Mutation Research, 235 (1990) 65-80 DNA Repair

65

Elsevier MUTDNA 06366

Chromatin-associated D N A endonucleases from xeroderma pigmentosum cells are defective in interaction with damaged nucleosomal D N A David D. Parrish and Muriel W. Lambert Department of Pathology, UMDNJ - New Jersey Medical School and The Graduate School of Biomedical Sciences, 185 South Orange Avenue, Newark, NJ 07103 (U.S.A.)

(Received 19 June 1989) (Revision received 17 August 1989) (Accepted 25 August 1989)

Keywords: DNA endonuclease; Psoralen plus UVA light; Histones; Chromatin structure; Xeroderma pigrnentosum

Summary The influence of nucleosome structure on the activity of 2 chromatin-associated D N A endonucleases, p l s 4.6 and 7.6, from normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells was examined on D N A containing either psoralen monoadducts or cross-links. As substrate a reconstituted nucleosomal system was utilized consisting of a plasmid D N A and either core (H2A, H2B, H3, H4), or total (core plus H1) histones from normal or XPA cells. Both non-nucleosomal and nucleosomal D N A were treated with 8-methoxypsoralen (8-MOP) plus long-wavelength ultraviolet radiation (UVA), which produces monoadducts and D N A interstrand cross-links, and angelicin plus UVA, which produces monoadducts. Both normal endonucleases were over 2-fold more active on both types of psoralen-plus-UVA-damaged core nucleosomal D N A than on damaged non-nucleosomal D N A . Addition of histone H I to the system reduced but did not abolish this increase. By contrast, neither XPA endonuclease showed any increase on psoralen-treated nucleosomal D N A , with or without historic H1. Mixing the normal with the XPA endonucleases led to complementation of the XPA defect. These results indicate that interaction of these endonucleases with chromatin is of critical importance and that it is at this level that a defect exists in XPA endonucleases.

Chromatin has been shown to be an important factor in determining the accessibility of D N A to damage by various types of agents as well as to repair of this damage (Lan and Smerdon, 1985; Bohr et al., 1987; Smerdon, 1989). Studies which

Correspondence: Dr. Muriel W. Lambert, Department of Pathology, UMDNJ - New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103 (U.S.A.).

have examined the involvement of chromatin in repair of different lesions have approached the problem from several different aspects. Chromatin has been isolated from cells which have been allowed to undergo repair after exposure to various types of damaging agents and the distribution of repair sites has been examined (Lan and Smerdon, 1985; Smerdon, 1989). Preferential repair of transcriptionally active versus inactive D N A sequences in the m a m m a l i a n genome has been studied (Mellon et al., 1986; Bohr et al.,

0921-8777/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

66 1987; Leadon and Snowden, 1988; Hanawalt et al., 1989) and the role, in D N A repair, of higherorder chromatin loops attached to the nuclear matrix has also been examined (Mullenders et al., 1986, 1988). However, little is known, in mammalian systems, regarding the influence of nucleosome structure on the activity of specific, isolated D N A enzymes on particular types of D N A adducts. Repair of D N A interstrand cross-links has been shown to occur in vitro in mammalian cells, but the mechanism of this repair is poorly understood. One of the most definitive agents for producing cross-links is psoralen plus long wavelength (366 nm) ultraviolet radiation (UVA) (Ben-Hur and Song, 1984; Cimino et al., 1985; Vigny et al., 1985). Psoralens are a group of naturally occurring furocoumarins which are widely distributed in the environment (Scott et al., 1976). They are used clinically as photosensitizing agents for the treatment of various hyperproliferative diseases, cutaneous pigmentary disorders and lymphomatous conditions (Parrish et al., 1974; Scott et al., 1976; Anderson and Voorhees, 1980; Ben-Hur and Song, 1984; Edelson et al., 1987). Psoralens interact with D N A by first intercalating between adjacent base pairs in the dark and then, when photoreacted with UVA, by forming both monoadducts and interstrand cross-links (Ben-Hur, 1984; Cimino et al., 1985; Vigny et al., 1985). We have developed a system which has enabled us to extract a series of D N A endonucleases from human chromatin and to detect individual selective activities of these enzymes on a number of different types of damaged D N A (Okorodudu et al., 1982; M.W. Lambert et al., 1983, 1988). Utilizing this system, we have isolated and partially purified several D N A endonucleases, from the chromatin of normal human lymphoblastoid cells, which are active on different types of damaged D N A (M.W. Lambert et al., 1983, 1988; M.W. Lambert and Parrish, 1989). Two of these endonucleases, p l s 4.6 and 7.6, are selectively active on D N A treated with psoralen plus UVA (M.W. Lambert et al., 1988). Results from our laboratory suggest that the endonuclease, p I 7.6, recognizes the monoadduct and the endonuclease, p I 4.6, recognizes the intercalation and possibly the cross-link (M.W. Lambert et al., 1988).

We have also examined cells from patients with the recessively transmitted, repair-deficient disorder, xeroderma pigmentosum (XP), for the presence of these endonuclease activities. XP patients are extremely sensitive to the action of sunlight and tend to develop skin cancers in sun-exposed parts of the body (reviewed in W.C. Lambert and Lambert, 1987; Kraemer et al., 1987). Of the 8 known complementation groups of excision-deficient XP patients, complementation group A (XPA), examined in the present study, is one of those most severely affected and has been shown to be deficient in repair of damage produced by sunlight or short-wavelength (UVC) radiation, psoralen plus UVA, and a variety of chemical agents (reviewed in W.C. Lambert and Lambert, 1987; Kraemer et al., 1987). We have found that the 2 endonucleases, p l s 4.6 and 7.6, active on psoralen plus UVA, are present in cells from XPA patients and have levels of activity on damaged naked DNA similar to those found in normal cells (M.W. Lambert et al., 1988). Since XPA cells are defective in repair of psoralen adducts, the question thus arises as to the nature of the repair defect in XPA cells and the level at which the defect is manifested. The present study examines whether nucleosome structure influences the activity of these 2 specific chromatin-associated normal human D N A endonucleases on psoralen-plus-UVA-induced adducts in DNA, and whether these same endonucleases from XPA cells can interact with the damaged D N A when nucleosomes are present. We have utilized a reconstituted nucleosomal system, consisting of a plasmid D N A and either normal or XPA histones, which provides us with a well defined substrate and allows us to regulate the composition and source of the histones (Kaysen et al., 1986, 1987; M.W. Lambert et al., 1988). Using this system, we now show that the presence of nucleosomes markedly enhances the activity of both normal enzymes, but the 2 XPA endonucleases are defective in their ability to act on psoralen-plus-UVA-damaged nucleosomal DNA. When the normal endonucleases examined in the present study were mixed with the XPA endonucleases, the XPA defect in ability to incise psoralen-plus-UVA-damaged nucleosomal DNA was corrected. Thus the defect in XPA cells re-

67 sides in the endonuclease activities we have isolated, since the corresponding normal endonucleases can complement this defect. We have previously found that 2 chromatin-associated XPA endonucleases, different from those examined in the present study, with selective activity against partially apurinic/apyrimidinic (AP) DNA, show defective interaction with chromatin (Kaysen et al., 1986). Considered altogether, our findings suggest that this failure to interact normally with nucleosomes may represent a general defect in endonucleases from XPA cells involved in repair. Materials and methods

Cell lines and culture conditions Normal (GM 1989 and GM 3299) and xeroderma pigmentosum, complementation group A (XPA) (GM 2345 and GM 2250A) lymphoblastoid cell lines (transformed with Epstein-Barr virus) were obtained from the Coriell Institute for Medical Research, Camden, NJ. The cells were grown in suspension culture in RPMI 1640 medium, supplemented with 12.5% fetal calf serum (Grand Island Biological Co.), as previously described, and harvested under conditions of maximal proliferation (Okorodudu et al., 1982). Cell cultures were routinely tested for mycoplasma, and steps were taken to insure that cells were not exposed to UV light and that other light exposure was minimal (Okorodudu et al., 1982). DNA endonuclease extraction Cell nuclei were isolated as previously described (M.W. Lambert et al., 1982). The chromatin-associated proteins were extracted from the nuclei and electrophoresed on an isoelectric focusing column with carrier ampholytes (LKB Instruments Inc.), pH 3.0-10.0 (M.W. Lambert et al., 1982). Fractions collected from the column were assayed for DNA endonuclease and exonuclease activity against calf-thymus DNA (M.W. Lambert et al., 1982) and [3H]poly(dA-dT) (Lindahl et al., 1969), respectively. Peaks of endonuclease activity were pooled, dialyzed into 50 mM potassium phosphate (pH 7.1), 1 mM fl-mercaptoethanol, 1 mM Na-EDTA, 0.25 mM phenylmethylsulfonylfluoride (PMSF), and 40% ethylene glycol and stored unfrozen at - 2 0 °C (M.W. Lambert et al.,

1988). Protein concentrations were determined by the method of Lowry et al. (1951) and by the BioRad protein assay (BioRad Laboratories). Histone isolation Nuclei were isolated from normal and XPA lymphoblastoid cell lines and histones and nonhistone proteins (NHPs) were extracted and separated as previously described (Kaysen et al., 1986). Histone H1 was removed from the total histones by precipitation with 5% perchloric acid (Kaysen et al., 1986). All buffers contained 0.25 mM PMSF. The purity of core (H2A, H2B, H3, and H4) and total (core plus H1) histories was monitored by electrophoresis on 2.5 M urea, 15% polyacrylamide gels (Kaysen et al., 1986). Calfthymus histones (Boehringer-Mannheim Biochemicals) were used as standards for comparisons. Protein concentrations were determined by the BioRad protein assay (BioRad Laboratories) using total calf-thymus histones as a standard. Plasmid growth and purification Escherichia coil strain HB101 containing plasmid pWT830/pBR322 (a clone of the entire SV40 and pBR322 genomes) was grown, harvested and lysed as previously described (Kaysen et al., 1986). DNA was extracted with phenol, treated with ribonuclease I and electrophoresed on 0.9% agarose gels. The uncleaved, circular, form I DNA band was cut from the gel and the DNA was electroeluted using an Elutrap electrophoresis chamber (Schleicher and Schuell, Inc.). The eluted DNA was recovered by ethanol precipitation, resuspended in 10 mM Tris-HC1, pH 7.2, 1 mM EDTA, and 0.5 M NaCI and further purified on a NACS 37 (Bethesda Research Laboratory) column. The DNA was eluted from the column with a 0.5-0.7 M NaC1 gradient. Aliquots of fractions collected from the column were electrophoresed on a 0.9% agarose gel and those containing greater than 95% form I DNA were pooled. DNA was recovered by ethanol precipitation and resuspended in 10 mM Tris-HC1, pH 8.0, 1 mM EDTA. Nucleosome reconstitution Plasmid DNA was mixed with core histones from normal or XPA cells, with or without histone H1, at a histone:DNA weight ratio of 1.0 in a

68 buffer containing 2 M NaC1, 50 mM Tris-HC1 (pH 8.0), 0.1 M EDTA, and 0.25 mM PMSF as previously described (Kaysen et al., 1986). The NaC1 was progressively decreased by stepwise dialysis at 4 ° C over a 24-h period to 0.5 M NaC1. Finally the mixture was dialyzed against 50 mM Tris-HC1 (pH 8.0), 0.1 M EDTA, 0.2 mM PMSF, and 50 mM NaC1. Reconstitution was also carried out, as described above, with 5 M urea and the urea was removed at 0.75 M NaCI. Reaction of psoralen with DNA 8-Methoxypsoralen (8-MOP) (Sigma Chemical Co.) was recrystallized and purity checked by thin-layer chromatography (M.W. Lambert et al., 1988). Photoreaction of 8-MOP with non-nucleosomal and nucleosomal D N A was carried out utilizing a treatment protocol which involved exposing 8-MOP (4-15 /tg/ml) treated DNA to 2 doses of UVA, an initial one (10 W / m 2 for 10 rain) after the 8-MOP had intercalated into the D N A and a second one (10 W / m 2 for 10 rain) after the unbound 8-MOP had been removed by dialysis (M.W. Lambert et al., 1988). This procedure increases the number of 8-MOP D N A interstrand cross-links (Ben-Hur and Elkind, 1973; Bredberg, 1982) and is sufficient to produce cross-links in 99% of the non-nucleosomal D N A molecules (M.W. Lambert et al., 1988). Nonnucleosomal and nucleosomal D N A was reacted with angelicin (Elder Co.) (25/~g/ml) for 20 min and then exposed to UVA light (10.0 W / m 2) for 5 min (M.W. Lambert et al., 1988). Cross-linking of psoralen to non-nucleosomal and nucleosomal D N A was determined by alkaline gel electrophoresis. After reaction of the psoralen with DNA, the D N A was cut by the restriction enzyme KPN I (New England Biolabs), which cuts the pWT830/pBR322 molecule at one site, and the linearized D N A molecules were run on 1.0% alkaline agarose gels and the proportion of D N A molecules containing interstrand crosslinks was calculated (M.W. Lambert et al., 1988). The total number of 8-MOP adducts bound to D N A was determined by measuring the uptake of [3H]8-MOP (79.5 C i / m M ) (Amersham Corporation) into nucleosomal ( _ h i s t o n e H1) and nonnucleosomal DNA. Samples were treated with [3H]8-MOP (0.1-15/~g/ml) in the dark and then

irradiated with UVA as described above. Unbound [3H]8-MOP was removed by dialysis and samples were irradiated with a second dose of UVA. Samples were counted in Ecoscint A (National Diagnostics) in a scintillation counter and the average number of adducts per D N A molecule determined. Assay for DNA endonuclease activity Endonuclease activity on nucleosomal and non-nucleosomal D N A was measured as previously described (M.W. Lambert et al., 1982). Briefly, 0.10 /~g of D N A substrate was reacted with each D N A endonuclease activity, p I 4.6 or 7.6, from either normal or XPA cells in 5 mM MgC12, 20 mM KC1 and 10 m M Tris-maleate (pH 7.5) at 3 7 ° C for 3 h. The concentration of each D N A endonuclease (0.36-0.46 /xg) was adjusted to produce 0 . 2 5 _ 0.1 breaks per D N A molecule on damaged non-nucleosomal DNA. The enzymatic reaction was terminated with 0.1 M EDTA. D N A samples were treated with 0.4% Sarkosyl (Ciba-Geigy) and 50/~g/ml proteinase K (Sigma Chemical Co.) for 1 h at 37 ° C (Kaysen et al., 1986) and electrophoresed on 1.0% agarose gels (M.W. Lambert et al., 1982). Gels were subsequently stained with ethidium bromide, photographed, the negatives of the gels scanned and endonuclease activity, expressed as the number of enzyme-induced breaks per D N A molecule, determined as previously described (M.W. Lambert et al., 1983; W.C. Lambert and Lambert, 1989a). Results

8-MOP adducts on non-nucleosomal and nucleosomal DNA The number of psoralen adducts bound to reconstituted nucleosomal as well as non-nucleosomal D N A was determined. Either core (histones H2A, H2B, H3, H4) or total (core plus histone H1) nucleosomal D N A was used. Each of these D N A substrates was reacted with [3H]8-MOP plus UVA and the number of [3H]8-MOP a d d u c t s / D N A molecule calculated. Core nucleosomal D N A contained approximately one half the number of adducts found in non-nucleosomal D N A over a range of 8-MOP concentrations (Fig. 1). The number of adducts in total nucleosomal D N A was

69

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CORE

..,L TOTAL

I"

0~. 0

l 15

l I I 30 45 60 3H-8MOP CONCENTRATION(#M)

75

Fig. 1. Quantitation of [3H]8-MOP adducts on nucleosomal and non-nucleosomal plasmid DNA. Varying concentrations of [3H]8-MOP were incubated with 0.1 #g of either nonnucleosomal plasmid DNA or plasmid DNA reconstituted into nucleosomes with core (minus histone H1) or total (with histone H1)histones as indicated. The DNA was then photoreacted with 2 doses of UVA and the number of [3H]8-MOP adducts bound per DNA molecule calculated.

multiples of activity of each enzyme on undamaged or damaged non-nucleosomal DNA. The solid horizontal line represents the activity of each enzyme on non-nucleosomal DNA. Neither the normal (Fig. 2A) nor the XPA (Fig. 2B) endonucleases, p l s 4.6 and 7.6, displayed any difference in activity on undamaged core or total nucleosomal DNA. However, both normal activities, pls 4.6 and 7.6, showed an approximately 2.5-fold increase ( p < 0.001) in activity on damaged nucleosomal D N A consisting of D N A and core histones (Fig. 2A). When histone H1 was added to the system, this increase was reduced, but remained approximately 1.5-fold greater ( p < 0.001) than the activity on damaged non-nucleosomal DNA. In marked contrast, when the same 2 endonuclease activities from XPA cells were tested on 8-MOP-plus-UVA-treated nucleosomal D N A consisting of core histones, there was no increase in endonuclease activity (Fig. 2B). The addition of histone H1 significantly decreased activity of both endonucleases to about 65% of their level on

A approximately 10% lower than in core D N A (Fig. 1). Endonuclease activity on non-nucleosomal nucleosomal 8-MOP-treated DNA

B

NORMAL

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_UNDAMAGED DAMAGED 3.0

m

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and

We have previously reported that there are 2 chromatin-associated DNA endonucleases, pls 4.6 and 7.6, in normal human lymphoblastoid cells which incise 8-MOP-plus-UVA-treated nonnucleosomal D N A with similar levels of activity (M.W. Lambert et al., 1988). These same 2 activities are present at similar levels in XPA cells (M.W., Lambert et al., 1988). The activity of these 2 endonucleases from normal and XPA cells was examined on 8-MOP core and total nucleosomal D N A (i.e., nucleosomal DNA with and without histone H1). The concentration of each endonuclease was adjusted to produce 0.25 + 0.1 breaks/molecule on damaged non-nucleosomal DNA. Fig. 2 shows the influence of nucleosome structure on the activities of these 2 endonucleases from normal and XPA cells on 8-MOP-plusUVA-treated DNA. The results are expressed as

2.0

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pI4.6 pT7.6 pI4,6 pI7.6 ENDONUCLEASES

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Fig. 2. Activity of DNA endonucleases, p l s 4.6 and 7.6, from normal and XPA cells on nucleosomal plasmid DNA treated with 8-MOP plus UVA. (A) Normal and (B) XPA endonucleases, p l s 4.6 and 7.6 (0.41+0.05 #g enzyme) were incubated with undamaged or 8-MOP (69 # m)-plus-UVA-treated plasmid DNA reconstituted with total or core histories. Endonuclease activity, expressed as multiples of activity on nonnucleosomal DNA, was adjusted to produce 0.25 +0.1 breaks/molecule on damaged non-nucleosomal DNA. The solid horizontal line represents enzyme activity on nonnucleosomal DNA. Vertical lines represent -t-S.E.M.

70

damaged non-nucleosomal DNA. These results were obtained over a range of 8-MOP concentrations (20-70/~M) for both normal and XPA endonucleases. 2 different normal and 2 different XPA cell lines were used and data were obtained from 1 or 2 different extractions from each cell line. No detectable difference was observed in the activities of either of the endonucleases from the 2 normal cell lines, or in either of those from the 2 XPA cell lines; therefore the data from the normal cell lines were pooled as were the data from the XPA cell lines in the results shown. Data presented represent the averages of 15-20 different experiments. These results were the same regardless of whether urea was utilized in the reconstitution system. Endonuclease actioity on non-nucleosomal and nucleosomal angelicin-treated DNA We have previously shown that the endonuclease activities, p l s 4.6 and 7.6, from both normal and XPA cells also incise DNA treated with angelicin plus UVA and that the endonuclease, p I 4.6, has less activity on angelicintreated D N A than the one at p I 7.6 (M.W. Lambert et al., 1988). Angelicin, after it intercalates, produces mainly monoadducts upon photoactivation rather than interstrand cross-links with DNA, due to the angular nature of its structure (Ben-Hur and Song, 1984; Cimino et al., 1985). In our system no cross-links were detected in the angelicin-treated DNA. The lesser activity of the endonuclease, p l 4.6, on angelicin-plus-UVA-treated D N A is consistent with our concept that this enzyme recognizes the intercalation and possibly the interstrand cross-link produced by 8-MOP plus UVA whereas the endonuclease, p I 7.6, recognizes the monoadduct (M.W. Lambert et al., 1988). Fig. 3 shows the influence of nucleosome structure on enzyme activity on angelicin-treated DNA. Endo-

A

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l

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0.3

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Fig. 3. Activity of DNA endonucleases, p l s 4.6 and 7.6, from normal and XPA cells on angelicin-plus-UVA-treated naked and nucleosomal plasmid DNA. (A) Normal and (B) XPA endonucleases, p l s 4.6 and 7.6 (0.41 +_0.05/~g), were incubated with angelicin (25 #m)-plus-UVA-treated naked (non-nucleosomal) plasmid DNA or plasmid DNA reconstituted with total or core (minus H1) histones. These values have had subtracted from them the enzyme activity on undamaged DNA. Vertical lines represent + S.E.M.

nuclease activity is expressed as the mean number of breaks per D N A molecule. This was done so that the differences between the activity of the endonuclease, p l 4.6, and that at p I 7.6 would not be masked. Both normal endonucleases showed an over 2-fold increase in activity on angelicin-treated nucleosomal D N A consisting of D N A and core histones as compared with damaged non-nucleosomal D N A (Fig. 3A). When histone H1 was added this increase was reduced, but activity remained greater than that on damaged nonnucleosomal D N A (Fig. 3A). The differences in activity between the 2 normal endonucleases was still observed. By contrast, neither XPA endonuclease showed any increase in activity on angelicin-treated core nucleosomal D N A (Fig. 3B).

Fig. 4. Complementation of the XPA repair defect on 8-MOP-plus-UVA-treated nucleosomal DNA. (A) Normal DNA endonuclease (N), p I 4.6, was mixed with XPA DNA endonuclease (X), p I 4.6, and (B) normal endonuclease, p l 7.6, was mixed with XPA endonuclease, p I 7.6, and assayed for activity on non-nucleosomal (left frame), nucleosomal (minus histone H1) (middle frame) and nucleosomal (plus histone H1) (right frame) plasmid DNA treated with 8-MOP (36/~m) plus UVA. Mixing experiments were carried out using 1/2 the amount (0.20+0.03/xg) of either of the normal or XPA endonucleases routinely used. The striped area on top of the bars indicates enzyme activity above that contributed by an additive effect. Vertical lines represent 5: S.E.M.

71

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73

When histone H1 was added to the system, the activity of the XPA endonucleases decreased below their level of activity on damaged nonnucleosomal DNA (Fig. 3B).

Complementation of the XPA repair defect at the nucleosomal level Experiments were carried out to determine whether mixing the normal and XPA endonucleases can complement, or correct, the defect seen in the XPA endonucleases on psoralen-plusUVA-damaged nucleosomal DNA. Half the amount of either of the normal and XPA endonucleases, pI 4.6 or 7.6, were mixed together. Fig. 4A shows that when the normal and XPA endonucleases, p l 4.6, were mixed together, the number of enzyme-mediated incisions produced on 8-MOP-plus-UVA-damaged non-nucleosomal DNA was equal to the combined activity of the 2 endonucleases. However, when the activity of the mixture was tested on damaged core or total nucleosomal DNA, the total number of breaks per DNA molecule produced was significantly greater (p < 0.001) than could be accounted for by an additive effect between the 2 endonucleases, and was, in fact, almost equal to the effect of twice the amount of normal endonuclease (Fig. 4A). Similar results were obtained when the normal and XPA endonucleases, p l 7.6, were mixed together and tested on 8-MOP-plus-UVA-treated DNA (Fig. 4B) and when either of the normal and XPA endonucleases, pI 4.6 or pI 7.6, were mixed separately and tested on angelicin-plus-UVA-treated DNA (Fig. 5A and B). In addition, the normal endonuclease, p l 4.6, was able to complement the XPA endonuclease, pI 7.6, and the normal endonuclease, p l 7.6, was able to complement the XPA endonuclease, pI 4.6 (Table 1). However, the XPA endonuclease, p l 4.6, could not complement the XPA endonuclease, pI 7.6 (Table 1). Each of these experiments was repeated at least 3 times. We have previously shown that there is an AP endonuclease, pI 9.8, in normal cells which is

TABLE 1 ACTIVITY O F D N A E N D O N U C L E A S E S F R O M N O R M A L H U M A N A N D X P A L Y M P H O B L A S T O I D CELLS ON 8 - M O P - P L U S - U V A - T R E A T E D N U C L E O S O M A L A N D NON-NUCLEOSOMAL DNA D N A endonuclease (pl) a

N u m b e r of enzyme-induced scissions per D N A molecule (xl0) b

Normal

XPA

Non-nucleosomal DNA

Core nucleosomal DNA

4.6 7.6 9.8 c

_

0.61 0.61 0.06

1.61 1.64 0.08

-

4.6 7.6

0.59 0.46

0.53 0.49

9.8 9.8

4.6 7.6

0.61 0.48

0.58 0.44

4.6 7.6

7.6 4.6

1.18 1.04

3.06 2.75

-

4.6 + 7.6

1.10

1.00

a 0.024 ~t8 of the normal AP endonuclease ( p l 9.8) and 0.20-t-0.03 /xg of normal and X P A endonucleases, p l s 4.6 and 7.6, were used. b Each endonuclease was incubated with 0.10 /~g 8 - M O P + UVA-treated plasmid D N A for 3 h at 37 * C. Each value has had subtracted from it the n u m b e r of breaks the enzyme produced on u n d a m a g e d D N A . c This AP endonuclease produced 0.35 and 0.96 breaks, respectively, on non-nucleosomal and core nucleosomal A P DNA.

specifically active on partially AP DNA and which also shows a greater than 2-fold increased activity on AP core nucleosomal DNA (Kaysen et al., 1986). This endonuclease was mixed with either the XPA endonuclease, pI 4.6, or that at 7.6, and tested to see whether it could correct the XP defect on 8-MOP-phis-UVA-treated core nucleosomal DNA. The results showed that the normal AP endonuclease could not correct the defect in the ability of the 2 XPA endonucleases to interact

Fig. 5. Complementation of the XPA repair defect on angelicin-plus-UVA-treated nuclensomal D N A . (A) Normal D N A endonuclease (N), p I 4.6, was mixed with X P A D N A endonuclease (X), p I 4.6, and (B) normal endonuclease, p I 7.6, was mixed with X P A endonuclease, p l 7.6, and assayed for activity on non-nuclensomal (left frame), nuclensomal (minus histone H1) (middle frame) and nucleosomal (plus histone H1) (right frame) plasmid D N A treated with angelicin (25 txg/ml) plus UVA. Mixing experiments were carried out as described in Fig. 4. Vertical lines represent + S.E.M.

74 with the psoralen-plus-UVA-damaged somal DNA (Table 1).

nucleo-

Discussion

We have previously shown that 2 nuclear chromatin-associated endonucleases, p l s 4.6 and 7.6, from XPA cells have levels of activity similar to those from normal cells on psoralen-plus-UVAdamaged non-nucleosomal DNA (M.W. Lambert et al., 1988). Cell culture studies, however, have shown that XPA cells, unlike normal cells, are defective in the repair of both monoadducts and cross-links in psoralen-plus-UVA-treated D N A (Baden et al., 1972; Kaye et al., 1980; Bredberg et al., 1982; Gruenert and Cleaver, 1985; W.C. Lambert and Lambert, 1987). The current investigation was undertaken to ascertain whether the repair defect in XPA cells could be at the level of the interaction of the endonucleases with damaged nucleosomal DNA.

Normal endonuclease incision of psoralen-plusUVA-damaged nucleosomal DNA The reconstituted nucleosome system utilized here, which we have previously characterized (Kaysen et al., 1986, 1987; Amari et al., 1986), allows direct analysis of the incision repair process at the molecular level, using a well-defined substrate in which the influence of normal or XPA histones, with and without H1, can be determined. The results obtained indicate that nucleosome structure enhances the activities of both of the 2 endonucleases, p l s 4.6 and 7.6, from normal cells on DNA containing 8-MOP-plus-UVA-induced monoadducts and cross-links or angelicin-plusUVA-induced monoadducts. This increase was approximately 2.5-fold on damaged nucleosomal D N A containing core histones (minus H1). When histone H1 was added to the reconstituted system the increase in endonuclease activity was reduced approximately 35%. This finding agrees with the proposed role of histone H1 in the condensation of chromatin (McGhee and Felsenfeld, 1980; IgoKemenes et al., 1982; Klingholz and Stratling, 1982; Watanabe, 1984), making it less accessible to endonucleolytic attack. Ishimi et al. (1981) found somewhat similar findings in a study using rion-damaged calf-thymus chromatin; they showed

that depletion of the chromatin of histone H1 caused nucleosomal DNA to unfold and to become sensitive to micrococcal nuclease at a particular site on the DNA which was protected from nuclease attack by the presence of HI. In the present study the presence of nucleosomes did not affect the activity of these endonucleases on undamaged DNA. This increase does not correlate with a change in the number of 8-MOP D N A adducts, since the number of adducts was reduced approximately 50% on core nucleosomal D N A and 60% when H1 was present. Gia et al. (1987) have also reported reduced binding of psoralen to extracted cellular chromatin versus binding to naked DNA, although in that report the chromatin substrate was less well defined than in the present study. Nucleosome assembly was necessary for the increase in endonuclease activity observed in the present study since simple addition of histones to the reaction mixture did not increase enzyme activity on the damaged DNA. It may be hypothesized that the presence of nucleosomes makes the sites of damage more accessible to endonucleolytic activity or that there is associated with the endonucleases an "accessibility factor" which increases accessibility of the 8-MOP and angelicin adducts on nucleosomal D N A to endonucleolytic attack. Alternatively, a direct interaction between the endonucleases and the histones may lead to increased ability of the endonucleases to incise the sites of psoralen adducts. Sollner-Webb et al. (1986) found that the apparent affinity of staphylococcal nuclease for chromatin was greater than for protein-free DNA, however, the enzymatic activity of staphylococcal nuclease was less on chromatin than on the protein-free DNA. Kinetic analysis of our 2 normal endonucleases indicates that their affinity for damaged nucleosomal D N A is greater than for damaged naked DNA, but that the maximum turnover number of the enzymes on both substrates is similar (Parrish et al., in preparation). The nucleosomal histones may also sequester the endonucleases and, in effect, increase their local effective concentration so as to produce increased activity on damaged nucleosomal DNA. Nucleosomal histones have been shown to sequester a nonhistone chromatin protein, the H2A-specific protease, but in that case this interaction led to

75 inhibition of enzyme activity (Davie et al., 1986; Elia and Moudrianakis, 1988). The precise nature of the interaction of these endonucleases with nucleosomal histones is currently under investigation. Xeroderma pigmentosum endonuclease activities on nucleosomai DNA In contrast to the normal endonucleases, the present results demonstrate that the 2 corresponding XPA endonuclease activities do not show any increase in activity on angelicin- or 8-MOP-plusUVA-treated core nucleosomal DNA and that they actually show a significant decrease in activity when histone H1 is added (p < 0.01). Kinetic analysis of these XPA endonucleases on both damaged nucleosomal and non-nucleosomal DNA indicates that the differences between the normal and XPA endonuclease activities we have observed are attributable to alterations in enzyme affinity for damaged substrate; the maximum turnover numbers of the enzymes on the damaged nucleosomal and non-nucleosomal substrates are similar (Parrish et al., in preparation). The differences between normal and XPA endonuclease activities on damaged nucleosomal DNA were not due to different initial levels of enzyme activity, since the concentrations of both XPA and normal endonucleases were adjusted to produce a similar number of breaks on nonnucleosomal damaged DNA. The differences were also not due to differences between normal or XPA histones used in the reconstituted system since we have previously shown that there are no quantitative or qualitative differences between normal or XPA histones (Amari et al., 1986). In addition, in the present study the results were the same regardless of whether normal or XPA histones were used. The differences between the normal and XPA endonucleases were also not due to idiosyncratic differences between cell lines, since these experiments were carried out using 2 different XPA and 2 different normal cell lines, each from individuals of different ethnic origins, with the same results. We have previously shown that 2 chromatin-associated AP DNA endonucleases, pls 9.2 and 9.8, from normal cells also exhibit increased activity on partially AP core nucleosomal DNA compared

with non-nucleosomal AP DNA, and that this increase is decreased in the presence of histone H1 (Kaysen et al., 1986). The 2 XPA AP endonucleases did not show this increase in activity (Kaysen et al., 1986). These findings and those of the current study together indicate that in 2 different groups of XPA endonucleases (AP and psoralen plus UVA) a defect exists in their ability to interact with damaged DNA when nucleosomes are present. These findings suggest that the defect observed in XPA cells, which prevents them from showing increased activity on psoralen-plus-UVAtreated or AP nucleosomal DNA, exists in the endonucleases themselves or in a closely associated co-factor. Substrate specificity of endonucleases We have previously demonstrated that while both endonucleases, pls 4.6 and 7.6, from both XPA and normal cells, show similar levels of activity on 8-MOP-plus-UVA non-nucleosomal DNA, the major activity against angelicin-treated non-nucleosomal DNA in both cell types was found in the endonuclease, pI 7.6 (M.W. Lambert et al., 1988). The angelicin-treated DNA, unlike the 8-MOP-plus-UVA DNA, did not contain any DNA interstrand cross-links. In both normal and XPA cells the activity of the endonuclease, p! 4.6, was only approximately 30% of that observed on 8-MOP-plus-UVA non-nucleosomal DNA (M.W. Lambert et al., 1988). The lower level of activity of the endonuclease, pI 4.6, on angelicin-treated DNA was also observed when nucleosomes were present. We have found that the endonuclease, pI 4.6, in normal cells is also active on DNA intercalated with adriamycin, whereas the other endonuclease, pI 7.6, is active in addition on UVC (254 nm) light-irradiated DNA (unpublished observations). It has been postulated that a UV endonuclease may be involved in the initial stages of repair of DNA adducts produced by photoreaction of psoralens, thus simulating pyrimidine dimer excision (Song and Tapley, 1979; Kaye et al., 1980; Gruenert and Cleaver, 1985). The covalent attachment of the psoralen molecule to D N A involves a cyclobutyl linkage just as does the pyrimidine dimer (Song and Tapley, 1979; Ben-Hur and Song, 1984; Vigny et al., 1985; Cimino et al., 1985).

76

These observations, along with the results from the present study, suggest that the endonuclease, p l 7.6, recognizes the psoralen monoadduct. In support of this is our preliminary finding that a monoclonal antibody for 8-MOP-plus-UVA monoadducts (Santella et al., 1985) inhibited the activity of this endonuclease on 8-MOP-plus-UVA D N A treated with the monoclonal antibody (M.W. Lambert et al., in preparation). The activity of the endonuclease, p I 4.6, was only slightly affected. We have shown that the endonuclease, p l 4.6, recognizes the intercalation (M.W. Lambert et al., 1988); whether it also recognizes the cross-link is presently under further investigation. In addition, we have recently found that in Fanconi anemia cells, complementation group A, which has been reported to be defective in the repair of D N A interstrand cross-links (Papadopoulo et al., 1987; Moustacchi et al., 1989), the endonuclease, p I 4.6, is quantitatively reduced in activity, and that in Fanconi anemia cells, complementation group B, which have been reported to be defective in the repair of psoralen-plus-UVA-induced monoadducts (Averbeck et al., 1988; Moustacchi et al., 1989), the activity of the endonuclease, p I 7.6, is reduced (in preparation). It thus appears that, of the 3 types of adducts produced by psoralen plus UVA: intercalation, monoadducts and cross-links, the second is recognized by the endonuclease, p I 7.6, and the first and possibly also the last are recognized by the endonuclease, p I 4.6. Our finding of 2 endonucleases active on psoralen-plus-UVA-treated D N A is consistent with the hypothesis proposed by Gruenert and Cleaver (1985) that more than one enzyme may be involved in repair of D N A interstrand cross-links in mammalian cells. Further purification of these 2 endonucleases will show whether there are 2 separate endonucleases, or 2 similar enzymes which separate on the basis of a postsynthetic modification.

Complementation of the defect in XPA endonucleases The results reported here demonstrate that mixing either of the 2 normal endonucleases with either of the corresponding XPA endonucleases, p I 4.6 or 7.6, can complement the defect in the ability of the XPA endonucleases to incise psora-

len-plus-UVA-damaged nucleosomal DNA. The activity of the XPA endonucleases on psoralendamaged nucleosomal DNA, when mixed with the normal endonucleases, was inceased to normal or near-normal levels and was significantly greater ( p < 0.001) than can be accounted for by a simple additive effect. This indicates that the XPA endonucleases lack something which can be complemented by the normal endonucleases. We have introduced each of the 2 normal endonucleases into 8-MOP-plus-UVA-damaged XPA cells in culture by electroporation and have been able to correct the XPA repair defect with both (Tsongalis et al., submitted). These results demonstrate that the repair defect in XPA cells resides in the endonucleases we have isolated since this defect can be corrected, at both the enzymatic and cellular levels, by corresponding endonucleases from normal cells. Previous studies from our laboratory indicate that an AP endonuclease, p I 9.8, from XPA cells also lacks a factor(s), which is present in the corresponding normal AP endonuclease, needed for interaction with AP nucleosomal D N A (Kaysen et al., 1987). Experiments were carried out to ascertain whether the "correcting factor" present in the normal AP endonuclease could complement the activity of either of the XPA endonucleases, p I 4.6 or 7.6, on psoralen-plus-UVA-damaged nucleosomal DNA. Mixing the normal AP endonuclease, p l 9.8, however, with either of the XPA endonucleases failed to complement the activity of either of the latter on psoralen-plus-UVA-damaged nucleosomal DNA. There are several possible explanations for these findings. There may be a factor associated with the enzymes active on psoralen-plus-UVA-treated D N A and a different factor, or the same one in a modified form, associated with the enzyme active on AP DNA; both factors may interact with chromatin and allow the respective XPA endonucleases to incise the appropriately damaged DNA. If, on the other hand, the correcting factor associated with the 3 normal endonucleases is the same, then there must be an explanation for its failure to complement the XPA endonucleases, pls 4.6 and 7.6, when these enzymes are mixed with the normal endonuclease, p l 9.8. A number of more or less complex hypothetical intermolecular interactions may be proposed to account for these findings; it may be that some

77 type of enzyme subunit system exists in human cells with at least 2 components, one recognizing the specific adduct and a different subunit enhancing its interaction with chromatin. Thus the human endonucleases may, like the UvrABC nuclease in E. coli (reviewed in Sancar and Sancar, 1988; Grossman, 1988), consist of interacting subunits, each with different specific functions, and it may be that recognition of the specific damaged site by the appropriate endonuclease must occur before the chromatin factor (or factors) associated with it can exert its effect. Thus, according to this hypothesis, since the normal AP endonuclease does not recognize psoralen-plusUVA DNA adducts, the "correcting factor" associated with it cannot be effective. Comparisons with other systems Our findings indicate that the defect observed in XPA cells which prevents them from showing increased activity on psoralen-phis-UVA-treated nucleosomal DNA exists in the endonucleases themselves or in a closely associated co-factor. The studies of Mortelmans et al. (1986) and of Kano and Fujiwara (1983), which utilized crude cell extracts, also suggest that XPA cells are defective in a factor which renders the DNA in UVirradiated chromatin accessible to endonucleolytic attack by cellular enzymes. In XPA cells a defect in localized decondensation of chromatin, associated with excision repair following exposure to UV irradiation, has been suggested by the studies of Hittelman (1986). An XP "correcting factor" has been isolated and partially purified from calf thymus which, upon microinjection into UVC-irradiated XPA cells, corrects the XPA repair defect (De Jonge et al., 1983). These studies indicate that the factor is a protein. Microinjection of cell extracts from human placenta or HeLa cells into XPA cells restored DNA repair synthesis in these cells after UVC irradiation (Yamaizumi et al., 1986). Any similarities between these factors and the normal human endonucleases isolated in the present study will have to await further purification of the enzymes. The calf thymus, human placenta and HeLa cell factors have not been tested in an isolated nucleosomal system so the exact level or nature of the correction cannot be ascertained at present.

Wood et al. (1988) have utilized a system in which soluble extracts from human cells can perform repair synthesis on damaged plasmid DNA. Those studies indicated that whole-cell extracts from several XP complementation groups, including XPA, were deficient in repair replication of closed circular plasmid DNA damaged by UVAactivated psoralen. They interpret their results as not supporting the hypothesis proposed by Mortelmans et al. (1976) and Kano and Fujiwara (1983) that XPA cells are deficient in the gene products necessary to make UV lesions in chromatin accessible to repair enzymes. The conditions for their DNA repair reactions, however, included an incubation time of 6 h of the cell extracts with the damaged DNA substrate. Their cell extracts and reaction conditions were according to the method of Manley et al. (1980). Hough et al. (1982), however, have shown at the ultrastructural level that incubation of these soluble whole-cell extracts (Manley et al., 1980) with DNA fragments for only 30 min leads to formation of nucleosomes or nucleosome-like structures, indicating that histones are present in these extracts. It is, therefore, unlikely, after the 6-h incubation with cell extracts used by Wood et al. (1988), that the plasmid DNA remained "naked". Thus an alternate interpretation of the results reported by Wood et al. (1988), which is not in conflict with either their results, with the results presented in the present paper, or those of Mortelmans et al. (1976) and Kano and Fujiwara (1983) is that the XPA endonuclease(s) are defective in their ability to interact with damaged DNA when it is in the form of chromatin. This failure to interact normally with nucleosomes may be a general defect in endonucleases from XPA cells active in DNA repair processes. General implications The existence of 2 different endonucleases in human chromatin, with specificity for psoralenplus-UVA-damaged DNA, which are defective in XPA cells and of 2 additional chromatin-associated endonucleases with specificity for AP DNA; which are also defective in XPA cells, may be a manifestation of more than one genetic deficiency in XPA. The ability of some but not all combinations of normal and XP endonucleases to comple-

78

ment the respective XPA defect is also consistent with this hypothesis. Such combinations of defective genes in the same complementation group of XP have been hypothesized to occur in the "co-recessive model" which we have proposed to explain a number of paradoxical findings in both clinical and laboratory studies of XP (W.C. Lambert and Lambert, 1985, 1989b). However, it is also possible that a single factor may be defective in all of the endonucleases we have examined. This factor, if determined by a single gene, might then represent the only XPA genetic abnormality, as predicted by autosomal recessive inheritance, or be one of several abnormalities encoded by different genes, as predicted by the co-recessive inheritance model. Further purification and characterization of the endonucleases we are examining should elucidate this question. Acknowledgments We would like to thank Dr. W. Clark Lambert for critical reading of the manuscript and Robert Lockwood for culturing the human cell lines and for isolating and purifying plasmid D N A . This work was supported by Grant AM 35148 from the National Institutes of Health and Grant 86-490-CCR from the New Jersey State Commission on Cancer Research. References Amari, N.M.B., W.C. Lambert and M.W. Lambert (1986) Comparison of histones in normal and xeroderma pigmentosum lymphoblastoid cells, Cell. Biol. Int. Rep., 10, 875-880. Anderson, T.F., and J.J. Vorhees (1980) Psoralen photochemotherapy of cutaneous disorders, Annu. Rev. Pharmacol. Toxicol., 20, 235-257. Averbeck, D., D. Papadopoulo and E. Moustacchi (1988) Repair of 4,5,8-trimethylpsoralen plus light-induced DNA damage in normal and Fancorti's anemia cells, Cancer Res., 48, 2015. Baden, H.P., J.M. Parrington, J.M. Delhanty and M.A. Pathak (1972) DNA synthesis in normal and xeroderma pigmentosum fibroblasts following treatment with 8methoxypsoralen and long wave ultraviolet light, Biochim. Biophys. Acta, 262, 247-255. Ben-Hur, E., and M.M. Elkind (1973) Psoralen plus near ultraviolet light inactivation of cultured Chinese hamster cells and its relation to DNA cross-links, Mutation Res., 18, 315-324.

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80 (1974) Photochemotherapy of psoriasis with oral methoxsalen and longwave ultraviolet light, N. Engl. J. Med., 291, 1207-1211. Sancar, A., and G.B. Sancar (1988) DNA repair enzymes, Annu. Rev. Biochem., 51, 29-68. Santella, R.M., N. Dharmaraja, F.P. Gasparro and R.L. Edelson (1985) Monoclonal antibodies to DNA modified by 8-methoxypsoralen and ultraviolet A light, Nucleic Acids Res., 13, 2533-2544. Scott, B.A., M.A. Pathak and t3.R. Mohn (1976) Molecular and genetic basis of furocoumarin reactions, Mutation Res., 39, 29-74. Smerdon, M.J. (1989) DNA excision repair at the nucleosomal level of chromatin, in: M.W. Lambert and J. Laval (Eds.), DNA Repair Mechanisms and Their Biological Implications in Mammalian Cells, Plenum, New York, in press. Sollner-Webb, B., R.D. Camerirti-Otero and G. Felsenfeid (1976) Chromatin structure as probed by nucleases and

proteases: Evidence for the central role of histones H3 and H4, Cell, 9, 179-193. Song, P.-S., and K.J. Tapley Jr. (1979) Photochemistry and photobiology of psoralens, Photochem. Photobiol., 29, 1177-1197. Vigny, P., F. Gaboriau, L. Voituriez and J. Cadets (1985) Chemical structure of psoralen-nucleic acid photoadducts, Biochimie, 67, 317-325. Watanabe, F. (1984) Condensation of polynucleosomes by histone H1 binding, FEBS Lett., 170, 19-22. Wood, R.D., P. Robins and T. Lindahl (1988) Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extracts, Cell, 53, 97-106. Yamaizumi, M., T. Sugano, H. Asahina, Y. Okada and T. Uchida (1986) Microinjection of partially purified protein factor restores DNA damage specifically in group A of xeroderma pigmentosum cells, Proc. Natl. Acad. Sci. (U.S.A.), 83, 1476-1479.