The endogenous nuclease sensitivity of repaired DNA in human fibroblasts

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Mutation Research, 184 (1987) 169-178 DNA Repair Reports Elsevier MTR 06240

The endogenous nuclease sensitivity of repaired DNA in human fibroblasts Audrey N. Player and G.J. Kantor Department of Biological Sciences, Wright State Unioersity, Dayton, OH 45435 (U.S.A.) (Received 13 March 1987) (Accepted 20 April 1987)

Keywords: DNA excision repair; Xeroderma pigmentosum; Nuclease sensitivity; Repaired DNA; Localized genomic repair.

Summary The limited DNA excision repair that occurs in the chromatin of UV-irradiated growth arrested cells isolated from a xeroderma pigmentosum (XP) complementation group C patient is clustered in localized regions. The repaired DNA was found to be more sensitive to nicking by endogenous nucleases than the bulk of the DNA. The extra-sensitivity does not change with increasing amounts of DNA damage or repair activity in the locally-repaired regions and is retained through a 24-h chase period. We suggest that these results are due to the occurrence of DNA repair limited to pre-existing, non-transient chromatin fractions that contain actively transcribed DNA. A similar extra-sensitivity of repaired DNA was not detected in cells of normal or XP complementation group A strains that exhibit either normal or limited repair located randomly throughout their genomes. The association between endogenous nuclease sensitivity and clustered repair probably defines a normal excision repair pathway that is specific for selected chromatin domains. The repair defect in XP-C strains may be one in pathways targeted for other endogenous nuclease-resistant domains.

The limited DNA repair in the chromatin of ultraviolet (UV, 254 nm) irradiated cultured fibroblasts from individuals with the genetic disease xeroderma pigmentosum (XP) and belonging to complementation group C (XP-C) occurs nonrandomly, with repaired sites clustered in localized regions (Mansbridge and Hanawalt, 1983). These localized regions are estimated to represent about 25% of the total genomic DNA in growth-arrested cells (Cleaver, 1986). The rate of excision repair in these regions is nearly the same as the overall genomic repair rate found in normal cells (Cleaver,

Correspondence: Dr. G.J. Kantor, Department of Biological Sciences, Wright State University, Dayton, OH 45435 (U.S.A.).

1986) although, because of little or no repair in other chromatin fractions, the average genomic repair rate is considerably less than that of normal cells (Kantor and Hull, 1984). The nonrandom clustered type of repair observed in XP-C cells is not observed in normal human cells when a similar experimental protocol is employed, although evidence from other kinds of experiments (Cohn and Lieberman, 1984; Kantor and Setlow, 1981; Mellon et al., 1986) suggests that some nonrandom preferential DNA repair of specific chromatin locations may occur. To further characterize the DNA that is repaired in XP-C cells, we examined its sensitivity to cleavage by activated endogenous nucleases. Sensitivity of the native chromatin to these nucleases provides a means of

016%8817/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

170

assessing whether or not chromatin conformation is a factor related to the location of repaired sites in DNA (Weintraub and Groudine, 1976; Vanderbilt, et al., 1982). Nucleases present in nuclei do not normally cleave chromatin DNA rapidly, but can be activated by incubating isolated nuclei in appropriate cation-containing buffers (Vanderbilt et al., 1982). The rate and extent of autodigestion can be controlled by employing different buffers so that in vitro conditions can be selected to produce minimal DNA cleavage or more extensive digestion to nucleosomal-sized DNA (Hibino and Sugano, 1985; Vanderbilt et al., 1982). The endogenous nucleases activated in buffers with low divalent cation concentrations have a preferential activity for actively transcribed chromatin regions (Vanderbilt et al., 1982; Anderson et al., 1983) indicating that these autodigestion conditions are useful for defining different chromatin domains (for a review of chromatin structure and domains, see Eissenberg et al., 1985). Results reported here, obtained using autodigestion conditions that permit only limited cleavage of DNA, demonstrate that preferentially repaired chromatin DNA in UV-irradiated XP-C cells maintained in a growth-arrested state is more sensitive to endogenous nuclease cleavage than is the bulk of the DNA. The sensitivity does not increase with the occurrence of more excision repair in the localized regions and retains its original sensitivity thru at least a 24-h incubation period. Thus the extrasensitivity of the preferentially-repaired DNA in XP-C is probably not the result of UV-induced chromatin damage and events associated with the DNA-repair process but is a result of an original nontransient conformation of the chromatin DNA. Materials and methods

Cell strains and culture conditions. All cell strains were human diploid fibroblasts in the phase II stage of the proliferative life span (Hayflick, 1965) at the time of use. They are identified by their American Type Culture Collection (ATCC, Rockville, MD) identification number and by a letter identifying the XP complementation group. WS-1 (CRL 1502) is a normal strain obtained

from R.J. Hay. XP8LO (CRL 1376, XP-A) was obtained from E.A. de Weerd-Kastelein. XPI2BE (CRL 1223, XP-A) and XP4RO (CRL 1260, XP-C) were obtained from the ATCC. Cultures were initiated from frozen stocks. Cells were cultured routinely at 37 °C in a humidified incubator using Eagle's minimum essential medium supplemented with 2 mM L-glutamic acid and non-essential amino acids (Grand Island Biological Company, Grand Island, NY; No. 410-1600 powdered medium), 30 mM 4-(2-hydroxyethyl)-lpiperazine-ethanesulfonic acid buffer (Hepes, pH 7.4), 10% fetal calf serum (Grand Island Biological Company, Grand Island, NY), streptomycin (50 mg/ml) and penicillin (61 mg/ml). Cell populations used for experiments were in a growth-arrested state achieved by culturing cells in the above-described medium supplemented instead with 0.5% fetal calf serum. Detailed procedures for preparing arrested cultures have been described previously (Dell'Orco et al., 1973; Kantor et al., 1977; Kantor, 1986). Briefly, cells inoculated into normal 10% serum medium were incubated for 24 h at 37°C, transferred to 0.5% serum medium and incubated further at 37°C. The medium was replaced with fresh 0.5% serum medium every 5 days. Cell populations were used for experiments after 10-12 days in the low serum medium, a time beyond which no further increase in cell number is observed (Kantor, 1986). Arrested populations with uniformly radiolabeled DNA were obtained by culturing actively proliferating cells in 10% serum medium containing [Me-a4C]thymidine (0.02 btCi/ml, 40-.60 mCi/mmole, New England Nuclear, Boston, MA).

Experimental protocol and irradiation techniques. Growth arrested cells with 14C-labeled DNA were used in all experiments. Prior to irradiation, the culture medium was removed and saved for later use. Cells were rinsed with Hepes-buffered saline and irradiated using a Westinghouse 15W germicidal lamp (Westinghouse Electric Corp, Pittsburgh, PA). Incident intensities were determined using a photovoltaic sensor from a Blak-Ray J-225 meter (Ultra-Violet Products, Inc., San Gabriel, CA) connected to a Keithley 160B digital multimeter (Keithley Instruments, Cleveland, OH). This system was calibrated using a Yellow Springs

171 Instruments Model 65 radiometer (Yellow Springs Instruments, Yellow Springs, OH) and also by determining the number of pyrimidine dimers induced in DNA of UV-irradiated human cells using an enzymatic assay (Paterson et al., 1973; Kantor and Setlow, 1981). After irradiation, the used medium was returned to the cultures. To radiolabel the repaired DNA, [Me-3H]thymidine (3HdThd, 20 #Ci/ml, 73 Ci/mmole, ICN Radiochemicals, Irvine, CA) was added to each culture. For some experiments, hydroxyurea (HU, freshly prepared in water; final concentration, 10 mM) was added to cultures 30 min prior to UV-irradiation; the same medium was used for post-UV incubation periods. When determinations of the occurrence of DNA semiconservative replication were required, bromodeoxyuridine (BrdUrd; 1.6 × 10-5 M) was added 30 min prior to UV-irradiation; post-UV incubations were in the same medium.

Endogenous nuclease digestions. Nuclei were isolated using procedures described by Williams and Friedberg (1979). Cells (1 × 106) released from a culture dish by trypsin digestion were collected, washed in B1 buffer (50 mM Tris-HC1, pH 7.8, 5 mM NaHSO3), resuspended in 1 ml of B2 buffer (B1 plus 3 m M MgC12, 0.1% Triton X-100 and 100 mM sucrose) and incubated on ice for 5 rain. Following Dounce homogenization of cells at 0 ° C, nuclei were collected by centrifugation at 3184 × g for 5 rain at 2 ° C. Nuclei were washed with 1 ml and finally resuspended in 100 ~1 of autodigestion buffer (0.35 M sucrose, 10 mM Tris-HC1, pH 7.4, 25 mM KC1, 5 mM MgC12 and 0.5 mM CaC12 (Vanderbilt et al., 1982). Samples were incubated at 37 °C for defined periods to autodigest DNA. The amount of DNA solubilized was measured by determining the amount of radioactivity that was trichloroacetic acid (10%)- and ethanol-precipitable using liquid-scintillation counting procedures. Gradient analyses. Aliquots of digested nuclei (10 #1) were diluted to 100/~1 in an ice-cold buffer (0.03 M Tris-HC1, pH 8.0, 0.01 M NaC1, 0.01 M EDTA) and layered onto 4 ml 5-20% alkaline sucrose gradients (2 M NaC1, 0.33 M NaOH) containing a top layering solution (0.2 ml, 1 M NaOH, 0.01 M EDTA, 0.1% sodium lauryl sulfate).

After a I h lysis period, gradients were centrifuged at 20°C, 28000 rpm using a Beckman SW-60 rotor and ultracentrifuge (model L5-75, Beckman Instruments, Palo Alto, CA) for desired times. After gradient fractionation onto paper strips (Carrier and Setlow, 1971) and subsequent washing (5% TCA, twice with 100% ethanol) of the strips, fractions were counted using standard techniques and a liquid-scintillation spectrometer (model 460C Tri-Carb, Packard Instruments Company, Inc., Downers Grove, IL). The number average molecular weights (Mn) of the DNAs were calculated from the distribution of radioactivity in each gradient using procedures previously described (Ahmed and Setlow, 1977; Regan et al., 1971). The occurrence of DNA semiconservative replication was detected and quantitated using alkaline cesium chloride density gradients as previously described (Kantor and Player, 1986; Smith et al., 1981). Gradients were centrifuged for 40 h at 20 ° C, 27 000 rpm using the Beckman SW-60 rotor and ultracentrifuge described above. Fractionation and radioactive detection were as above. Results

Endogenous nuclease digestion of chromatin DNA. The nature and kinetics of endogenous nudease digestion of bulk chromatin DNA in normal and XP4RO fibroblasts are defined in part by the data presented in Fig. 1 and Table 1. Incubation of isolated nuclei in the autodigestion buffer at 37 °C leads to a decrease in the singlestranded DNA size as detected in alkaline sucrose gradients. Sample DNA profiles are shown in Figs. 2 and 4. Data presented in Fig. I illustrate that the average molecular weights (Mn) calculated from the DNA profiles decrease with incubation time from about 50-90 × 106 dalton to about 10-30 × 106 dalton in 1 h. While variability in both the original size of the DNA and the size after digestion was observed, some properties of the DNA-cleavage reactions served as useful guides in other experiments. Further decreases in size for incubation times up to 4 h were not observed. No differences in the relative extents of digestion between the two strains were readily apparent. Chro-

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nuclear suspensions were lysed on the gradients. Although the size of the DNA decreased with autodigestion time, no loss of acid-precipitable radioactivity compared to the initial amount was observed. This observation is confirmed by the data presented in Table 1. No detectable amounts of DNA in either irradiated or unirradiated cells were solubilized during a 4-h incubation period.

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Fig. 1. Rate of autodigestion of DNA in nuclei of human fibroblasts. Nuclei from growth-arrested normal (WS-1) and XP-C (XP4RO) fibroblasts were incubated in autodigestion buffer at 37°C for the times indicated. Samples lysed on alkaline sucrose gradients were subjected to centrifugationto size-fractionate the DNA. (&, O, II), WS-1; (×, +, ©, D, zx), XP4RO; different symbolsrepresent independentexperiments. Nuclei from 3H-labeled unirradiated and 14C-labeled UVirradiated (20 J/m 2) WS-1 (A) or XP4RO cells (zx)were mixed prior to autodigestionand sized on the same gradient. In both cases, the 3H and 14C DNAs cosedimented.The line is drawn through one set of data (e) to illustrate a trend. matin DNA from both unirradiated and UVirradiated cells was digested at the same rate. For an individual experiment, equal aliquots of the TABLE 1 ACID-PRECIPITABLE DNA-ASSOCIATED RADIOACTIVITY FROM AUTODIGESTED WS-1 NUCLEI a

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Nuclei from XP4RO cells exposed to UV and incubated for a period to permit DNA repair were suspended in autodigestion buffer at 37 ° C for 60 min to determine the relative nuclease sensitivity of the repaired DNA. Gradient profiles resulting from sedimentation of DNA in alkaline sucrose gradients are shown in Fig. 2. Bulk 14C-labeled DNA contains 3H-labeled repair patches resulting from a 3-h incubation period in the presence of 3H-dThd after exposure of cells to 10 J / m 2 UV. The resulting 3H-labeled patches are nonrandomly located (Kantor and Player, 1986; Player, 1986). Prior to autodigestion, the 3H and 14C cosediment, indicating that these isotopes are in DNAs of equal size (part A.1). Incubation of nuclei at 37 ° C for 60 min results in an unequal sedimentation rate for the two DNAs, indicating unequal nicking, with the 3H-repair labeled DNA exhibiting more nicks than the bulk DNA (part A.2). The 3H-DNA represents repaired DNA as defined by its association with bulk 14C-DNA in alkaline cesium chloride density gradients and by the absence of any detectable DNA semiconservative replication (Fig. 3) (Pettijohn and Hanawalt, 1964). Thus the repaired DNA in XP4RO cells is more sensitive to endogenous nuclease digestion than is the bulk of the DNA. Results of a chase experiment to examine the nuclease sensitivity of the initially repaired DNA 24 h later are presented in Fig. 2 (B.1, B.2). No readily observable changes in the initial sensitivity are detected. Calculations of the number of breaks in the respective DNAs indicate that the repaired DNA has nearly the same relative sensitivity 24 h later as it has initially. The differences in the number of breaks, calculated as 1/Mn (Ahmed and Setlow, 1977) per 108 dalton, between the bulk and repaired DNAs initially and 24 h later are 4.3 and 4.1, respectively.

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The CsC1 density gradient results presented in Fig. 3 are necessary to demonstrate aspects of DNA metabolism that are critical to the interpretation of the nuclease digestion experiments. The aH incorporation is the result of DNA-repair synthesis. The addition of HU to the culture medium is required to inhibit the small but experimentally significant m o u n t of DNA replicative synthesis that occurs in the UV-irradiated cells in

the 3-h incubation period. HU apparently does not influence the subsequent nuclease digestion results. Cells irradiated with 20 J / m E and incubated for 3 h in the absence of HU to repairlabel DNA exhibit no semi-conservative synthesis; however, the repaired DNA is more sensitive to autodigestion than is the bulk DNA (data not shown). The HU concentration used (10 raM) did not affect the number of pyrimidine dimers ex-

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Fig. 3. DNA excision-repair synthesis in XP4RO. DNA was extracted from an aliquot of the nuclei isolated for the experiments described in Fig. 2 and centrifuged to equilibrium in alkaline cesium chloride gradients. XP4RO cells were incubated for a 3-h period immediately following exposure to UV (10 J / m 2) in the presence of 3H-dThd and BrdUrd with (part A) no HU, (part B) HU or (part C) HU followed by an additional 24-h incubation period in 0.5% serum medium. The part B gradient is the companion to Fig. 2A; the part C gradient, companion to Fig. 2B. (a), 3H; (O), 14C-bulk-labeled DNA. Density increases from right to left.

cised in a 24-h period in our experimental conditions, as determined by a UV-endonuclease-alkaline sucrose gradient assay (data not shown; Player, 1986). A similar sensitivity of repaired DNA to autodigestion is not observed in normal or XP-A cells. Results of similar experiments with normal WS-1 and XP8LO (XP-A) cells are shown in Fig. 4. The DNA synthesis that occurs in UV-irradiated cultures of both of these strains using the experimental conditions defined is repair synthesis that is randomly located throughout the genome (Kantor and Player, 1986). Results presented in Fig. 4 for both strains illustrate that following autodigestion, the repaired and bulk DNAs have equal sizes indicating equivalent extents of digestion. Both bulk and repair-labeled DNAs from undigested nuclei co-sediment as large molecular weight DNA (~ 100X 106 dalton) from both strains, in a

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Fig. 4. Relative cleavage rate of repaired and bulk DNA in normal and XP-A cells. Growth arrested normal (WS-1; part A) and XP-A (XP8LO; part B) cultures labeled in DNA with 14C were UV-irradiated (10 J / m 2) and incubated with 3H-dThd and HU (10 mM) for 3 h to label the repaired DNA. Isolated nuclei were incubated for 60 min at 37 ° C. Samples lysed on alkaline sucrose gradients were subjected to centrifugation to size-fractionate the DNA. (O), 14C-bulk DNA; (zx), 3H-repairlabeled DNA. M n values are listed in the figure. Radioactivity values for part A were 10500 cpm of 3H, and 4200 cpm of 14C; for part B, 4600 cpm of 3H and 1700 cpm of 14C. Centrifugation time was 270 min for both samples.

manner similar to that observed for XP4RO, as depicted in Fig. 2 (data not shown).

Effect of UV dose on endogenous nuclease sensitivity of repaired DNA. Both the total amount of DNA excision-repair synthesis and the amount in localized chromatin regions increase linearly with UV dose to about 20 J / m 2 in XP-C cells, even though the overall amount of repair is much less than that observed for normal cells (Kantor and Elking, in preparation). Since the repair activity changes with the amount of DNA damage, within these limits, we examined the nuclease sensitivity of the partially repaired DNA in XP4RO cells as a function of UV dose. Results are summarized in Fig. 5. Cells irradiated with either 5, 10 or 20 J / m 2 were incubated for a 3-h period in medium containing 3H-dThd and HU. Isolated nuclei were

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same for the 3 samples. The extra number of strand breaks in the repaired DNA was 5-6 per 108 dalton compared to the bulk DNA, regardless of UV dose to the cells.

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Time (minutes) Fig. 5. Effectof UV dose on the extent of endogenousnuclease digestion of repaired and bulk DNA. Growth-arrestedXP4RO cells labeledin DNA with 14C were UV-irradiated as indicated and incubated for 3 h with 3H-dThd and HU (10 mlV0 to label repaired DNA. Nuclei were autodigested for the indicated times. Samples lysed on alkaline sucrose gradients were subjeeted to eentrifugationto size-fractionatethe DNA. (O, e), 14C-bulk DNA; (zx,&), 3H-repair-labeledDNA. The open and closed symbols are for two independent experiments. The number of DNA strand breaks was calculated as 1/M,. The differences (average of 2 Expts.) in the number of strand breaks are: A, 5.0/108 dalton; B, 5.5; C, 6.0.

autodigested and the resulting DNA sized on alkaline sucrose gradients. The relative sensitivity of the repaired DNA compared to bulk DNA is the

Data are presented that demonstrate that repaired DNA in XP-C cells has an extra-sensitivity to endogenous nucleases compared to the bulk of the DNA. The sensitivity is detected as breaks in single-stranded DNA with no detectable solubilization. A similar extra-sensitivity is not observed in normal or XP-A cells. Vanderbilt et al. (1982) have demonstrated that the DNA most sensitive to endogenous nucleases represents actively transcribed regions. Thus we suggest that the preferentially repaired DNA in XP-C may represent at least in part actively transcribed DNA. The repaired DNA in UV-irradiated XP-C cells is preferentially situated near attachment sites of DNA loops to the nuclear matrix (Mullenders et al., 1984), a location found also to have enriched levels of transcribed DNA (Robinson et al., 1983). This relationship prompted Mullenders and coworkers to suggest that the limited repair in XP°C cells is located primarily in transcribed DNA regions (MuUenders et al., 1984, 1986). Our evidence is consistent with this suggestion. Apparently not all transcriptionally active genes are preferentially repaired in XP-C cells. The dihydrofolate reductase gene is repaired no more rapidly than the bulk DNA (Bohr et al., 1986). The repaired DNA that is sensitive to autodigestion may therefore represent a subset of transcriptionally active genes defined as nuclear matrix-associated and endogenous nuclease sensitive. DNA repair in normal cells is relatively rapid compared to XP-C cells and seems to occur proficiently throughout the genome (Mansbridge and Hanawalt, 1983; Mullenders et al., 1984). While repair may occur in endogenous nuclease sensitive regions, it may not be detected as such because the sensitivity is masked in the assay by significant repair activity elsewhere. This leads us to suggest that the repair in XP-C represents a specific subset of normal repair and thus is indicative of a normal pathway. The repair defect in XP-C may be one that eliminates excision-repair pathways specific

176

for other kinds of chromatin structural domains. XP8LO is an exceptional complementation group A strain that is relatively resistant to the lethal effects of UV and exhibits a considerable amount of repair compared to classical XP-A strains. DNA is repaired initially at a normal rate at doses of 5 J / m 2 or less (Kantor and Hull, 1984). It has been suggested, based on an analysis of overall repair rates, that it is defective in a repair mechanism responsible for the second slower component of the normal excision rate curve (Kantor and Hull, 1984). The limited repair in XP-A strains, including XP8LO, is randomly located throughout the genome (Kantor and Player, 1986). Extra-sensitivity of the repaired DNA to endogenous nucleases is not detected (Fig. 4). This implies that repair occurs in some chromatin fractions that differ from those in XP-C cells. Since the repair rate in XP8LO is saturated at about 5 J / m 2 compared to 20 J / m 2 in normal cells, we suggested previously that the defect in XP8LO is partly concerned with the number of repair enzymes. The limited repair in XP-C is saturated at 20 J / m 2 (Kantor and Elking, in preparation). The defect in XP-C may thus not be one of number of repair enzymes, but as already suggested, a defect in specific repair enzymes essential for selected fractions of chromatjn. The data presented in Fig. 1 show that damage induced in DNA by UV does not change the sensitivity of the DNA to endogenous nucleases. Autodigested chromatin from mixed unirradiated and irradiated cells fractionated on the same gradients have identical sizes, illustrating the identical sensitivities of the two DNAs. Cells exposed to UV doses ranging from 1 to 20 J / m 2 exhibit a linear increase in repair activity, as determined by quantitative measures of repair synthesis, that is limited to localized regions (Kantor and Elking, in preparation). This is interpreted to mean that sufficient localized repair potential exists to handle the increased damage accompanying the higher UV doses. This increased repair activity in the localized regions does not create a greater sensitivity of these regions to endogenous nucleases (Fig. 5). We suggest, based on these results, that repair occurs in a pre-existing subset of chromatin that is

sensitive to endogenous nucleases regardless of the amount of DNA damage or repair activity. This suggestion is consistent with our previous one that the chromatin subset, as a pre-existing one, is actively transcribed chromatin. The extra-sensitivity of the repaired XP-C DNA observed immediately after a period of repair is the same 24 h later (Fig. 2). The endogenous nuclease-sensitive regions are thus not structurally transient regions. This result is consistent with the hypothesis that repair occurs in a pre-existing endogenous nuclease-sensitive region. This result also serves to distinguish these nuclease-sensitive regions from micrococcal nuclease-sensitive repaired regions that lose their extrasensitivity during a chase period (Bodell and Cleaver, 1981; Smerdon and Lieberman, 1978, 1980; Williams and Friedberg, 1979). We suggest that the growth-arrested cells used here, in a G O stage, have their chromatin in a non-transient state that has both endogenous nuclease-sensitive and -resistant regions that do not change from day to day. This non-transient state would not be expected in cells traversing a cell division cycle. In summary, the endogenous nuclease sensitivity of repaired DNA in XP-C cells defines a specific non-transient subset of chromatin DNA that contains the preferentially repaired DNA detected in these cells. This subset is sensitive to endogenous nucleases, not because of DNA damage or processes of repair, but because of its pre-existence in sensitive structural domains, ones that retain their sensitivity at least through a 24 h period. This subset may include nuclear matrix associated and transcriptionally active DNA. The repair in XP-C cells probably involves a normal pathway for these specific chromatin domains. The repair defect in XP-C cells is thus in a repair pathway that is targeted for endogenous nuclease-resistant domains. In this respect, the defect is differentiated from that in XP-A cells where overall repair processes make no distinction between nuclease-sensitive and -resistant regions. This differentiation is in addition to the previous observation of randomly and non-randomly located repair in XP-A and XP-C cells, respectively.

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Acknowledgements This research was supported by funds from the Biological Sciences Department and the Biomedical Sciences Ph.D. Program of Wright State University and from the State of Ohio through a Research Challenge Program award (No.660721) to G.J. Kantor.

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