Xeroderma pigmentosum endonuclease complexes show reduced activity on and affinity for psoralen cross-linked nucleosomal DNA

Mutation Research, DNA Repair, 273 (19t12) 157-170 © 1992 Elsevier Science Publishers B.V. All rights reserved 0921-8777/92/$05.110

157

MUTDNA 00187

Xeroderma pigmentosum endonuclease complexes show reduced activity on and affinity for psoralen cross-linked nucleosomal D N A David D. Parrish *, W. Clark Lambert and Muriel W. Lambert Department of Laboratory Medicine and Pathology, UMDNJ, New Jersey Medical School and Graduate School of Biomedical Sciences, 185 South OrangeAt'enue, Newark, NJ 07103.2757 (U.S.A.) (Accepted 1 July 1991)

Keywords: DNA endonucleases; Xeroderma pigmentosum; Nucleosomal DNA; Interstrand cross-links

Summary Two DNA endonuclease complexes have been isolated from the chromatin of normal human and xeroderma pigmentosum, eomplementation group A (XPA), lymphoblastoid cells which are active on DNA damaged with psoralen plus long wavelength ultraviolet radiation (UVA). In both normal and XPA cells, one endonuclease complex, p l 4.6, recognizes the psoralen cross-link and the other endonuclease complex, p l 7.6, recognizes the psoralen monoadduct. The levels of activity of these complexes from both normal and XPA cells are similar on damaged naked DNA. Kinetic analysis of assays using graduated concentrations of substrate revealed that selective activity of these endonuclease complexes on 8-MOP plus UVA treated D N A correlates with a reduction in Km of these complexes, indicating an increased affinity for, or rate of association with, damaged naked DNA. When the damaged substrates were reconstituted into core nuclcosomes (without histone HI), both normal endonuclease complexes showed a 2.5-fold enhancement of activity, which correlated kinetically with a further increase in affinity, or rate of association (decreased Kin), for this damaged nucleosomal substrate. This increase in activity and in affinity was reduced but not eliminated when histone HI was present. By contrast, neither XPA endonuclease complex showed this enhanced activity on, or affinity for, damaged core nucleosomal DNA, and actually showed decreased activity, and affinity, when histone H1 was present. Introduction, via electroporation, of either of the normal complexes into 8-MOP plus UVA treated XPA cells in culture corrected their DNA-repair defect, further confirming the role of these complexes in the repair process.

07103-2757(U.S.A,I.

Chromatin structure has been shown to play an important role in determining the distribution Ol repair sites on DNA as well as the accessibility of damaged DNA to enzymatic attack (Lan and Smerdoa, 1985; Bohr et al., 1987, Smerdon, 1989).

* Present address: Ciba-Geigy Corporation, Experimental Toxicology Division.Summit,NJ (U.S.A.).

Depending upon the type of damage, a particular lesion may be nonrandomly located and repaired

Correspondence: Muriel W. Lambert, Ph.D., Departmenl of

Laboratory Medicineand Pathology, UMDNJ, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ

158 in the core or linker regions of the nucleosome (Cerutti et al., 1980; Smerdon, 1989). Preferential repair of DNA has been demonstrated to occur in transcriptionally active DNA sequences, in which chromatin is in a more open configuration, compared to transcriptionally inactive DNA sequenees (Mellon et al., 1986; Bohr et al., 1987; Leadon and Snowden, 1988; Hanawalt et al., 1989). Chromatin configuration has also been shown to play a role in repair processes occurring in higher order chromatin loops in the nuclear matrix (Mullenders et al., 1986, 1989). However, the influence of nucleosome structure on the activity and binding affinity of isolated specific DNA-repair enzymes on damaged DNA remains virtually unexamined, We have isolated two ehromatin-associated DNA endonuclease complexes from normal human lymphoblastoid cells, p l s 4.6 and p/ 7.6, which are selectively active on DNA treated with psoralen plus long wavelength (365 rim) ultraviolet radiation (UVA) (Lambert et al., 1988). Our results indicate that the endonuclease complex, pl 4.6, recognizes the psoralen intercalation and also the psoralen cross-link and that the endonuclease complex, pl 7.6, recognizes the psoralen monoadduet (Lambert et al., 1988; Parrish and Lambert, 1990). We have shown that the activity of these normal endonuelease complexes on psoralen.damagcd DNA increases approximately 23-fold when the damaged DNA is reconstituted into nucleosomes containing core histones (H2A, H2B, H3 and H4)(Parrish and Lambert, 1990), Addition ol histone HI to the system reduces but does not eliminate this increase in activity on damaged nueleosomal DNA compared to damaged naked DNA (Parrish and Lambert, 1990). We have previously shown that these two chromatin-associated endonuclease complexes, pls 4.6 and 7.6, are present in lymphoblastoid cells derived from patients with the inherited, cancerprone, DNA-repair-deficient disease, xerodcrma pigmentosum, complementation group A (XPA), and have levels of activity similar to those of the normal endonuclease complexes on DNA damaged by psoralen plus UVA (Lambert et al., 1988). By contrast, neither XPA endonuclease complex shows any increase in activity on damaged core nucleosomal DNA, and actually shows

decreased activity when histone H1 is present (Parrish and Lambert, 1990). We have also shown that the normal human endonuclease complexes can complement both of the XPA complexes on damaged nucleosomal DNA in a cell-free system (Parrish and Lambert, 1990) and can also correct the XPA-repair defect when introduced via electroporation into XPA cells in culture (Tsongalis et al., 1990). These results indicate that a defect exists in the ability of the XPA endonuclease complexes to interact with damaged nueleosomal DNA. In order to elucidate the nature of the interaction of these endonuclease complexes with psoralen plus UVA damaged non-nucleosomai and nucleosomal (+ histone HI) DNA, a kinetic analysis of activities of these complexes on graduated concentrations of substrate has now beer, carried out. The results indicate that both normal but neither of the XPA endonuclease complexes have an increased affinity for, or rate of association with, damaged nucleosomal DNA, compared with damaged non-nucleosomal DNA. The affinity of the normal and XPA endonuelease complexes for damaged nucleosomal DNA correlates with their ability, or inability, to incise this damaged substrate. Materials and methods

Cell lines and culture conditions Normal human (GM 1989 and GM 3299) and xeroderma pigmentosum, complementation group A (XPA), (GM 2345 and GM 2250A) lyrephoblastoid cell lines (transformed with Epstein-Barr Virus) were obtained from the C',iell 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.) and harvested under conditions of maximal proliferation, as previously described (Okorodudu et al., 1982), Cell cultures were routinely tested for mycoplasma (Okorodudu et al., 1982). DNA endonuclease extraction Cell nuclei were isolated and the chromatinassociated proteins were separated from the nucleoplasmic proteins in a series of steps, passed

159 though a CM sephadex column and electrophoresed on an isoelectric focusing column (Lambert et al., 1982, 1988). Fractions collected from the column were assayed for DNA endonuclease and exonuclease activities (Lambert et al., 1982). Peaks of endonuclease activity were pooled, dialyzed into 50 mM potassium phosphate (pH 7.1), 1 mM fl-mercaptoethanol, 1 mM NaEDTA, 0.25 mM phenylmethylsulfonyifluoride (PMSF) and 40% ethylene glycol and stored unfrozen at - 2 0 ° C (Lambert et al., 1988). Protein concentrations were determined by the BioRad protein assay (BioRad Laboratories).

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; Lambert et al., 1988). DNA was extracted with phenol, treated with ribonuclease l and eleetrophoresed on 0.9% agarose gels. The uncleaved, circular, Form I DNA band was cut from the gel, electroeluted and recovered by ethanol precipitation (Parrish and Lambert, 1990). The DNA was further purified on a NACS 37 (Bethesda Research Laboratory) column (Parrish and Lambert, 1990). DNA eluting from the column, consisting of greater than 95% Form l DNA, was recovered by ethanol precipitation and resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.

Histone isolation Nuclei were isolated from normal and from XPA lymphoblastoid cell lines and histones were extracted and separated as previously described (Kaysen et al,, 1986). Histone HI was removed from total histones by precipitation with 5% perchloric acid (Kaysen et al., 1986). The purity of core (H2A, H2B, H3 and H4) and total (core plus H1) histones was monitored by gel electrophoresis (Kaysen et al., 1986). Protein concentrations were determined by the BioRad protein assay (BioRad Laboratories)using total calf-thymus histones as a standard.

Nucleosome reconstitution Plasmid DNA was mixed with histones (+ histone H1) from normal or XPA cells at a histone : DNA weight ratio of 1.0 in a buffer containing 2 M NaCI, 50 mM Tris-HCl (pH 8.0), 0.1 M EDTA and 0.25 mM PMSF (Kaysen et al., 1986, 1987). The NaCI concentration was progressively decreased by stepwise dialysis at 4°C over a 28-h period to 50 mM NaCI (Kaysen et al., 1986; Parrish and Lambert, 1990), Reactionofpsoralen with DNA 8-Methoxypsoralen (8-MOP) (Sigma Chemical Co.) was recrystallized and purity checked by thin-layer chromatography (Lambert et al., 1988). Photoreaction of 8-MOP with non-nucleosomal and nucleosomal DNA was carried out utilizing a treatment protocol which involved exposing 8MOP (7-15/zg/ml) treated DNA to two doses of UVA radiation, an initial dose (10 W / m 2 for 10 min) after the 8-MOP has intercalated into the DNA and a second dose (10 W / m 2 for l0 rain) after the unbound 8-MOP has been removed by dialysis (Lambert et al., 1988). This procedure has been shown to increase the number of DNA interstrand cross-links (Ben-Hur and EIkind, 1973; Bredberg, 1982) and produced cross-links in 99% of the non-nucleosomai DNA molecules (Lambert et al., 1988). Angelicin (Elder Co.) (25 /zg/ml) was reacted with non-nucleosomal and nucleosomal DNA for 20 rain and then exposed to UVA light (10 W/m-') for 5 rain (Lambert et ai., 1988). Control DNA for the psoralen-treatcd DNAs was exposed to UVA irradiation only. Cross-linking of psoralen to non-nucleosomal and nucleosomal DNA was determined by alkaline gel electroI~horesis (Lambert et al., 1988). Assay for DNA endonucl~'ase actit'ity Endonuclease activity on nueleosomal and non-nucleosomai DNA was measured using a gel electrophoretic assay which measures the conversion of circular, supercoiled Form ! DNA to nicked, relaxed circular Form I! DNA (Lambert et al., 1988; Parrish and Lambert, 1990). Briefly, 0.10 p~g of DNA substrate was reacted with each DNA endonuclease complex from either normal or XPA cells in 10 mM MgCl,, and 10 mM Tris-maleate (pH 7.5) at 37°C for 3 h. The con-

160

centration of each DNA endonuclease complex was adjusted, at similar levels of protein, to produce 0.05 + 0.01 breaks per DNA molecule on non-damaged non-nucleosomai DNA in these assays. The enzymatic reaction was te~.ainated with 0.1 M EDTA and the DNA 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; Parrish and Lambert, 1990). "Samples were electrophoresed on 1.0% agarose gels whieh were subsequently stained with 0.5/zg/ml ethidium bromide and photographed, The negatives of the gels were scanned and endonuelease activity, expressed as the number of enzyme induced breaks per DNA molecule, determined as previously described (Lambert et al., 1988; Parrish and Lambert, 1990). At the low concentration used here, the binding of ethidium bromide to superhelical versus non-superhelical forms of DNA was completely equivalent and did not influence the calculations of the various forms of DNA. In addition, in the exposure ranges used, the response of the film used in photographing the gels was linear. Kinetic analysis For analysis of the kinetics of these endonuclease-mediated reactions, assays were performed on undamaged and damaged, non-nucleosomal and nucleosomal (±histone HI) DNA as described above, but with graduated reductions in substrate concentration (0.1-0.025/zg), These assays were carried out over a range encompassing at least a 4-fold decrease in substrate concentration. Over this range all components of the assay system, described above, remained linear. Results were plotted as [S]-i versus c-~ according to Lineweaver and Burke and also as t, versus c/[S] according to Eadie and Hofstee where [S] and r represent initial molar substrate concentration and velocity of the enzymatic reaction, respectively. Velocity of the reaction was computed based on cleavages produced per substrate molecule and on the number of substrate molecules present in the assay solution. Values of Vmax (maximum velocity) and K,, (Michaelis constant) were determined from linear extrapolations on these graphic representations using standard methods based on the Michaclis-Menten equa-

tion. Values of turnover number, K~,., were computed from these values for Vm~x. Results Activity of the normal and XPA endonuclease complexes on psoralen plus UVA irradiated naked and nucleosomal DNA We have shown that there are two DNA endonuelease complexes, p/s 4.6 and 7.6, in the chromatin of normal human lymphoblastoid cells which are selectively active against DNA containing interstrand cross-links and monoadducts produced by 8-MOP plus UVA (Fig. 1A) (Lambert et al., 1988). One of these complexes, p l 7.6, is also active on DNA monoadduets produced by angelicin plus UVA (Fig. 1B) (Lambert et al., 1988). These same two endonuelease complexes are present in XPA cells and have levels of activity on 8-MOP or angelicin plus UVA treated DNA which are similar to those of the normal complexes (Fig. 1A and 1B) (Lambert et al., 1988). The activity of the normal and XPA complexes was examined on damaged nucleosomal DNA. The reconstituted nucleosomal system, which utilizes a plasmid containing the SV40 genome and normal or XPA histones, gave standard patterns of digestion with micrococcal nuclease and DNAase ! and showed positioning of nucleosprees in a region near the $V40 origin of replication (Kaysen et al, 1986; Amari et al., 1986; Kaysen et al., 1987). The activity of the two normal endonuclease complexes, pls 4.6 and 7.6, on core (minus histone H1) nucleosomal DNA treated with 8-MOP plus UVA was increased approximately 2.5-fold compared to their activity on damaged non-nucleosomal DNA (Fig. 2) (Parrish and Lambert, 1990). This increase was reduced, but not eliminated, when histone HI was added to the system, remaining approximately 1.5-fold greater than the activity on damaged naked DNA (Fig. 2) (Parrish and Lambert, 1990). These increases in activity, which were not observed on undamaged nueleosomal DNA, were also present on angelicin plus UVA light treated DNA (Parrish and Lambert, 1990). In marked contrast, these same two endonuclease complexes

161

from XPA cells did not show any increase in activity on either 8-MOP or angelicin plus UVA treated core nucleosomal DNA and showed a significant decrease in activity when histone HI was added (Fig. 2) (Parrish and Lambert, 1990). These difference were not due to t h : source of

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8-MOP + UVA LIGHT

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the histones, since using either normal or XPA histones in the system made no difference. In addition, we have previously examined normal and XPA histones and have found no differences quantitatively, qualitatively or in DNA binding affinity (Amari et al., 1986).

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pI OF ENOONUCLEASEFRACTIONS Fig, 1, Activity of chromatin-associated DNA endonuclease complexes from normal human and XPA lymphoblastoid cells on non-nucleosomal DNA treated with (A) 15 #g/ml 8-MOP plus two doses of UVA and (B) 25 p,g/ml angelicin plus UVA, These values have had subtracted from them the enzyme activity on undamaged DNA, Vertical lines represent_+S,E,M, (Lambert et al,, 1988).

162

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slightly reduced Vma, and Kcat (20-25% reduclion) on 8-MOP plus UVA treated DNA compared to their activi"es on control DNA, but activities of none of these 4 c o m p l e x e s s h o w e d a significant change in these values on damaged or control nucleosomal versus similarly treated nonnucleosomal DNA (Figs. 4 and 5). By contrast, the activities of the normal and of the XPA endonuclease complex, p l 7.6, both showed an increase of approximately 80% in Vmax and Kcat

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0 pI 4.6 pt 7.6 p14.6pI 7.6 pT4.6 pI 7.6 pX4.6 p17.6 0 EN00NUCLEASES [NDONUCLEASES Fig. 2. Activity of DNA endonuclease complexes, pls 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 endonuclease complexes (0.4] 4-0.05 p,g) were incubated with undamaged or 8-MOP (15 p,g/ml) plus U V A treated DNA reconstituted with total or core histones. Endonuclease activity is expressed as multiples of activity on non.nucleosomal DNA. The solid horizontal line represents enzyme activity on non-nucleosomal DNA. Vertical lines represent + S,E.M, (Parrish and Lambert, 1990),

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Analysis of V,,,ax and K,.a, of normal and XPA endonuclease complexes on psoralen plus UVA damaged non.nucleosomal and nucleosomal DIVAs Both DNA endonucleas¢ complexes from both cell types were assayed against a range of differoat substrate concentrations (Fig, 3) so that kinetic analysis of their activities could be carried out. Kinetic analysis of the activities of both of the normal and both of the XPA endonuclease complexes, pls 4.6 and 7.6, assayed against untreated, control (with UVA, without psoralen) and psoralen plus UVA damaged DNAs, both naked and reconstituted into nucleosomes ( + hist o n e HI) all produced linear results o n both Lineweaver-Burke and Eadie-Hofstee graphic representations. The coefficient of correlation (R)

was at least 0.98 and 0.93, respectively, on each of these analyses. The results presented here reprosent pooled data from two normal or from two XPA cell lines. Values represent the average of 3-5 separate experiments, Activities of both of the normal and of both of the XPA endonuelease complexes showed a

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0 z 4 s a ~0 ~Z 0 2 4 S e i0 llz SUBSTRATECONCENTRATIONAS NUMBER OF MOLECULESx I09 Fig, 3. Activity of the normal and XPA DNA endonuclease complexes, (A) p l 4.6 and (B) p l 7,6, on increasing concentralions of naked (i.e., non-nucleosomal), core (without histone H I ) and total (with histone H I ) nucleosomal plasmid DNA treated with S-MOP ('/ ~e/ml) plus two doses of UVA, Values are expressed as total number of enzyme mediated cleavages per minute and are representative for one set of

experiments.Assay volumewas 40/~1 containing0.34+0.04 ~,g endonuclease complex, Values in replicate experiments varied by :!:5%, These values have had subtracted from them the enzyme activity on control DNA, exposed to two doses of UVA radiation, as above, without treatment with 8-MOP.

163

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Fig. 4. Maximum velocity (Vmax) (maximum number of cleavages produced per minute) and Kcat (maximum number of cleavages produced per , i n per /zg endonuclease) of the normal and XPA endonuclease complex, p l 4.6, on naked, core or total nucleosomal DNA treated with 8-MOP(Tp.g/ml) plus two doses of UVA irradiation (10 W/m 2 for 10 min). Control DNA was exposed to two doses of UVA irradiation, as above, without treatment with 8-MOP. Vertical bars represent standard errors of the mean for 5 or more Expts. Assay volume was 40/LI containing 0.34+0.04/~g of endonuclease complex.

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on angelicin plus UVA damaged DNA compared to control DNA, but again no significant change in these values was noted on damaged or control nucleosomal versus similarly treated non-nucleosomal DNA (Fig. 6). Activity of the normal and XPA

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Fig. 6. l/max and Kcat of the normal and XPA endonuclease complex,p l 7.6, on non-nucleosomal or core and total nudeosomal DNA treated with angelicin (25 pg/ml) plus UVAirradiation (10 W/m 2 for 5 min). Control DNA was exposed to UVA-irradiation, as above, without treatment with angelicin. Assay volume was 40/~1 containing 0.34_+0.04 #g of endonucleasecomplex. Vertical bars represent standard errors of the mean for 5 or more Expts.

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Fig. 5. Ymax and Kca~ of the normal and XPA endonuclease complex, p l 7.6, on non-nucleosomal or core and total nucleosomal DNA treated with 8-MOP plus two doses of UVAirradiation. Control DNA was exposed to two doses of UVA irradiation without treatment with 8-MOP. Reaction conditions and vertical bars are as for Fig. 4.

endonuclease complexes, p l

4.6, on an-

gelicin plus UVA damaged DNA was not examined kinetically since we have found that both complexes have only a low level of activity on their substrates (Lambert et a]., 1988; Parrish and Lambert, 1990). All 4 endonuclease complexes showed a Vm,,x and Kc,, approximately 17% higher on control (UVA treated) DNA compared to untreated DNA (data not shown). The K m of the normal and XPA endonuclease complexes on psoralen plus UVA damaged nonnucleosomai and nucleosomal DNA Both normal and both XPA endonuclease complexes showed a reduction in K m, to approximately 50% of their value on control DNA, on non-nucleosomal DNA treated with 8-MOP plus U V A (Figs. 7 and 8). A further decrease was noted in the K m o f both normal complexes o n damaged DNA when nucleosomes were present

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~°,EO ~oC.0,O.,~ DNa50BSTRME Fig. 7. Michaelis constants (Km). expressed as numbers of substrate molecules within the assay solution (40/~1). of the normal and XPA endonuclease complex, p/4.6, assayed on naked (non-nucleosomal), core and total nucleosomal DNA treated with 8-MOP plus UVA. Reaction conditions are as for Fig. 4. Vertical bars refer to the standard errors of the mean of results obtained graphically for K m from at least 5 Expts.

(Figs. 7 and 8). This additional decrease was not seen on nucleosomal versus naked DNA treated only with UVA. The Kms of both normal endonuclease complexes on 8-MOP plus UVA dam-

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aged core nucleosomal D N A were approximately 60% of those observed for the same enzymes on damaged naked D N A ( p < 0.001). This decrease in K m was also present, but was not as great, with the K m approximately 80% of that observed on damaged naked DNA, on damaged total nucleosomal (+histone H1) D N A ( p <0.005). Again, no decrease in K m o n total nucleosomal versus naked D N A treated only with U V A was observed. By contrast, neither XPA endonuclease complex showed these additional decreases, and instead showed increases, in K m on 8-MOP plus UVA

damaged

nucleosomal

DNA,

compared

n o difference in K m o f either XP endonuclease c o m p l e x o n either type of control nucleosomal

D N A compared with that against control naked DNA. The normal and XPA endonuclease cornplexes, pl 7.6, showed no change in their K m on angelicin plus U V A treated naked D N A compared to control naked D N A (Fig. 9). However, the normal complex showed a 50% reduction in

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Fig. 8. Michaelis constants (K.,) of the normal and XPA endonuclease complex, pl 7.6, on non-nucleosomal and on core and total nu¢leosomal DNA, treated with 8-MOP plus two doses of UVA-irradiation, Control DNA was exposed to two doses of UVA irradiation without treatment with 8-MOP, Reaction conditions and vertical burs are as for Figs. 4 and 7, respectively,

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similarly damaged non-nucleosomal D N A (Figs. 7 and 8). A significantly greater increase was noted for both XPA complexes against damaged nucleosomal D N A containing histone HI. There w a s

8~10~ I ~l~tO

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ONnSuB$roatt Fig, 9, Michaelis constants (K m) of the normal and XPA endonuelease, pl 7.6, on non-nucleosomal or core and total nucleosomal DNA treated with angelicin plus UVA irradiation, Reaction conditions are as for Fig. 6. Vertical bars represent the standard errors of the mean of results obtained graphically for Km from at least 4 Expts.

165

similarly damaged core nucleosomal DNA compared to damaged naked DNA. When histone H1 was present this decrease in K m w a s n o t as great; the K m w a s 80% of the value on damaged naked DNA. In contrast, activity of the XPA endonuclease complex, p I 7.6, did not show any significant change in K m o n angelicin plus UVA treated core nucleosomal DNA and instead showed a slight but significant increase in its K m on total nucleosomal DNA compared to its K m on damaged naked DNA. Activities of each of these four endonuclease complexes showed a reduction of approximately 7% in K m on DNA irradiated with the dosages of UVA used here (control DNA) versus untreated DNA.

K m on

Complementation of the XPA repair defect by the normal human endonuclease complexes We have introduced each of these normal endonuclease complexes, via electroporation, into XPA cells in culture treated with 8-MOP plus UVA and have corrected the XPA repair defect (Tsongalis et al., 1990). Each of the normal but neither of the XPA complexes restored UDS in treated XPA cells to higher than normal levels (Fig. 10) (Tsongalis et al., 1990). In addition both normal and XPA endonuclease complexes inr " l ~ t,D0,uc~.s~ ~,-E~ooNuc~.sE II,"'~LE~DO~"C~"SE

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B

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125-

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m ~s -

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Fig. 10. UDS in XPA lymphoblastoid cells in culture treated with 8-MOP plus UVA and electroporated with the normal or XPA DNA endonuclease complexes. Normal or XPA cornplexes (A) p l 4.6 or (B) p i 7.6 (I.4 pg) were introduced into XPA cells via electroporation. Results are expressed as percent of normal UDS + S.E.M. for 4-5 separate Expts. with a total of 1.5 x 10 "~ to 2.5 x 10"~ cells counted. (Tsongalis et al..

1990)

creased UDS in normal cells to higher than normal levels (Tsongalis et al., 1990). Similarly, mixing the no~mal and XPA endonucleases together and examining their activity on 8-MOP or angelicin plus UVA treated nucleosomal DNA led to complementation of the XPA defect in our cell-free system (Parrish and Lambert, 1990). Discussion We have isolated two chromatin-associated DNA endonuclease complexes from normal human lymphoblastoid cells which have specificity for psoralen plus UVA light damaged DNA (Lambert et al., 1988; Parrish and Lambert, 1990). These two complexes, pls 4.6 and 7.6, which recognize psoralen interstrand cross-links and monoadducts, respectively, show increased activity on damaged DNA when nucleosomes are present (Parrish and Lambert, 1990; Lambert et al., 1991). These complexes thus perform three functions: damage recognition, endonucleolytic incision and chromatin interaction. Each contains both a DNA endonuclease and a protein needed for endonuclease interaction with damaged nucleosomal DNA (Parrish and Lambert, 1990). Whether damage recognition is associated with a s e p a r a t e protein or is performed by the endonuclease is under investigation. These same two endonuclease complexes are present in XPA cells but are defective in their ability to interact with psoralen plus UVA damaged nucleosomal DNA (Parrish and Lambert, 1990). Thus both normal and XPA cells have the endonuclease, but the XPA cells are defective in the protein needed for interaction with damaged nucleosomal DNA (Parrish and Lambert, 1990). The nature of the defective interaction of the XPAendonuclease complex with psoralen damaged nucleosomal DNA has been explored further by carrying out a kinetic analysis of this interaction utilizing graduated concentrations of substrate.

Kineticanalysis of the normal and XPA endomwlecomplexes on psoralen plus UVA damaged DNA K i n e t i c analysis of assays using graduated doses of substrate indicates that the selective activity against 8-MOP plus UVA treated DNA shown by

ase

166

both normal and both XPA endonuclease complexes is associated with increased affiniw, or rate of association, of enzyme with the substrate, as indicated by the decreased Km. In addition, the turnover number (Kcat) is actually slightly decreased on this damaged substrate compared to that on control DNA. The reduced rate of cleavage (Kcat), following association of the cornplex with DNA damaged with 8-MOP plus UVA, may be due to the marked distortion which occurs in DNA following formation of a psoralen DNA cross-link (Vigny et al., 1985; Cimino et al., 1985). By contrast, the increased activity of both the normal and the XPA endonuclease complex, p l 7.6, on angelicin plus UVA treated DNA is associated with an increase in Kcat, compared to activity on control DNA, with no decrease in K m detected. It is possible, however, that an increase in affinity is present which is masked by the increase in Kc.~, which may, itself, increase Km. The increased activity of the normal endonuclease complexes on both 8-MOP (both cornplexes) and angelicin (the complex, p l 7.6) plus UVA treated DNA when they are reconstituted into nucleosomes is associated only with an increase in affinity, or rate of association, of the complex for the substrate, as indicated by decreases in Km with no corresponding changes in Kc,,t. This increase in affinity is greatest on d a m aged core nucleosomal DNA and is diminished but not abolished when histone H1 is present, This increased affinity correlates with a 2.5-fold enhancement of the activity of both normal cornplexes on 8-MOP or angelicin plus UVA damaged core nucleosomal DNA and only a 1.5-fold increase when histone HI is present. By contrast, the XPA endonuclease complexes fail to show this decrease in K m (increase in affinity), and actually show an increase in K m (decrease in affinity) when histone H1 is present, indicating that the XPA endonucleases are defective in their ability to interact with chromatin because of reduced affinity, or rate of association, with damaged nucleosomal substrates, This correlates, in turn, with failure of the XPA endonuclease complexes to show any increase in activity on damaged core nueleosomal DNA and with a reduced activity of these complexes when histone HI was present,

The increases in affinity and/or rate of association shown by the normal endonuclease cornplexes are damage-specific, since they are not seen on undamaged nucleosomal versus nonnucleosomal DNA. These increases on damaged nucleosomal DNAs are not due to an increased number of DNA adducts, since we have determined that the number of 8-MOP adducts is reduced approximately 50% on core nucleosomal DNA and 60% when histone H1 is present (Parrish and Lambert, 1990). The nucleosomal system in relationship to endonuclease activity. The enhanced activity of the normal human but not the XPA endonuclease complexes for damaged nucleosomal DNA appears to be related to the presence of histones H3 and H4 (Parrish and Lambert, in preparation). This could either be due to direct interaction of the endonucleases with these histones or due to the fact that these histones can form nucleosome-like structures. The reduced affinity and activity of the normal and XPA endonuclease complexes for damaged nucleosomal DNA when histone H1 is present is in agreement with the proposed role of histone H1 in condensation of chromatin, making it less accessible to endonucleolytic attack (McOhee and Felsenfeld, 1980; leo. Kemenes et al., 1982; Klingholz and $tratling, 1982; Watanabe, 1984). These results are corroborated, in part, by those obtained by Ishimi et al. (1981), who found that histone H1 protected a site in undamaged calf-thymus DNA from micrococcal nuclease. Enhancement of the affinity of an endonuclease for DNA when it is present in chromatin has also been reported by SoUner-Webb et al. (1986). They found that the apparent affinity of staphylo. coccal nuclease for ehromatin was greater than that for protein-free DNA; however, the activity of the nuclease was less on chromatin than on protein-free DNA. This differs from the present results in which the increased affinities of the endonuclease complexes for damaged nucleosoreal DNA correlate with an increase in their activity on this substrate. We have also found that two normal but not XPA chromatin-associated endonuclease complexes, pls 9.2 and 9.8, which selectively recognize apurinic/apyrimidinic (AP)

167

sites, show increased activity on AP nucleosomal DNA compared to AP naked DNA (Kaysen et al., 1986). It is possible that all of the normai endonuclease complexes have associated with them the same or a similar chromatin protein which makes damage on nucleosomal DNA more accessible to endonucleolyt!c attack than on naked DNA. Relationship with other systems. Our studies indicate that the defect in XPA cells in the incision step of repair of psoralen plus UVA treated DNA exists in the inability of the XPA endvnuclease complex to interact with damaged nucleosomal DNA (Kaysen et al., 1986; Parrish and Lambert, 1990). The studies of Morteimans et al. (1976), and of Kano and Fujiwara (1983), using crude cell extracts, also suggest that XPA cells are defective in a factor which renders the DNA in UVC-irradiated chromatin accessible to endonucleolytic attack. The work of Hittelman (1986) suggests that XPA cells have a defect in decondensation of chromatin associated with excision repair following UV-irradiation. All of these studies support the concept that a defect related to endonuclease interaction with chromatin is present in XPA cells, The human endonuclease complex may have some similarities with the UvrABC system in E. coil in which the interaction of different protein subunits is needed for endonucleolytic incision of damaged DNA (Sancar and Sancar, 1988; Grossman, 1988; Orren and Sancar, 1989). in the human endonuelease complex, a chromatin-interacting protein is present which is needed for endonuelease incision of damaged nucleosomal DNA. There is specificity associated with the interaction of each complex with damaged nucleosomal DNA. For example, adding the normal AP endonuclease complex, p l 9.8, does n o t c o r rect the defect in the ability of the XPA endonuclease complex, pl 7.6, to incise 8-MOP plus UVA damaged nucleosomal DNA (Parrish and Lambert, 1990). it may be that recognition of the specific damaged site by the appropriate endonuclease is needed before the chromatin-interacting protein can exert its effect. The UvrABC system is different from the human endonuclease complexes in that it does not show enhanced activity

on damaged nucleosomal DNA (Parrish and Lambert, in preparation). This is not surprising since E. coli does not have the complex chromatin structure of human cells. Studies reported by Wood et al. (1988) and by Sibghat-Uliah et al. (1989), appear to contradict the concept that the defect in XPA cells resides at the level of interaction of the endonucleases with damaged chromatin. In those studies soluble cell extracts of normal, but not XPA, cells were reported to be able to remove adducts from exogenous naked DNA irradiated with UVC. Such extracts of mammalian cells, however, have been shown to reconstitute naked plasmid DNA into structures, visualized ultrastructurally, which resemble nucleosomes (Manley et al., 1980; Hough et al., 1982). The defect observed by Wood et al. using these extracts, therefore, may also have been due to an inability of a XP.,~ endonuclease to interact with some type of damaged nucleosome-like structure rather than a failure to act on damaged non-nucleosomal DNA. An XP "correcting factor" has been isolated from calf thymus which, upon microinjection into XPA cells, corrects their repair defect (de Jonge et al., 1983). Correction of the DNA-repair defect after UVC-irradiation has also been reported in XPA cells following microinjection of crude cell extracts from human placenta or HeLa cells (Yamaizumi et al., 1986). Tanaka et al. (1990) have recently cloned a human XPA eDNA which, when transfected into UVC-irradiated XPA cells from Japanese patients, corrects the repair defeet. This eDNA encodes a protein with a zincfinger motif, suggesting that it is a DNA-binding protein (Tanaka et al., 1990; Satokata et al., 1990). This is consistent with our studies, which indicate that the XPA-correcting factor which we have isolated is a protein which makes damaged nucleosomal DNA accessible to damage-specific endonucleases (Lambert and Parrish, 1989; Parrish and Lambert, 1990). Whether the protein in our studies is similar to that encoded by the gene identified by Tanaka and co-workers is under investigation. XPA is a complex disease, the etiology of which has not yet been worked out (Cleaver, 1990) and it may be associated with more than one defective gene (Lambert and Lambert, 1985,

168 1989). O u r studies indicate that a defect exists in the interaction o f X P A e n d o n u c l e a s e complexes with d a m a g e d nucleosomal D N A ( L a m b e r t and Parrish, 1989; Parrish a n d L a m b e r t , 1990; Tsongalis, 1990); the defect in the ability o f the X P A e n d o n u c l e a s e complexes to incise d a m a g e d nucleosomal D N A correlates with a d e c r e a s e d affinity, o r rate of association, of these complexes

with this damaged substrate. This defect appears tO be related to a protein n e e d e d for interaction of the e n d o n u c l e a s e s with d a m a g e d nucleosomal D N A . X P has long b e e n c o n s i d e r e d one of the best h u m a n models for carcinogenesis due to an e n v i r o n m e n t a l agent. Identification o f a molecular defect in affinity, or rate of association, of X P A e n d o n u c l e a s e complexes for d a m a g e d nucleosomal versus non-nucleosomal D N A suggests that X P may now also be c o n s i d e r e d as a m o d e l

for the study of the intermolecular protein/DNA interactions in chromatin.

Acknowledgments We would like to thank Dr. S u r i e n d e r K u m a r

for critical reading of the manuscript and Mr. R o I ' ¢ ~ Lockwood for culturing the h u m a n cell

lines and for isolating and purifying plasmid DNA, Grant A M 35148 from the National Institutes of Health.

This work was s u p p o r t e d by

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