Carcinogenesis vol.11 no.3 pp.499-503, 1990

SHORT COMMUNICATION

Eledtroporatiom off mormal tamami DNA eedomiiiiclleases into ceis corrects tlheir EDNA repair defect

Gregory J.Tsongalis, W.Clark Lambert and Muriel W.Lambert1 Department of Pathology, UMDNJ, New Jersey Medical School and the Graduate School of Biomedical Sciences, 185 South Orange Avenue, Newark, NJ, USA 'To whom correspondence should be addressed

Xeroderma pigmentosum (XP*) is a genetically transmitted disease characterized by extreme sensitivity to ultraviolet radiation and a high incidence of cancer in tissues exposed to sunlight (1,2). In the eight currently accepted classical XP complementation groups there is a defect in the excision-repair pathway which acts on pyrimidine dimers and other adducts induced in DNA by short wavelength ultraviolet (UVC) radiation as well as on lesions produced by a variety of different agents ( 1 - 3 ) . Although the defect has been shown to occur at the level of the initial endonuclease mediated incision step, the precise molecular mechanisms responsible remain unclear. We have isolated several chromatin-associated DNA endonucleases from the nuclei of normal human lymphoblastoid cells which are selectively active on DNA containing different types of lesions (4-6). We have shown that these endonucleases are also present in the nuclei of lymphoblastoid cells from XP patients belonging to complementation group A (XPA), one of the most severely affected of the excision deficient XP complementation groups (4-6). Two of these endonucleases, pi 4.6 and 7.6, in normal cells are active on DNA damaged by psoralen plus long •Abbreviations: XP, xeroderma pigmentosum; XPA, XP complementation group A; UDS, unscheduled DNA synthesis; uvc, short wavelength ultraviolet.

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Cells from patients with the cancer-prone inherited disease, xeroderma pigmentosum (XP) are known to be defective in the endonuclease-mediated incision step in excision repair of a number of different types of DNA adducts, but the molecular events responsible have not been delineated. We have previously reported isolation of two DNA endonucleases, pi 4.6 and 7.6, from normal human chromatin which recognize adducts produced by psoralen plus long wavelength ultraviolet radiation (UVA). These endonucleases are both present in XP complementation group A (XPA) cells even though these cells are hypersensitive to this type of damage. We now report that introduction by electroporation of either normal endonuclease into XPA cells restored their markedly deficient DNA repair-related unscheduled DNA synthesis (UOS) to higher than normal levels following exposure to psoralen plus UVA. Introduction of XPA endonucleases into similarly treated XPA cells had little or no restorative effect on UDS. However, both normal and XPA endonucleases increased UDS in normal cells to higher than normal levels. These results indicate that XPA cells have endonucleases which can repair these adducts but which cannot function in intact cells unless a factor(s), which they lack is provided by normal cells.

wavelength UV radiation (UVA) (4), which produces intercalation of the psoralen molecules between DNA base-pairs as well as covalent monoadducts and DNA interstrand cross-links (7,8). Our results indicate that the endonuclease, pi 7.6, recognizes the psoralen monoadduct and that the one at pi 4.6 is recognizing the intercalation and possibly the cross-link (4,9). Although XPA cells have been shown to be defective in repair of damage produced by psoralen plus UVA (10 — 12), we have found that these same two endonucleases, assayed on damaged naked DNA, are present at normal levels in XPA cells (4). This indicates that the deficiency in XPA cells does not reside in the ability of these endonucleases to act on damaged naked DNA. We have also recently shown that a defect exists in the ability of these XPA endonucleases to interact with the psoralen plus UVA damaged DNA when it is in the form of nucleosomes (9). In addition, we have found a similar defect in interaction with damaged chromatin in yet another chromatin-associated endonuclease, an apurinic/apyrimidinic DNA endonuclease, from XPA cells (13). In the present paper we address the question of whether the two endonucleases active on psoralen plus UVA treated DNA, which we have recently isolated, are responsible for the cellular defect in repair of psoralen plus UVA damage to DNA observed in XPA cells in culture. We accomplished this by introducing the two normal endonucleases into psoralen plus UVA treated XPA cells via electroporation and observing whether they could correct the XPA repair defect. Normal human (GM 1989 and GM 3299) and XPA (GM 2345A and GM 2250A) lymphoblastoid cells, transformed with Epstein-Barr virus (Coriell Institute for Medical Research, Camden, NJ) were grown in suspension culture as previously described (14). Cell nuclei were isolated and the chromatinassociated proteins extracted from the nuclei and electrophoresed on an isoelectric focusing column with carrier ampholytes (LKB Instruments Inc.), pH 3.0—10.0 (4,15). Fractions collected from the column were assayed for DNA endonuclease and exonuclease activity (15). Peaks of endonuclease activity, which contained no exonuclease activity, were pooled, diaJyzed into 50 mM potassium phosphate (pH 7.1), 1 mM /3-mercaptoethanol, 1 mM Na-EDTA, 0.25 mM phenylmethylsulfonylfluoride, and 40% ethylene glycol and stored unfrozen at -20°C (4,15). XP complementation group A cells were suspended in phosphate-buffered saline (PBS) (0.15 M) and treated with 8-methoxypsoralen (8-MOP) (35 jtM) (Sigma Chemical Company) for 20 min in the dark; they were then irradiated with UVA light (principally 366 nm) at 10 W/m2/s for 10 min; then they were washed twice with PBS and reirradiated with 10 W/m2/s; finally the cells were washed once with ice-cold PBS and resuspended in 0.9 ml PBS at a density of 3 X 106 cells/ml. This dual radiation procedure has been shown to increase the proportion of DNA interstrand cross-links compared with monoadducts (16,17). The normal or XPA endonuclease, pi 4.6 or7.6(0.1 ml) (0.4-1.8 /ig), were then added to the cell suspension and a high voltage electric pulse was applied using a BTX

G.J.Tsongalis, W.C.Lambert and M.W.Lambert

Transfector 300 System (Biotechnologies, Inc.). The cells were exposed to a field strength of 1.7 kV/cm over a distance of 3.5 mm for ~4.0 ms. They were then incubated on ice for 10 min and resuspended in 2 ml of RPMI 1640 medium buffered with HEPES buffer and supplemented with 7.5% fetal calf serum and 7.5% horse serum prewarmed to 37°C. Ability of the cells [

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Fig. 1. UDS in XPA lymphoblastoid cells treated with 8-MOP plus UVA light and electroporated with the normal or XPA DNA endonuclease, pi 4.6 or 7.6. Normal or XPA endonuclease (A) pi 4.6 or (B) pi 7.6 (1.4 ng) were introduced into XPA cells via electroporation. Results are expressed as percent of normal UDS ± SE for four to five separate experiments with a total of 1.5 x 10 3 -2.5 x I03 cells counted.

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to perform excision repair of psoralen plus UVA induced damage in their DNA was then ascertained by monitoring incorporation of 3H labeled thymidine into DNA repair patches during a 2-h pulse, i.e. unscheduled DNA synthesis (UDS). The cells were pulsed for 2 h with 10 /iCi/ml [3H]thymidine (specific activity 61 Ci/mmol) (ICN Radiochemicals) at 37°C. The cells were washed twice with ice-cold PBS, smeared onto glass slides and prepared for autoradiography using Kodak NTB3 emulsion. Autoradiographic grain counts were carried out on slides stained with Giemsa. Any cell with 40—100 silver grains per nucleus was classified as undergoing UDS in our system. Electroporation of XPA cells, treated with 8-MOP plus UVA, with either of the normal endonucleases, pi 4.6 (Figure 1 A) or pi 7.6 (Figure IB), resulted in correction of their DNA repair defect. Unscheduled DNA synthesis in these XPA cells was over 100% of that observed in normal cells similarly treated but without addition of enzyme. The increases in UDS were dependent on electroporation of the endonucleases and on damaging the cells since no increase was observed in undamaged cells or in cells electroporated without enzyme. They were also dependent upon the concentration of the enzyme. Pretreatment of the endonucleases with proteinase K prior to electroporation abolished the correction of the XPA repair defect, suggesting that the correcting factor is a protein. Correction of the XPA repair defect depended upon electroporation of an endonuclease which specifically recognizes 8-MOP plus UVA damage, since a normal human AP endonuclease, pi 9.8 (13), when introduced into 8-MOP plus UVA damaged XPA cells did not significantly

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treated with 8-MOP plus UVA had no effect on UDS levels. Furthermore, electroporation of either of the XPA endonucleases also produced UDS levels above normal [Figure 3 (A and B)]. These increases were concentration dependent, with either too much or too little (of either normal or XPA) enzyme producing lower increases in UDS than the optimum [Figures 4 (A and B) and 5 (A and B)]. More normal endonuclease was needed to provide peak levels of UDS in XPA cells than XPA endonuclease in normal cells. This is consistent with the concept that a factor is present in normal cells and normal enzyme preparations which is defective in XPA cells and which in normal cells can add to the total effect of the normal endonuclease. Decreases in UDS produced by higher doses of enzyme may be due to an increase in nonspecific cleavages introduced into the cellular DNA of these cells by the partially purified endonucleases. These results indicate

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Fig. 4. Influence of enzyme concentration on UDS in normal cells treated with 8-MOP plus UVA light and electroporated with XPA DNA endonucleases, pi 4.6 or 7.6. Normal cells were treated with 8-MOP plus UVA light. Varying concentrations of the XPA endonuclease (A) pi 4.6 and (B) pi 7.6 were added to the cell suspension and electroporation was carried out. Results are expressed as percent of normal UDS ± SE for three to 3 3 five separate experiments with a total of 0.9 x 10 -2.5 x 10 cells counted.

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Fig. 3. UDS in normal lymphoblastoid cells treated with 8-MOP plus UVA and electroporated with normal or XPA DNA endonucleases, pi 4.6 or 7.6. Normal cells were treated wtih 8-MOP plus UVA and either normal or XPA endonuclease, (A) pi 4.6 or (B) pi 7.6 (0.7 #ig) was added to the cell suspension and electroporation was carried out. Results are expressed as percent of normal UDS (100%) ± SE for three to five separate experiments with a total of 0.9 X 10 3 -2.5 x 103 cells counted.

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Fig. 5. Influence of enzyme concentration on UDS in XPA cells treated with 8-MOP plus UVA light and electroporated with normal DNA endonucleases, pi 4.6 or 7.6. XPA cells were treated with 8-MOP plus UVA light. Varying concentrations of the normal endonuclease (A) pi. 46 and (B) pi 7.6 were added to the cell suspension and electroporation was carried out. Results are expressed as percent of normal UDS ± SE for three to five separate experiments with a total of 0.9 x 10 3 -2.5 X 103 cells counted.

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increase UDS above background levels. In contrast, electroporation of endonucleases, pi 4.6 or 7.6, obtained from XPA cells into psoralen plus UVA treated XPA cells did not significantly increase UDS levels above background levels observed in these cells [Figure 1 (A and B)]. Unscheduled DNA synthesis levels in XPA cells either electroporated without enzyme or not electroporated were —10% of those observed in normal cells following psoralen plus UVA treatment. The electroporation process did not detectably effect UDS in any cell line tested. Figure 2 shows examples of these autoradiograms. Heavily labeled cells in this figure are in S phase. We have previously shown that both of these endonucleases from normal lymphoblastoid cells show a greater than 2-fold increase in activity when the psoralen plus UVA damaged DNA is in the form of reconstituted nucleosomes, as compared with their activities on damaged non-nucleosomal DNA (9). By contrast, these same endonucleases derived from XPA cells fail to show this increase on damaged nucleosomal DNA, but show instead a significant decrease in activity, even though their activity against damaged non-nucleosomal DNA is similar to that of the normal cells (9). However, mixing either XPA endonuclease with either of the two normal endonucleases corrects this defect (9). Thus there is a correcting factor associated with both normal endonucleases which is capable of correcting the defect in either XPA endonuclease. These results indicate that XPA cells have endonucleases which can recognize and incise non-nucleosomal DNA containing psoralen plus UVA adducts, but lack a factor needed for interaction with damaged DNA when it is in the form of nucleosomes. Electroporation of either of the normal endonucleases into normal cells treated with 8-MOP plus UVA also resulted in an increase in UDS above that observed in normal cells electroporated without enzyme [Figure 3 (A and B)]. This effect was specific for these two endonucleases; DNase I did not produce any increase in UDS in normal cells treated with 8-MOP plus UVA. In addition, a normal human AP endonuclease, pi 9.8 (13) upon electroporation into normal cells treated with 8-MOP plus UVA also did not increase UDS above the normal levels. Electroporation of either normal endonuclease into normal cells not

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enzymes into cells and studying their role in cellular DNA repair processes. Proteins can be introduced into a large number of cells, these cells are in suspension and do not have to be adherent to a substratum, detergents are not used and this procedure can be carried out on a large number of cells in a matter of milliseconds. In addition, as we show in the current paper, the levels of unscheduled DNA repair synthesis (UDS) in normal cells, treated with a DNA damaging agent, and electroporated are 100% of those in similarly treated non-electroporated cells. Therefore, electroporation, by itself, has no detectable detrimental effect on DNA repair synthesis. The results reported here confirm that the DNA endonucleases we have isolated from normal human lymphoblastoid cell chromatin are, indeed, capable of functioning in DNA repair processes in intact cells in culture. Together with our previous results (9,12), they clearly show that XPA cells have these endonucleases but lack one or more closely associated factors that allow them to act in intact cells, presumably by enabling them to interact with damaged cellular DNA in the form of chromatin. The results obtained also suggest that a similar approach, combining this methodology for isolation of active chromatinassociated DNA endonucleases with electroporation, will make possible the further elucidation not only of this defect but also of deficiencies in other XP complementation groups. It should also be an effective method for studying the etiology of other diseases in which defective DNA repair is known or suspected to be present. Acknowledgements We would like to thank Robert Lockwood for culturing the human cell lines. This work was supported by Grant AM 35148 from the National Institutes of Health.

References 1. Kraemer,K.H., Lee.M.M. and Scotto,J. (1987) Xeroderma pigmentosum: cutaneous, ocular, and neurological abnormalities in 830 published cases. Arch. Dermatol, 123, 241-250. 2. Umbert,W.C. and Lambert,M.W. (1987) DNA repair deficiency and cancer in xeroderma pigmentosum. Cancer Rev., 7, 1—25. 3. Bootsma.D., Keijzer.W., Jung,E.G. and Bohnart.E. (1989) Xeroderma pigmentosum complementation group XP-I withdrawn. Mutat. Res., 218. 149-152. 4. Lambert.M.W., Fenkart.D. and Clarke.M. (1988) Two DNA endonuclease activities from normal human and xeroderma pigmentosum chromatin active on psoralen plus ultraviolet light treated DNA. Mutat. Res., 193, 65—73. 5. Lambert.M.W., Lambert,W.C. and Okorodudu.A.O. (1983) Nuclear DNA endonuclease activities on partially apurinic/apyrimidinic DNA in normal human and xeroderma pigmentosum lymphoblastoid cells and mouse melanoma cells. Chem. Biol. Interact., 46, 109-120. 6. Lambert.M.W. and Parrish,D.D. (1989) Modulation of human chromatinassociated endonuclease activity on damaged DNA by nucleosome structure. In: Lambert.M.W. and Laval,J. (eds), DNA Repair Mechanisms and Their Biological Implications in Mammalian Cells. Plenum Press, New York (in press). 7. Ben-Hur,E. and Song,P.-S. (1984) The photochemistry and photobiology of furocoumarins (psoralens). Adv. Radial. Biol., 11, 131-177. 8. Cimino.G.D., Gamper,H.B., Isaacs,S.T. and Hearst,.).E. (1985) Psoralens as photoactive probes of nucleic acid structure and function: organic chemistry, photochemistry. Annu. Rev. Biochem., 54, 1151-1193. 9. Parrish,D.D. and Lambert.M.W. (1990) Chromatin-associated DNA endonucleases from xeroderma pigmentosum cells are defective in interaction with damaged nucleosomal DNA. Muiat. Res. (in press). 10. Kaye.J., Smith.C.A. and Hanawalt,P.C. (1980) DNA repair in human cells containing photoadducts of 8-methoxypsoralen or angelicin. Cancer Res., 40. 696-702. 11. Bredberg.A., Lambert.B. and Soderhall.S. (1982) Induction and repair of psoralen cross-links in DNA of normal human and xeroderma pigmentosum fibroblasts. Mutat. Res., 93. 221-234. 12. Gruenert.D.C. and Cleaver.J.E. (1985) Repair of psoralen-induced cross-

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that introduction of additional normal enzyme or of XPA enzyme into damaged normal cells can increase the efficiency of repair in these cells and further support the hypothesis that XPA cells contain endonucleases which can repair 8-MOP plus UVA damage in normal cells but lack a factor necessary to allow these XPA endonucleases to function in their own cells. This factor can be supplied by normal cells when XPA endonucleases are introduced into them by electroporation. Gianelli et al. (18) have postulated, based on their experimental findings in heterokaryons, that normal cells have a surplus of the factor needed to complement the repair defect in XPA cells, which they termed 'factor A'. Our results support this hypothesis and, further, indicate that this surplus factor, in our system, allows the XPA endonucleases to repair damaged chromosomal DNA in normal human cells. We do not know whether these two endonucleases share a common closely associated co-factor, have different closely associated co-factors which can substitute for each other in endonuclease and UDS assays, or have an activity present in the same protein molecule which both has endonuclease activity and performs this function. Although a number of cell-free systems have been employed to attempt to elucidate the XPA defect, the results have been conflicting. Mortlemans et al. (19), and later Kano and Fujiwara (20), obtained results from crude cell extracts suggesting that XPA cells were not deficient in an endonuclease but rather in a factor or factors required for interaction of an endonuclease with damaged DNA in the form of chromatin. On the other hand, Wood et al. have reported defective DNA repair synthesis in damaged naked plasmid DNA by soluble extracts of XPA cells (21). Such extracts of mammalian cells, however, have been shown to reconstitute naked plasmid DNA into nucleosomes or nucleosome-like structures, that can be visualized ultrastructurally (22). Therefore the defect observed by this latter group in XPA cell extracts may also have been due to an inability of an XPA endonuclease or endonucleases to interact with damaged DNA in the form of nucleosomes or nucleosome-like structures rather than a failure to act on damaged non-nucleosomal DNA. This alternative interpretation of their results would then be in agreement with our own results (9) and those of Mortlemans et al. (23) and Kano and Fujiwara (24). A definitive test for any factor said to be responsible for the XPA defect, however, is its ability to restore DNA repair functions to normal levels in XPA cells in culture. A number of methods have been utilized for introduction of cell extracts or isolated enzymes into cells. These include microinjection (23-26) and permeabilization of cells (27-34). Restoration of UV-induced UDS in XPA cells has been accomplished by microinjection of extracts of HeLa cells and human placenta into these cells (23—25) and by introduction of the T4 phage endonuclease V and Micrococcus luteus UV specific DNA endonucleases into XPA cells via microinjection, permeabilization of the cell membranes and fusion of cell membranes with inactivated sendai virus (27—29,33,35). Recently electroporation has been utilized for introducing macromolecules into cells. Exposure of cells to a brief, high voltage electric field temporarily and reversibly permeabilizes cells to exogenous molecules (36 — 38). Although the mechanism of electroporation is not known, it is thought that the electric field produces a voltage across the cell membrane leading to membrane breaks or openings in localized areas which close when the electric field is removed. This method has recently been used for the introduction of DNA (39-41) and of restriction enzymes into cells (42). There are several extremely important advantages of using electroporation for introducing selected

Electroporation of DNA endonucleases into XP electrofusion. Rev. Physiol. Biochem. Pharmacol., 105, 175—256. 37. Potter.H. (1988) Electroporation in biology: methods, applications, and instrumentation. Anal. Biochem., 174, 361-373. 38. Shigekawa,K. and Dower.W.J. (1988) Electroporation of eukaryotes and prokaryotes: a general approach to the introduction of macromolecules into cells. Biotechniques, 6, 742-775. 39. Potter.H., Weir,L. and Leder.P. (1984) Enhancer-dependent expression of human K immunogloublin genes introduced into mouse pre-B lymphocytes by electroporation. Proc. Natl. Acad. Sci. USA, 81, 7161-7165. 40. Chu,G., Hayakawa,H. and Berg,P. (1987) Electroporation for the efficient transfection of mammalian cells with DNA. Nucl. Acids. Res., 15, 1311-1326. 41. Andreason,G.L. and Evans.G.A. (1988) Introduction and expression of DNA molecules in eukaryotic cells by electroporation. Biotechniques, 6, 650—660. 42. Winegar,R.A., PhillipsJ.W., Youngblom,J.H. and Morgan.W.F. (1989) Cell electroporation is a highly efficient method for introducing restriction endonucleases into cells. Mutat. Res., 225, 49—53. Received on September 22, 1989; revised on December 1, 1989; accepted on December 14, 1989 Downloaded from http://carcin.oxfordjournals.org/ at Florida Atlantic University on July 16, 2015

links and mono-adducts in normal and repair-deficient human fibroblasts. Cancer Res., 45, 5399-5404. 13. KaysenJ.H., Amari,N.M.B. and Lambert,M.W. (1986) Enhancement of two apurinic/apyrimidinic endonuclease activities from normal but not xeroderma pigmentosum lymphoblastoid cells by nucleosome structure. Mutas. Res., 165, 221-231. 14. Okorodudu.A.O., Lambert,W.C. and Lambert,M.W. (1982) Nuclear deoxyribonuclease activities in normal and xeroderma pigmentosum lymphoblastoid cells. Biochem. Biophys. Res. Commun., 108, 576 — 584. 15. Lambert,M.W., Lee.D.E., Okorodudu,A.O. and Lambert.W.C. (1982) Nuclear deoxyribonuclease activities in human lymphoblastoid and mouse melanoma cells: a comparative study. Biochim. Biophys. Acta, 69, 192—203. 16. Ben-Hur,E. and Elkind.M.M. (1973) Psoralen plus near ultraviolet light inactivation of cultured Chinese hamster cells and its relation to DNA crosslinks. Mutat. Res., 18, 315-324. 17. Bredberg.A. (1982) Genetic toxicity of psoralen and ultraviolet radiation in human cells. Acta Dermato-Venereol., 104, 1—40. 18. Giannelli,F., Pawsey.S.A. and A very ,J.A. (1982) Differences in patterns of complementation of the more common groups of xeroderma pigmentosum: possible implications. Cell, 29, 451—458. 19. Mortelmans.K., Friedberg.E.C, Slor,H., Thomas.G. and CleaverJ.E. (1976) Defective thymine dimer excision by cell-free extracts of xeroderma pigmentosum cells. Proc. Natl. Acad. Sci. USA, 73, 2757-2761. 20. Kano,Y. and Fujiwara,Y. (1983) Defective thymine dimer excision from xeroderma pigmentosum chromatin and its characteristic catalysis by cellfree extracts. Carcinogenesis, 4, 1419—1424. 21. Wood.R.D., Robins.P. and Lindahl.T. (1988) Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extracts. Cell, 53, 97-106. 22. Hough,P.V.C, Mastrangelo.I.A., Wall, J.S., Hainfeld,J.F., Simon,M.N. and Manley.J.L. (1982) DNA —protein complexes spread on N2-discharged carbon film and characterized by molecular weight and its projected distribution. J. Mol. Bioi, 160, 375-386. 23. De Jonge.A.J.R., Vermeulen.W., Klein,B. and Hoeijmakers,J.H.J. (1983) Microinjection of human cell extracts corrects xeroderma pigmentosum defect. EMBOJ., 29, 637-641. 24. Vermeulen.W., Osseweijer,P., de Jonge,A.J.R. and Hoeijmakers,J.H.J. (1986) Transient correction of excision repair defects in fibroblasts of 9 xeroderma pigmentosum complementation groups by microinjection of crude human cell extracts. Mutat. Res., 165, 199-206. 25. Yamaizumi,M., Sagano,T., Asahina.H., Okada.Y. and Uchida.T. (1986) Microinjection of partially purified protein factor restores DNA damage specifically in group A of xeroderma pigmentosum cells. Proc. Natl. Acad. Sci. USA, 83, 1476-1479. 26. Yamaizumi,M., lnaoka,T., Uchida,T. and Ohtsuka.E. (1989) Microinjection of T4 endonuclease V produced by a synthetic denV gene stimulates unscheduled DNA synthesis in both xeroderma pigmentosum and normal cells. Mutat. Res., 217, 135-140. 27. Smith.C.A. and Hanawalt,P.C. (1978) Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts. Proc. Natl. Acad. Sci. USA, 75, 2598-2602. 28. Ciarrocchi.G., Jose.J.G. and Linn.S. (1979) Further characterization of a cell-free sytem for measuring replicative and repair DNA synthesis with cultured human fibroblasts and evidence for the involvement of DNA polymerase a in DNA repair. Nucl. Acids Res., 7, 1205-1219. 29. Dresler.S.L., Roberts,.!.D. and Lieberman,M.W. (1982) Characterization of deoxyribonucleic acid repair synthesis in permeable human fibroblasts. Biochemistry, 21, 2557-2564. 30. Natarajan.A.T. and Obe.G. (1984) Molecular mechanisms involved in the production of chromosomal aberrations. Chromosoma, 90, 120-127. 31. Bryant,P,E. (1984) Enzymatic restriction of mammalian cell DNA using PvuU and BamHl: evidence for the double-strand break origin of chromosomal aberrations. Int. J. Radial. Bioi, 46, 5 7 - 6 5 . 32.0be,G., Palitti,F., Tanzarella.C, Degrassi,F. and DeSalvia.R. (1985) Chromosomal aberrations induced by restriction endonucleases. Mutat. Res., 150, 359-368. 33. Kaufmann.W.K. and Briley.L.P. (1987) Reparative strand incision in saponinpermeabilized human fibroblasts. Mutat. Res., 184, 237-243. 34. Winegar,R.A. and Preston,R.J. (1988) The induction of chromosome aberrations by restriction endonucleases that produce blunt-end or cohesiveend double-strand breaks. Mutat. Res., 197, 141-149. 35. Tanaka,K., Hayakawa.H., Sekiguchi.M. and Okada,Y. (1975) Restoration of ultraviolet-induced DNA synthesis of xeroderma pigmentosum cells by the contact treatment with bacteriophage T4 endonuclease V (Sendai virus). Proc. Natl. Acad. Sci. USA, 72, 4071-4075. 36. Zimmermann.U. (1986) Electrical breakdown, electropermeabilization, and

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