucleus, counted over sections of this organelle, followed closely the number of AP sites in D N A during the first 36 h of germination. This suggests that most of the damage to embryo cell D N A in the stored seeds leads to AP sites as an intermediary step during their repair.

The large number of silver grains over the cytoplasm is probably due to the replication of

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TABLE 1

DNA REPAIR SYNTHESIS IN Zea mays RADICLE CELLS AFTER 19 h OF GERMINATION

DNAase

- + A

Nucleus 1.58+0.13 0.12+0.03 1.46+0.13 Cytoplasm 7.45 + 0.43 0.64 + 0.08 6.81 + 0.44

After 19 h of germination, Zea mays embryos were exposed for 5 h to [3H]thymidine. The radioactivity incorporated into DNA was measured by autoradiography of 1 #m-thick sec- tions. The mean number of silver gains per cell, either over the nucleus or the cytoplasm, was corrected for background; 50 cells were examined in each of 6 different preparations and the error is the standard deviation on the mean.

Controls were treated with DNAase before autoradi- ography. Control values are subtracted from the values of the specimen not treated with DNAase to give the net results (A) used in Fig. 1.

m i t o c h o n d r i a l a n d p r o p l a s t i d i a l D N A wh ich oc-

curs i n d e p e n d e n t l y f r o m the r ep l i ca t i on o f nuc l ea r

D N A . Th i s i n t e r p r e t a t i o n is s u p p o r t e d by the

o b s e r v a t i o n tha t the r e l a t ive v o l u m e s o f m i t o c h o n -

d r i a a n d p r o p l a s t i d s inc rease by 35% a n d 138%

respec t ive ly d u r i n g the f irs t 48 h o f g e r m i n a t i o n

( u n p u b l i s h e d results) .

A c k n o w l e d g e m e n t s

Th i s w o r k was s u p p o r t e d by a g ran t f r o m the

F o n d s de la R e c h e r c h e F o n d a m e n t a l e Co l l ec t ive

o f B e l g i u m a n d b y the " A c t i o n s conce r t6e s " No .

8 0 / 8 5 - 1 8 of the Be lg ian g o v e r n m e n t .

R e f e r e n c e s

Cheah, K.S.E., and D.J. Osborne (1978) DNA lesions occur with loss of viability in embryos of ageing rye seed, Nature (London), 272, 593-599.

Deltour, R. (1985) Nuclear activation during early germination of the higher plant embryo, J. Cell Sci., 75, 43-83.

Lindahl, T., and O. KarlstriSm (1973) Heat-induced depyri- midination of DNA in neutral solution, Biochemistry, 12, 5151-5154.

Lindahl, T., and B. Nyberg (1972) Rate of depurination of native deoxyribonucleic acid, Biochemistry, 11, 3610-3618.

Osborne, D.J. (1980) Senescence in seeds, Chap. 2 in: K.V. Thimann (Ed.), Senescence in Plants, CRC Press.

Talpaert-Bod6, M., and M. Liuzzi (1983) Reaction of apurinic/apyrimidinic sites with [14C]methoxyamine, A method for the quantitative assay for AP sites in DNA, Biochim. Biophys. Acta, 740, 410-416.

Thomas, P.S., and M.N. Farquhar (1977) Specific measure- ment of DNA in nuclei and nucleic acids using diamino- benzoic acid, Anal. Biochem., 89, 35-44.