dna damage induced by uv light affects restriction endonuclease
TRANSCRIPT
Mutagenesis vol.11 no.5 pp.463-466, 1996
DNA damage induced by UV light affects restriction endonucleaserecognition sites: correlation between effects at chromosomal leveland naked DNA
A-Sgura1, RJMeschini2, AAntoccia3, F.Palitti2, G.Obe4
and C.TanzareUa3-5
'Department of Genetics and Molecular Biology, University 'La Sapienza',Rome, ^Department of Agrobiology and Agrochemistry, University ofTuscia, Viterbo, 3Department of Biology, III University of Rome, Rome,Italy and 4Department of Genetics, University of Essen, Essen, Germany5To whom correspondence should be addressed at: Dip. di Genetica eBiologia Molecolare, Universita 'La Sapienza', P.le A. Moro 5,00185-Roma, Italy
Cytogenetic and molecular analyses were performed in GjChinese hamster ovary cells treated with 254 nm UV light,to test the hypothesis that UV may affect the recognitionsite of specific restriction endonucleases (RE). Since short-wavelength UV light induces mainly cyclobutane dinners(CPD) at TT and CT sequences, RE were selected accordingto the presence or absence of thymine at the recognitionsite (Dral TTT/AAA, A/i/I AG/CT and HaeJU GG/CC). Adrastic reduction of Dral- and A/uI-induced chromosome-type aberrations was found in cells pretreated with UV.Conversely, such a reduction was not observed withHaelll. To better understand this phenomenon, a mole-cular analysis was carried out at both the genome leveland at the hypoxanthine phosphoribosyl transferase genelevel, showing that the cutting pattern of Dral on isolatedDNA from UV-irradiated cells was strongly reduced com-pared with an untreated sample, whereas Haelll was notable to modify the cutting pattern of irradiated cells. Ourdata demonstrate a good correlation between the resultsobtained with cytogenetic and molecular approaches, sug-gesting that cyclobutane dimers are the main lesionsresponsible for the observed reduction of the cleavingactivities of RE, at both the chromatin and naked DNAlevels.
IntroductionThe treatment of living cells with both chemical and physicalagents may modify the structure of chromatin (Smerdon andLiebertnan, 1978; Obe et al, 1987b). Chromatin structuralalterations can affect the yield of chromosomal aberrationsinduced by DNA stranded scission agents (Dettor et al., 1972;Sankaranarayanan et al, 1990; Vyas et al, 1991; SakamotoHojo et al., 1994). The mechanism responsible for UV-inducedmodifications of chromatin structure may include a transientstructural alteration (Ljungman et al, 1989) or the presenceof UV-induced specific lesions. In fact, UV-treatment inducescyclobutane dimers (CPD), pyrimidine-pyrimidone (6-4)photoproducts and DNA-protein cross-links. At low UV dosesthe prevalent lesions are the cyclobutane dimers, while the(6-4) photoproducts become the predominant photolesion atdoses >5000 J/m2 (Bianchi et al, 1991). CPD are formed atTT, TC-CT and CC sequences, with a frequency of 50, 40and 10% respectively (Ellison and Childs, 1981). Stucturalstudies indicate that the presence of CPD leads to a distortion
of the DNA double helix (Pearlman et al, 1985), which couldprevent DNA cleavage in the restriction site of a givenendonuclease when assessed at gene level on isolated DNA(Bianchi et al, 1990). So far, no data are available inthe literature on the effect of UV-treatment on restrictionendonuclease (RE)-induced chromosomal damage in livingcells where chromatin is organized in a high order structure.It is known that RE induces chromosomal aberrations (CA) inmammalian cells throughout the production of DNA double-strand breaks at specific recognition sites (Bryant, 1984;Natarajan and Obe, 1984).
In this context our aim was to gain more insights intothe relationship between UV-induced alteration of chromatinstructure, modification of RE recognition sites and productionof CA in living cells. CA analysis was performed in G] Chinesehamster ovary (CHO) cells exposed to short-wavelength UVlight and subsequently treated with Dral (TTT/AAA), Haelll(GG/CC) and AM (AG/CT), characterized by the presenceor absence of thymine at the recognition sites. Molecularexperiments were carried out to further correlate the effect ofUV damage in modifying RE cutting activity.
Materials and methods
Cytogenetic experimentsCHO cells were routinely grown in McCoy's 5A medium (Flow Laboratories,Scotland) supplemented with 10% fetal calf serum (Flow Laboratories),penicillin (5000 IU/ml) and streptomycin (5000 u.g/ml) at 37°C in 5% CO2.Cells (5X103) were plated as a monolayer on 35 mm plastic Petri dishes 3 hbefore treatment. Cells were washed once with phosphate-buffered saline(PBS) and then exposed to 4 and 8 J/m2 of 254 nm UV light (Philips UVG-D68, dose rate 0.68 J/s), which are expected to induce respectively 8.96X 104
and 1.792X105 cyclobutane dimers for the CHO haploid genome. Some ofthe dishes were subsequently incubated with 100 |il of a mixture containing1:1 glycerol and distilled water, as the permeabilizing agent (Johannes andObe, 1991), with McCoy's 5A medium and either 40 U Dral (BoehnngerMannheim), 7 U Haelll (Boehnnger) or 7 U of Alul (Promega, USA) for30 min at 37°C in a 5% CO2 incubator. The units of RE were selected so asto obtajn similar yields of chromosomal damage on the basis of pilotexperiments. Control cultures (untreated or UV irrradiated) were carried outby exposure of cells with a mixture containing glycerol, McCoy's 5A and theappropriate amount of storage buffer of the respective RE. Cells werewashed once with PBS and incubated with complete medium containingbromodeoxyuridine (5XI0~* M) for 19 h, including a 2 h treatment withcolchicine (10~5 M). The number of mitotic cells collected by the mitoticshake-off method was in the same range as the control and treated samples,suggesting that no mitotic delay occurred.
Metaphase chromosomes were stained using the fluorescence plus Giemsatechnique (Perry and Wolff, 1974), and 100 first metaphases were scored forchromosomal aberrations according to the guidelines of the IAEA (1986) foreach experimental point. Experiments were repeated at least three times.
Molecular experimentsCHO cells (2X103) were seeded on 135 mm Petri dishes and grown for 48 hto confluence. Before UV irradiation the cells were washed once with PBSand then exposed on ice to 1000 and 3000 J/m2 of 254 nm UV light. Cellswere immediately lysed in 10 mM Tris-HCl, pH 8.0, 150 mM NaCI, 1 mMEDTA, 0.5% SDS and 100 |lg/ml proteinase K (Boehringer, Mannheim) at37°C for 16 h. DNA was isolated following the standard extraction procedureand RNA was digested with 50 Jig/ml RNase A (Sigma, St Louis, USA).Purified DNA (15 ug) from each UV dose and from untreated cells was
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A_Sgura el al.
digested with either Dral (3 U enzyme/u.g DNA) or Haelll (3 U enzyme/p.gDNA) by incubation for 4 h at 37°C, and the reaction was quenched byadding 10 mM EDTA and heating at 65°C for 10 min. After digestion, DNAsamples were loaded onto a 0.8% neutral agarose gel and subjected toelectrophoresis at 25 V for 16 h in TAE (40 mM Tns-acetate plus 10 mMEDTA) buffer. The gel was photographed under UV illumination and theDNA was transferred to Hybond N+ (Amersham) by vacuum Southern blottingand hybridized with a 32P-labelled hypoxanthine phosphonbosyl transferase(HPRT) gene-specific c-DNA probe comprising exons 6-9. Membranes wereexposed to Kodak XOMAT-S films at -80°C using intensifying screens. Thedifference in the UV doses used in the molecular and cytogenetic expenmenLswas related to the different experimental procedures. In fact, the cytogeneticanalysis was carried out at the single cell level, whereas the molecularapproach was performed on DNA isolated from a large number of damagedand undamaged cells.
ResultsThe cytogenetic results shown in Table I represent data froma typical experiment chosen from the three experiments thatshowed a similar trend. The frequency of UV-inducedchromatid-type aberrations, ranging from 16.8 to 48.2%, wascorrelated with the UV doses used. Post-treatment with theRE buffer led to a high frequency of UV-induced aberrations.This may be ascribed to a general change in the superstructureof chromatin due to salts contained in the buffer. On the otherhand, the frequency of UV-induced chromatid-type aberrationswas not modified when cells were post-treated with RE.
RE alone induced only chromosome-type aberrations whichwere significantly reduced when cells were pre-treated withUV light and post-treated with Dral and Alul. No such effectwas observed with Haelll.
Figure 1 shows the influence of UV light on the frequenciesof dicentrics and rings in cells treated with RE. Chromosome-type aberrations showed a statistically significant reductiononly for Dral and Alul, with values ranging from 65 to 84%and 59 to 62% respectively.
In Figure 2 is shown the agarose gel electrophoresis per-formed on the DNA isolated from cells irradiated with 1000and 3000 J/m2 of UV light and digested with Dral and HaelU.The efficiency of Dral in cleaving UV-irradiated samples ascompared with the non-irradiated ones indicated a dose-related decrease. This was not observed with Haelll. Theautoradiogram obtained with the Southern blotting techniqueand hybridization with a radioactive HPRT gene-specific c-DNA probe (Figure 3) confirmed that there was a modificationof the cutting pattern at the single copy gene level. The effectof UV irradiation on the cutting pattern of Dral producedincomplete DNA digestion with a reduction in the intensity ofthe main band present in the non-irradiated sample, and theproduction of additional bands of higher molecular weight.This difference in the restriction pattern was not seen whenisolated cellular DNA was treated with Haelll.
Table I. Frequency
Treatment
ControlUV 4 J/m2
UV 8 J/m2
Buffer OralUV 4 J/m2
+ buffer DralUV 8 J/m2
+ buffer DralDralUV 4 J/m2
+ DralUV 8 J/m2
+DraIBuffer HaelllUV 4 J/m2
+ buffer HaelUUV 8 J/m2
+ buffer HaelllHaelllUV 4 J/m2
+ HaelllUV 8 J/m2
+ HaelllBuffer AlulUV 4 J/m2
+ buffer AlulUV 8 J/m2
+ buffer AlulAlulUV 4 J/m2
+ AlulUV 8 J/m2
+ Alul
of chromosomal
Aberrantmetaphases (%)
_
9.233.66.0
38.0
40822.5
24.3
41.47.0
27.6
36.021.0
346
50 336
183
44.018.6
17.6
38.0
aberrations induced by
Aberrations per
Chromatid type
AL
_
0.8_1 5
1 0
2.01 3
23
0.4_
0.4
0.80.3
_
1 02.3
33
3.01 3
1.0
23
100
B'
_
366.02.0
24.8
33.25.6
18.6
25.90.5
28.8
15.61 9
14.0
15.00.6
7.3
17.030
1.6
12.6
UV light and REs
metaphases
B"
_
2.02.81.2
5.6
5.623.0
9.6
9.63.5
3.2
7.214.6
16.3
14.61.0
1.6
6.32.0
3.0
20
in the G| phase
RB'
_
11 239 624
304
38.820
183
40.43.0
29.6
33.21.6
26.0
38.6-
21 0
58 320
25 3
53 3
of CHO cells
Total (- AL)
_
16848.2
56
608
77.630 6
65.1
75.97.0
61.6
56.018 1
56 3
68.21 6
29 9
81 670
29 9
67 9
Chromosome
Die.
_
-
1 00.4
2.5
2.212.3
5.0
4.01 0
1.2
4.611.8
74
1000.3
_
1 610.0
1 3
2.0
type
Rings
_
--_
_
-
1 3
0.6
1.20.5
0.4
1.22.6
23
2.0-
0.3
0.63.0
06
1 0
Total
_-1.004
25
2.213.6
5.6*
5.2*1.5
1.6
5.814.4
9.7
12.00 3
0.3
1.613.0
1.9*
3 0*
Abbreviations: AL. achromatic lesions: B'. chromatid breaks. B". iso-chromatid breaks; RB'. chromatid exchanges. Die. dicentnesSignificance according to Student's l-test *P < 0 05.
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Effects of UV light on RE recognition sites
Discussion
It has been reported that treatment conditions can affect thechromatin structure and consequently modify the rate ofchromosomal aberrations induced by ionizing radiation (Dettoretal., 1972; Sankaranarayanan ef ai, 1990; Vyas et ai, 1991)
16
14
12
10
os 6
• RH
l \
" IA
J4J+RI.
KJ.RI
wDRA
iii
I
1 HAE ! ALU I
Fig. 1. The frequencies of chromosome-type aberrations (Dicentrics, Rings)induced by RE alone or in combination with 4 or 8 J/m~ UV light. REversus RE + UV, significant according to Student's (-test: *P < 0.05.
and restriction endonucleases (Sakamoto Hojo et ai, 1994).Treatment of rodent and human cells with Alul in the presenceof hypertonic concentrations of various inorganic salts led toincreased frequencies of chromosomal aberrations (Obe andKamra, 1986; Obe et ai, 1986). The reverse effect wasobserved with heat shock treatment (Vasudev and Obe, 1987).Experimental evidence on specific chemical modifications ofthe RE recognition site indicate that substitution of thyminewith 5-bromodeoxyuridine in mammalian cells treated withRE containing thymine residues in the recognition site led toa decreased yield of chromosomal aberrations (Stoilov et ai,1986; Obe et ai, 1987a; Cortes and Ortiz, 1992). However,in this context the relationship between UV-induced chromatinalterations and RE-induced chromosomal aberrations havebeen poorly investigated. Since UV has been shown to inducea transient structural alteration and specific lesions, thesephenomena may affect the RE recognition site and conse-quently RE-induced chromosomal damage.
Our results show that pre-treatment with UV led to a lowerfrequency of chromosome-type aberrations when cells werepost-treated with either Dral or Alul. Interestingly, thereverse effect was observed when cells were UV-treated andimmediatly exposed to X-rays (Holemberg and Gumauskas,1986). Futhermore, enhancement is likely to occur as a resultof a synergic interaction between UV- and X-ray-inducedlesions during repair activities. Although double-strand breaksare recognized as the ultimate lesions leading to chromosomalaberrations as induced by X-rays and RE (Natarajan and Obe,1978; Bryant, 1988), the effects observed after post-treatmentwith X-rays may be ascribed to their ability to cut DNA in anon-specific manner.
It should be noted that in our experimental conditions a
UK \ I
I I K \ I II W III
Fig. 2. Cutting pattern by RE as shown by agarose gel electophoresis ofisolated DNA from CHO cells irradiated with 1000 and 3000 J/m2 of UVlight.
Fig. 3. Autoradiogram of DNA isolated from CHO cells irradiated with1000 and 3000 J/m2 of UV light and subsequently digested with Dral andHaelll.
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reduction in the frequency of chromosome-type aberrations wasobserved only when RE recognition sites were characterized bythe presence of thymine residues. The observation that therewas no dose response following UV exposure may be due tothe number of sites recognized by RE at the concentration used.
Treatment with HaelQ showed neither a reduction nor anenhancement of chromosome-type aberration frequency, andled to the following suggestions: (i) UV- and RE-inducedlesions do not interact in chromosomal damage induction;(ii) our observation indicates the importance of thymine in theRE recognition site; and (iii) the contribution of transitory andaspecific chromatin structural modification should be excluded.Furthermore, biochemical data indicate low production ofpyrimidine-pyrimidone (6-4) photoproducts in cellular DNAat doses <5000 J/m2 and lack of DNA-protein cross-links atshort wavelength (Bianchi et al, 1990). Consequently, ourcytogenetic data may indicate cyclobutane dimers as the maintype of lesion responsible for the decreased RE activity.
To further investigate our hypothesis and correlate the effectof UV damage in modifying RE cutting activity at cytogeneticand molecular levels, a molecular analysis was performed.The data obtained with Dral and HaeUl at the overall genomeand HPRT gene-specific levels support the cytogenetic findingsand rule out the contribution of other chromatin modifications,such as DNA-protein cross-links and temporary alterations,with only cyclobutane dimers playing a role in the observedreduction of the cleaving activity of RE on isolated DNA fromUV-irradiated cells. In fact, the presence of a cyclobutanedimer leads to a distortion of the DNA double helix of 19.7°and introduces a kink of 27.0° that protrudes into the majorgroove: at the site of the dimer the helical displacement is2.66 A (Pearlman et al., 1985). Such a distortion could produce:(i) the inability of RE to recognize its restriction site or (ii)the inability of RE to cut at its recognition site even if theenzyme is still able to recognize it. In conclusion, our dataindicate that cyclobutane dimers are the main lesions respons-ible for the observed reduction in RE cleaving activity inisolated DNA as well as in chromatin organized in a high-order chromosomal structure.
AcknowledgementsS.A. is the recipient of a fellowship from Consigho Nazionale delle Ricerche.Roma and A A. is a post-doctoral fellow of "III I'niversita di Roma'
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Received on September 26. 1995. accepted on April 15. 1996
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