Mutation Research, 244 (1990) 257-263

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Elsevier MUTLET 0372

Correction of the ultraviolet light induced DNA-repair defect in xeroderma pigmentosum cells by electroporation of a normal human endonuclease Gregory J. Tsongalis, W. Clark Lambert and Muriel W. Lambert Department of Pathology, UMDNJ-- New Jersey Medical School and the Graduate School of Biomedical Sciences, 185 South Orange Avenue, Newark, NJ07103 (U.S.A.)

(Accepted26 February 1990)

Keywords: DNA endonucleases;Electroporation;Ultravioletlight damage; Xerodermapigmentosum

Summary Cells from patients with xeroderma pigmentosum, complementation group A (XPA), are known to be defective in repair of pyrimidine dimers and other forms of damage produced by 254-nm ultraviolet (UVC) radiation. We have isolated a DNA endonuclease, pI 7.6, from the chromatin of normal human lymphoblastoid cells which recognizes damage produced by UVC light, and have introduced this endonuclease into UVC-irradiated XPA cells in culture to determine whether it can restore their markedly deficient DNA repair-related unscheduled DNA synthesis (UDS). Introduction of the normal endonuclease, which recognizes predominantly pyrimidine dimers, but not the corresponding XPA endonuclease into UVC-irradiated XPA cells restored their levels of UDS to approximately 80°70 of normal values. Electroporation of both the normal and the XPA endonuclease into normal human cells increases UDS in normal cells to higher than normal values. These results indicate that the normal endonuclease can restore UDS in UVC-irradiated XPA cells. They also indicate that XPA cells have an endonuclease capable of increasing the efficiency of repair of UVC damage in normal cells.

Exposure of cells to ultraviolet radiation produces a variety of lesions in cellular DNA, which, if unrepaired, may have lethal, mutagenic or carcinogenic consequences. The major lesion produced by short wavelength UV (254 nm) (UVC) radiation is the cyclobutane pyrimidine dimer (Patrick and Rahn, 1976). Another important leCorrespondence: Dr. Muriel W. Lambert, Department of Pathology, UMDNJ -- New JerseyMedicalSchool, 185 South Orange Avenue, Newark, NJ 07103 (U.S.A.).

sion is the pyrimidine-pyrimidine (6-4) photoproduct (Patrick and Rahn, 1976; Mitchell, 1988). These photoinduced lesions are removed in mammalian cells by an excision-repair pathway in which a series of specific enzymes recognizes and excises the defective DNA segment and replaces it with newly synthesized DNA (Lehmann and Karran, 1981; Lambert and Lambert, 1987). This process is readily detected by incorporation of [3H]thymidine into repair patches during non S-phase 'unscheduled' DNA synthesis (UDS).

0165-7992/90/$ 03.50 © 1990ElsevierSciencePublishers B.V. (BiomedicalDivision)

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The human genetic disorder, xeroderma pigmentosum (XP), is a recessively transmitted disease characterized by a hypersensitivity to UV radiation and a high incidence of cancer in tissues exposed to sunlight (for reviews see Cleaver, 1974; Kraemer et al., 1987; Lambert and Lambert, 1987). 8 complementation groups, each deficient in the excisionrepair pathway and in removal of UV photoproducts, are currently recognized for this disorder, a ninth having recently been withdrawn (Kraemer et al., 1987; Lambert and Lambert, 1987; Bootsma et al., 1989). XP, complementation group A (XPA), is one of the most severely affected of the excision-deficient X P complementation groups (Kraemer et al., 1987; Lambert and Lambert, 1987). In XPA cells this repair defect has been shown to occur at the level of the initial, endonuclease mediated, incision step (Cleaver, 1974; Setlow, 1978) yet the molecular mechanisms involved in this defect remain unclear. We have isolated several chromatin-associated DNA endonucleases from the nuclei of normal human lymphoblastoid cells which are selectively active on different types of DNA lesions (Lambert et al., 1983, 1988; Lambert and Parrish, 1989). One of these endonucleases, pI 7.6, is active on UVCirradiated DNA; our results indicate that it recognizes pyrimidine dimers (Lambert and Parrish, 1989). This same endonuclease is also present in the nuclei of lymphoblastoid cells from XPA patients and can incise UVC-irradiated non-nucleosomal DNA (Parrish et al., in preparation). These results indicate that the repair defect in XPA cells does not reside in the ability of this endonuclease to act on damaged non-nucleosomal DNA. We have recently shown that the defect resides in the ability of the endonuclease to interact with UVC-irradiated DNA when it is in the form of nucleosomes (Parrish et al., in preparation). Recently, electroporation has been utilized for the introduction of macromolecules into cells. Exposure of the cells to a brief, high voltage electronic pulse temporarily and reversibly permeabilizes ceils to exogenous molecules (Zimmermann, 1986; Potter, 1988; Shigekawa and Dower, 1988). This method has been used for the introduction of DNA

(Potter et al., 1984; Chu and Berg, 1987; Andreason and Evans, 1988) and of restriction enzymes into cells (Winegar et al., 1989). We now report the ability of a normal human DNA endonuclease, p i 7 . 6 , to complement the repair defect in X P A lymphoblastoid cells, in culture, treated with UVC radiation. This was accomplished by introducing this endonuclease into UVC-irradiated X P A cells via electroporation and observing correction of the X P A repair defect by monitoring UDS levels autoradiographically. Materials and methods

Normal human (GM 1989 and GM 3299) and X P A (GM 2345A and GM 2250A) lymphoblastoid cells, transformed with Epstein-Barr virus (Coriell Institute for Medical Research, Camden, N J) were grown in suspension culture in RPMI 1640 medium buffered with Hepes buffer and supplemented with 15°70 heat inactivated horse serum (Grand Island Biological Co.) (Okorodudu et al., 1982). The cells were maintained in logarithmic growth phase. The human DNA endonuclease, pI 7.6, was extracted from chromatin-associated proteins from isolated lymphoblastoid cell nuclei as previously described (Lambert et al., 1982, 1988). Normal or XPA lymphoblastoid cells, which were to be exposed to UVC radiation, were resuspended in 1 ml ice-cold phosphate-buffered saline (PBS), without added MgCI2 or CaCI2, at a density of 3 x 106 cells/ml in 35-mm 3 tissue-culture dishes. Cells were subjected to 35 J / m 2 UVC radiation from a germicidal lamp. After UVC irradiation, the cells were resuspended in 1 ml ice-cold PBS to which the normal or X P A endonuclease, pI 7.6, or Micrococcus luteus UV endonuclease (Applied Genetics, Inc., Freeport, NY) was added. The endonucleases were then introduced into the cells by electroporation using a BTX Transfector 300 System (Biotechnologies, Inc.). The cells were exposed to an electric pulse with a field strength of 1.7 kV/cm over a distance o f 3.5 mm for approximately 4.0 milliseconds. The cell suspension was then incubated on ice for 10 min and resuspended in 2 ml RPMI 1640 medium,

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buffered with Hepes buffer and supplemented with 15°70 horse serum, prewarmed to 37°C. The ability of the cells to perform excision repair of damage induced by UVC radiation was monitored by measuring levels of UDS. The cells were pulsed for 2 h with l0 ~tCi/ml [3H]thymidine (spec. act. 61 Ci/mole) (ICN Radiochemicals) at 37°C. The cells were then washed twice with ice-cold PBS, smeared onto glass slides, and prepared for autoradiography using Kodak NTB3 emulsion. Slides were stained with Giemsa and cells with 40-100 silver grains per nucleus were classified as undergoing UDS. Results

Following introduction by electroporation of the pyrimidine dimer specific endonuclease from M. luteus, UVC-irradiated XPA cells showed levels of 100

~NONE m ML .UTEUS m NORMAL

-- ~ X P A

80-

UDS 78°70 of those of UVC-irradiated normal cells (Fig. 1). UDS levels in XPA cells either electroporated without enzyme or not electroporated were approximately 12°70 or less of those observed in normal cells following UVC irradiation (Fig. 1). Less than 2°70 of XPA cells irradiated with UVC showed UDS. The electroporation process did not detectably effect UDS in untreated or treated cells. Electroporation of the normal human endonuclease, pI 7.6, into XPA cells treated with UVC radiation resulted in restoration to approximately 80°7o of normal levels. This was similar to the results we obtained with the M. luteus endonuclease (Fig. 1). When the corresponding XPA endonuclease was electroporated into UVCirradiated XPA cells, however, no significant increase in UDS was observed (Fig. 1). The increase in UDS levels induced in XPA cells by electroporation of either the M. luteus or the normal human endonuclease, pI 7.6, was concentration-dependent, with concentrations below or above optimal producing lesser increases in UDS in XPA cells (Fig. 2). For the M. luteus enzyme, the optimal concentration was 5.0/zg/ml, and for the normal human enzyme 1.4 #g/ml (Fig. 2). Thus the human endonuclease was somewhat more efficient in our system, since the degree of correction of UDS in XPA cells by both endonucleases was

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ENDONUCLEASE Fig. 1. UDS in XPA lymphoblastoid cells treated with 35 J / m 2 UVC radiation and electroporated either without enzyme or with the normal human or XPA DNA endonuclease, pI 7.6 (1.4 /Lg)or the M. luteus UV endonuclease (5/~g). Results are expressed as percent of normal UDS + SEM for 4-5 separate Expts. with a total of 1.5 x 103 to 2.5 × 103 cells counted.

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Fig. 2. Influence of enzyme concentration on UDS in XPA cells treated with 35 J / m 2 UVC radiation and electroporated with (A) M. luteus UV endonuclease or (B) normal human (= =) or XPA (O-----O) DNA endonuclease, pI 7.6. Results are expressed as percent of normal UDS + SEM for 3-5 separate Expts. with a total of 1.0x 103 to 2.5 x 103 cells counted.

260

about the same. Pretreatment of the normal human endonuclease with proteinase K, prior to electroporation, abolished the correction of the XPA defect (data not shown), indicating that the correcting factor is a protein. Electroporation of the normal endonuclease, pI 7.6, into normal cells resulted in an increase in UDS above that observed in normal cells electroporated without enzyme (Fig. 3). Electroporation of the XPA endonuclease, p! 7.6, into normal cells also produced UDS levels above normal (Fig. 3). All of these studies were carried out using two different normal and two different XPA cell lines with similar results obtained between cell lines of the same type. Discussion

UV-induced UDS has been reported to be par150 l D

NORMAL ENDONUCLEASE XPA ENDONUCLEASE

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Fig. 3. UDS in normal lymphoblastoid cells treated with 35 J / m 2 U V C radiation and electroporated with normal or X P A D N A endonuclease, p l 7.6 (0.7/~g). Results are expressed as percent of normal UDS (100%) _+ SEM for 4-5 separate Expts. with a total of 1.2 x 103 to 2.5 x 103 cells counted.

tially restored in XPA cells by the microinjection of HeLa cell and human placental extracts (de Jonge et al., 1983; Vermeulen et al., 1986; Yamaizumi et al., 1986). Introduction of T4 phage endonuclease V or M. luteus endonuclease into XPA cells via microinjection, permeabilization of the cell membranes, and fusion of cell membranes with inactivated Sendai virus has also been shown to restore UDS levels in these cells (Tanaka et al., 1975; Smith and Hanawalt, 1978; Ciarrocchi et al., 1979; Dresler et al., 1982; Obe et al., 1985; de Jonge et al., 1985; Yamaizumi et al., 1989). We have recently isolated and identified an endonuclease, pi7.6, from normal cells which recognizes pyrimidine dimers produced by UVC (Lambert and Parrish, 1989). This endonuclease also recognizes monoadducts produced by psoralen plus long wavelength (UVA) radiation (Lambert et al., 1988; Parrish and Lambert, 1990). Since the covalent addition of psoralen to DNA involves a cyclobutal linkage similar to that of the pyrimidine dimer (Vigny et al., 1985; Cimino et al., 1985), it is possible that this is the type of lesion recognized by this endonuclease. We wished to ascertain whether this normal human endonuclease functions in DNA repair of UVC radiation-induced damage and whether, when introduced into UVC irradiated XPA cells, it can correct their repair defect. The present study has utilized electroporation to introduce the endonuclease, pi7.6, into UVCirradiated XPA cells in culture. The results show that this endonuclease is capable of restoring UDS in UVC-irradiated XPA cells to 80°70 of the normal value. The M. luteus UV endonuclease electroporated into XPA cells also restored UDS to a similar level. This value is comparable to those reported by others utilizing different techniques for introduction of the M. luteus UV endonuclease into cells (Dresler et al., 1982; de Jonge et al., 1985). This is probably due to the fact that pyrimidine dimers do not constitute the only photoproduct formed by UVC radiation at low fluences (Patrick and Rahn, 1976; Mitchell, 1988) and both M. luteus and the normal human endonuclease incise DNA at sites of pyrimidine dimers. We have recently shown that

261 this same human endonuclease, pI 7.6, electroporated into XPA cells treated with 8-methoxypsoralen plus UVA, restores UDS levels to greater than 100% of levels observed in similarly damaged normal human cells (Tsongalis et al., 1990). Thus it is unlikely that the endonuclease, pI 7.6, fails to produce 100% correction in UDS in UVC-irradiated XPA cells due to artifactual or technical factors. We have previously shown that the endonuclease, pI 7.6, is also present in XPA lymphoblastoid cells and can incise both UVC-irradiated (Parrish et al., in preparation) and 8-MOP plus UVA treated (Lambert et al., 1988; Lambert and Parrish, 1990) DNA with levels of activity similar to those from normal cells. The present study, however, showed that electroporation of the XPA endonuclease into UVC-irradiated XPA cells did not increase levels of UDS. On the other hand, electroporation of the XPA or the normal endonuclease into normal cells irradiated with UVC resulted in an increase in UDS above that observed in normal cells electroporated without endonuclease. These results indicate that introduction of the normal or of the XPA endonuclease into UVCdamaged normal cells can increase the efficiency of repair in these cells and further supports the hypothesis that XPA cells contain an endonuclease which can repair UVC damage in normal cells, but lack a factor necessary to allow these endonucleases to function in their own cells. This factor is supplied by the normal cells when the XPA endonuclease is introduced into them by electroporation. A number of cell-free systems have been used to examine the XPA defect, but the results have been conflicting. Studies using crude cell extracts suggest that XPA cells are deficient in a factor(s) required for interaction of an endonuclease with damaged DNA in the form of chromatin (Mortelmans et al., 1976; Kano and Fujiwara, 1983). Wood et al. (1988), on the other hand, using soluble extracts of XPA cells, reported defective DNArepair synthesis on damaged non-nucleosomal plasmid DNA. These studies have recently been confirmed by Sibghat-Ullah et al. (1989). How-

ever, such extracts of mammalian cells have been shown to reconstitute naked plasmid DNA into nucleosomes or nucleosome-like structures, visualized ultrastructurally (Manley et al., 1980; Hough et al., 1982). Therefore the defect observed by Wood et al. (1988) and Sibghat-Ullah et al. (1989) may have been due to an inability of a XPA endonuclease(s), in their cell extracts, to interact with damaged DNA in the form of nucleosomes or nucleosome-like structures. This alternative interpretation of their results would be in agreement with our own results (Parrish and Lambert, 1990) and those of Mortelmans et al. (1976) and Kano and Fujiwara (1983). We have recently shown that there is a defect in the interaction of the XPA endonucleases, pls 4.6 and 7.6, with damaged nucleosomal DNA (Lambert and Parrish, 1989; Parrish and Lambert, 1990). Both normal endonucleases, pI 4.6 and 7.6, show greater affinity for and greater activity on UVC and/or on psoralen plus UVA irradiated DNA in the form of reconstituted nucleosomes than on similarly damaged non-nucleosomal DNA. By contrast, neither XPA endonuclease shows additional affinity for or activity on damaged nucleosomal DNA and shows decreased activity when histone H1 is present in the nucleosomes. These results thus indicate that XPA cells have endonucleases which can recognize and incise damaged non-nucleosomal DNA, but which lack the ability to interact with damaged DNA when it is in the form of nucleosomes. We have shown that this defect can be corrected at the nucleosomal level by mixing the XPA endonucleases with the respective normal endonucleases in our cell-flee assay system (Lambert and Parrish, 1989; Parrish and Lambert, 1990). These results, combined with those obtained in the present study, show that the normal endonuclease, pi 7. 6, can correct the XPA repair defect, both in a cell free nucleosomal system as well as in UVC irradiated XPA cells in culture. They also provide further evidence that XPA cells have an endonuclease capable of recognizing UVCdamaged DNA and that this endonuclease can increase efficiency of repair of UVC damage in normal cells.

262

Acknowledgements We would like to thank Robert Lockwood culturing the human

for

cell lines. T h i s w o r k is s u p -

ported by Grant AM 35148 from the National Institutes of Helath.

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Correction of the ultraviolet light induced DNA-repair defect in xeroderma pigmentosum cells by electroporation of a normal human endonuclease.

Cells from patients with xeroderma pigmentosum, complementation group A (XPA), are known to be defective in repair of pyrimidine dimers and other form...
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