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• W2125778284 abstract "The fate of nucleosomes during nucleotide excision repair is unclear. We have used organomercurial chromatography to capture accessible thiol groups of proteins at (or near) nascent repair sites in normal and xeroderma pigmentosum (group C) human cells. The reactive groups include cysteine 110 of histone H3, which is exposed in unfolded nucleosomes. Immediately after UV irradiation and a short pulse labeling of repair patches, intact nuclei were digested with restriction enzymes to release ∼18% of the chromatin into soluble fragments, which are enriched (∼4-fold) in a constitutively transcribed gene. Upon organomercurial affinity fractionation, ∼1.8% of the soluble chromatin remains bound in high salt (0.5m NaCl) and is released with dithiothreitol. In normal cell chromatin, this fraction is enriched in nascent repair patches (1.5–1.8-fold) over the unbound fraction. This enrichment decreases following short chase periods with a time course similar to the loss of enhanced nuclease sensitivity of these regions (t ½ ≈ 30 min). Much less enrichment of nascent repair patches is observed in the thiol-reactive fraction from XPC cells, which repair primarily the transcribed strand of active genes. These results suggest that transient nucleosome unfolding occurs during nucleotide excision repair in normal human cells, and this unfolding may require the XPC protein. The fate of nucleosomes during nucleotide excision repair is unclear. We have used organomercurial chromatography to capture accessible thiol groups of proteins at (or near) nascent repair sites in normal and xeroderma pigmentosum (group C) human cells. The reactive groups include cysteine 110 of histone H3, which is exposed in unfolded nucleosomes. Immediately after UV irradiation and a short pulse labeling of repair patches, intact nuclei were digested with restriction enzymes to release ∼18% of the chromatin into soluble fragments, which are enriched (∼4-fold) in a constitutively transcribed gene. Upon organomercurial affinity fractionation, ∼1.8% of the soluble chromatin remains bound in high salt (0.5m NaCl) and is released with dithiothreitol. In normal cell chromatin, this fraction is enriched in nascent repair patches (1.5–1.8-fold) over the unbound fraction. This enrichment decreases following short chase periods with a time course similar to the loss of enhanced nuclease sensitivity of these regions (t ½ ≈ 30 min). Much less enrichment of nascent repair patches is observed in the thiol-reactive fraction from XPC cells, which repair primarily the transcribed strand of active genes. These results suggest that transient nucleosome unfolding occurs during nucleotide excision repair in normal human cells, and this unfolding may require the XPC protein. In eukaryotic cells, DNA is wrapped around histone octamers, forming nucleosomes, which are the primary repeating units of chromatin (reviewed in Refs. 1van Holde K.E. Chromatin. Springer-Verlag New York Inc., New York1989Crossref Google Scholar and 2Wolffe A.P. Chromatin: Structure and Function. 2nd Ed. Academic Press, Inc., New York1995Google Scholar). In processes such as DNA replication, transcription, and repair, the proteins involved must access DNA buried within this structural hierarchy. This obstacle is formidable in the case of nucleotide excision repair (NER), 1The abbreviations used are: NER, nucleotide excision repair; XPA, XPC, and XPD, xeroderma pigmentosum complementation group A, C, and D, respectively; DTT, dithiothreitol; bp, base pair(s); PMSF, phenylmethylsulfonyl fluoride; EPNP, 1,2-epoxy-3-(p-nitrophenoxy)propane. as DNA lesions occur in all regions of chromatin (reviewed in Refs. 3Smerdon M.J. Lambert M.W. Laval J. DNA Repair Mechanisms and Their Biological Implications in Mammalian Cells. Plenum Publishing Corp., 1989: 271-294Crossref Google Scholar, 4Smerdon M.J. Thoma F. Hoekstra M.F. Nickoloff J.A. DNA Damage and Repair: Biochemistry, Genetics, and Cell Biology. Humana Press Inc., Totowa, NJ1998Google Scholar, 5Smerdon M.J. Conconi A. Moldave K. Progress in Nucleic Acids Research and Molecular Biology. Academic Press, Inc., 1998Google Scholar). Thus, damaged DNA within all levels of chromatin structure must be recognized, removed, and replaced by repair enzymes. Several human diseases, characterized by high cancer incidence, result from deficiencies in NER (reviewed in Refs. 6Cleaver J.E. Kraemer K.H. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. 6th Ed. Mcgraw-Hill, New York1989: 2949-2971Google Scholar and 7Friedberg E.C. Walker G.C. Wolfram S. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, DC1995Google Scholar). For example, xeroderma pigmentosum, which is manifested as extreme sensitivity to UV light, often results in skin cancer. The xeroderma pigmentosum locus heterogeneity has been divided into eight complementation groups (6Cleaver J.E. Kraemer K.H. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. 6th Ed. Mcgraw-Hill, New York1989: 2949-2971Google Scholar), indicating that the molecular basis of this disease may involve one of several defects in the DNA repair pathway. Some of these defects may prevent repair enzymes from accessing damaged DNA complexed with nucleosomes. Indeed, DNA damage-specific endonuclease activity in extracts from human XPA and XPD cells decreases when DNA is folded into nucleosomes, while this activity is enhanced in extracts from normal human cells (8Parrish D.D. Lambert M.W. Mutat. Res. 1990; 235: 65-80Crossref PubMed Scopus (20) Google Scholar, 9Parrish D.D. Feng X. Lambert M.W. Biochem. Biophys. Res. Commun. 1992; 189: 782-789Crossref PubMed Scopus (2) Google Scholar, 10Parrish D.D. Lambert W.C. Lambert M.W. Mutat. Res. 1992; 273: 157-170Crossref PubMed Scopus (12) Google Scholar). Furthermore, XPC cells repair primarily the template strands of transcriptionally active chromatin and are deficient in repair of inactive (or bulk) chromatin (11Kantor G.J. Barsalou L.S. Hanawalt P.C. Mutat. Res. 1990; 235: 171-180Crossref PubMed Scopus (80) Google Scholar, 12Venema J. van-Hoffen A. Natarajan A.T. van-Zeeland A.A. Mullenders L.H. Nucleic Acids Res. 1990; 18: 443-448Crossref PubMed Scopus (199) Google Scholar, 13Venema J. van-Hoffen A. Karcagi V. Natarajan A.T. van-Zeeland A.A. Mullenders L.H. Mol. Cell. Biol. 1991; 11: 4128-4134Crossref PubMed Scopus (289) Google Scholar). Since transcriptionally active chromatin has a number of distinguishing features compared with bulk chromatin (reviewed in Refs. 2Wolffe A.P. Chromatin: Structure and Function. 2nd Ed. Academic Press, Inc., New York1995Google Scholar and 14), factors may exist in cells that promote access to DNA lesions in condensed chromatin domains for DNA repair processing. It is clear that NER modulates nucleosome structure (for reviews, see Refs. 3Smerdon M.J. Lambert M.W. Laval J. DNA Repair Mechanisms and Their Biological Implications in Mammalian Cells. Plenum Publishing Corp., 1989: 271-294Crossref Google Scholar, 4Smerdon M.J. Thoma F. Hoekstra M.F. Nickoloff J.A. DNA Damage and Repair: Biochemistry, Genetics, and Cell Biology. Humana Press Inc., Totowa, NJ1998Google Scholar, 5Smerdon M.J. Conconi A. Moldave K. Progress in Nucleic Acids Research and Molecular Biology. Academic Press, Inc., 1998Google Scholar). During (and immediately after) repair synthesis, the association of histones with DNA appears to be locally disrupted. In early studies, nucleosome rearrangement during NER was operationally defined as (a) a change in nuclease digestion kinetics of newly repaired DNA (or “nuclease sensitivity”) and (b) a change in association of newly repaired DNA with nucleosome core particles (15Smerdon M.J. Lieberman M.W. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4238-4244Crossref PubMed Scopus (181) Google Scholar, 16Smerdon M.J. Lieberman M.W. Biochemistry. 1980; 19: 2992-3000Crossref PubMed Scopus (47) Google Scholar, 17Smerdon M.J. Kastan M.B. Lieberman M.W. Biochemistry. 1979; 18: 3732-3739Crossref PubMed Scopus (42) Google Scholar). Newly repaired DNA is almost absent from isolated nucleosome cores immediately after repair synthesis but rapidly associates with core histones (t½ ≈ 20 min) in intact human cells. Over longer times (1–24 h), there is a slow change in these features, resulting in the randomization of repair patches in nucleosomes, thought to reflect nucleosome migration onto newly repaired DNA (18Nissen K.A. Lan S.Y. Smerdon M.J. J. Biol. Chem. 1986; 261: 8585-8588Abstract Full Text PDF PubMed Google Scholar). Interestingly, XPC fibroblasts differ quantitatively in these features, in that nascent repair patches are less sensitive to nuclease digestion than in normal cells and completely randomize in chromatin by 1 h after labeling (i.e. are not associated with a slow rearrangement phase) (Ref. 17Smerdon M.J. Kastan M.B. Lieberman M.W. Biochemistry. 1979; 18: 3732-3739Crossref PubMed Scopus (42) Google Scholar; also see Ref. 5Smerdon M.J. Conconi A. Moldave K. Progress in Nucleic Acids Research and Molecular Biology. Academic Press, Inc., 1998Google Scholar). This suggests that nucleosome rearrangement, following repair, occurs more rapidly in XPC cells and that the newly repaired nucleosomes are more mobile than at least some of those repaired in normal cells. The changes observed in both nuclease sensitivity and nucleosome core association appear to be due to the same event, since the time courses of these changes are virtually identical (15Smerdon M.J. Lieberman M.W. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4238-4244Crossref PubMed Scopus (181) Google Scholar). This phenomenon was referred to as “nucleosome rearrangement” to avoid implication of a mechanism for this process (15Smerdon M.J. Lieberman M.W. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4238-4244Crossref PubMed Scopus (181) Google Scholar), and an unfolding-refolding model was proposed to explain these changes (19Lieberman M.W. Smerdon M.J. Tlsty T.D. Oleson F.B. Emmelot P. Kriek E. Environmental Carcinogenesis. Elsevier/North-Holland and Biochemical Press, 1979: 345-363Google Scholar). Following this rapid rearrangement phase, newly repaired nucleosomes appear to have typical structures that contain histone H1 (20Smerdon M.J. Watkins J.F. Lieberman M.W. Biochemistry. 1982; 21: 3879-3885Crossref PubMed Scopus (16) Google Scholar). This rapid rearrangement phase occurs regardless of the time after UV damage that repair synthesis takes place (16Smerdon M.J. Lieberman M.W. Biochemistry. 1980; 19: 2992-3000Crossref PubMed Scopus (47) Google Scholar). Furthermore, prior to the rapid rearrangement phase, nascent repair patches do not yield a DNase I footprint (16Smerdon M.J. Lieberman M.W. Biochemistry. 1980; 19: 2992-3000Crossref PubMed Scopus (47) Google Scholar), suggesting that newly repaired DNA is not tightly bound to a surface of core histones immediately after repair synthesis. In addition, repair patch ligation precedes nucleosome formation in human cells (21Hunting D.J. Dresler S.L. Lieberman M.W. Biochemistry. 1985; 24: 3219-3226Crossref PubMed Scopus (25) Google Scholar, 22Smerdon M.J. J. Biol. Chem. 1986; 261: 244-252Abstract Full Text PDF PubMed Google Scholar). Thus, completion of NER during the rapid phase of nucleosome rearrangement appears to progress from an unligated nonnucleosome structure to a ligated nonnucleosome structure and finally to a ligated nucleosome structure (3Smerdon M.J. Lambert M.W. Laval J. DNA Repair Mechanisms and Their Biological Implications in Mammalian Cells. Plenum Publishing Corp., 1989: 271-294Crossref Google Scholar, 4Smerdon M.J. Thoma F. Hoekstra M.F. Nickoloff J.A. DNA Damage and Repair: Biochemistry, Genetics, and Cell Biology. Humana Press Inc., Totowa, NJ1998Google Scholar, 5Smerdon M.J. Conconi A. Moldave K. Progress in Nucleic Acids Research and Molecular Biology. Academic Press, Inc., 1998Google Scholar). A number of studies implicate nucleosome disruption (or unfolding) during transcription (reviewed in Refs. 2Wolffe A.P. Chromatin: Structure and Function. 2nd Ed. Academic Press, Inc., New York1995Google Scholar and 23Thoma F. Trends Genet. 1991; 7: 175-177Abstract Full Text PDF PubMed Scopus (59) Google Scholar). Furthermore, Allfrey and co-workers (e.g. see Ref. 24Allfrey V.G. Chen T.A. Methods Cell Biol. 1991; 35: 315-335Crossref PubMed Scopus (19) Google Scholar) employed organomercurial affinity chromatography as a means to bind “open” nucleosomes at exposed thiols of disrupted (or unfolded) H3 histones to capture actively transcribing chromatin. Nucleosomes from a number of different species, including slime mold, yeast, mice, and humans, were fractionated with this method, and in each case active genes were preferentially bound to the organomercurial affinity matrix. In addition, the thiol reactivity of nucleosomes containing the genes c-fos and c-myc correlated with the expression of these genes when assayed by nuclear run-off transcription (25Chen T.A. Allfrey V.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5252-5256Crossref PubMed Scopus (116) Google Scholar). Thus, when genes are actively transcribed in cells, at least some of these sequences are preferentially associated with the thiol-reactive nucleosomes. This suggests that proteins at (or near) these genes have exposed sulfhydryl groups during transcription. Candidate proteins include H3 histones, which have one internal cysteine (at position 110) exposed in unfolded nucleosomes and other proteins with exposed thiols that are strongly bound to these genes (i.e. remain bound in 0.5 m salt). Perhaps the most direct evidence for the involvement of cysteine 110 of histone H3 in this assay comes from studies on Saccharomyces cerevisiae, which does not naturally possess a cysteine residue in histone H3 (26Chen T.A. Smith M.M. Le S. Sternglanz R. Allfrey V.G. J. Biol. Chem. 1991; 266: 6489-6498Abstract Full Text PDF PubMed Google Scholar, 27Chen-Cleland T.A. Smith M.M. Le S. Sternglanz R. Allfrey V.G. J. Biol. Chem. 1993; 268: 1118-1124Abstract Full Text PDF PubMed Google Scholar). A mutant yeast strain was constructed with a cysteine substitution for alanine at position 110, and this substitution yielded binding of transcriptionally active chromatin to the organomercurial matrix (27Chen-Cleland T.A. Smith M.M. Le S. Sternglanz R. Allfrey V.G. J. Biol. Chem. 1993; 268: 1118-1124Abstract Full Text PDF PubMed Google Scholar). This binding did not occur in wild type strains. Furthermore, electron spectroscopic imaging implicates the existence of U-shaped structures in the nucleosomes collected from thiol-reactive chromatin but not in the unbound fraction of the organomercurial matrix (28Basset-Jones D.P. Mendez E. Czarnota G.J. Ottensmeyer F.P. Allfrey V.G. Nucleic Acids Res. 1996; 24: 321-329Crossref PubMed Scopus (65) Google Scholar). In the present study, organomercurial chromatography has been used to fractionate labeled excision repair patches in human chromatin as an assay for nucleosome unfolding during NER. We have observed an enrichment of nascent repair patches associated with thiol-reactive nucleosomes of normal human cells, and this enrichment decreases in these cells as nucleosomes rearrange after the repair event. On the other hand, chromatin from XPC cells shows much less enrichment, suggesting that this transient change is more rapid in transcriptionally active chromatin and/or that this effect is due to NER occurring only in bulk (inactive) chromatin in normal human cells. Normal human diploid fibroblasts (strain AG1518) and human XPC fibroblasts (strain GMO2996) were purchased from Coriell Cell Repositories (Camden, NJ). The DHFR (dihydrofolate reductase) gene construct, pZH-15, was a gift from Drs. C. A. Smith and P. Hanawalt (Stanford University, Stanford, CA). This construct contains a 788-bp insert of genomic DNA from −315 bp into intron 2 of the human DHFR gene. 14C- and3H-labeled thymidine was obtained from Moravek Biochemicals (Brea, CA). All restriction enzymes were obtained from Life Technologies, Inc. Affi-Gel 501 organomercurial agarose was purchased from Bio-Rad, and agarose magnetic beads, activated byp-nitrophenyl chloroformate, were synthesized by Perceptive Diagnostics (Cambridge, MA). Cell culture conditions were as described by Smerdon et al. (17Smerdon M.J. Kastan M.B. Lieberman M.W. Biochemistry. 1979; 18: 3732-3739Crossref PubMed Scopus (42) Google Scholar). In all experiments, to ensure a minimum amount of background replication, the cells were grown to confluence and treated with 2 mm hydroxyurea 45 min prior to UV damage. To allow comparison of data between experiments and between samples within one experiment, the “double-label” procedure described in Smerdon et al. (17Smerdon M.J. Kastan M.B. Lieberman M.W. Biochemistry. 1979; 18: 3732-3739Crossref PubMed Scopus (42) Google Scholar) was used. Cells were pulse-labeled with 20 nCi/ml [14C]dThd during the growth phase to yield uniformly 14C-labeled DNA. At growth arrest (or confluence), cells were induced to repair by exposure to 12 J/m2 UV light in the presence of 10 μCi/ml [3H]dThd in conditioned media (or 20 μCi/ml [3H]dThd for XPC cells), which was incorporated into the nascent repair patches during specified incubation times. Therefore, the 3H/14C ratio accurately measures the relative repair activity in each of the organomercurial fractions. In some experiments, following this pulse label, the 3H was chased with 50 μm unlabeled thymidine for varying times (see Ref. 17Smerdon M.J. Kastan M.B. Lieberman M.W. Biochemistry. 1979; 18: 3732-3739Crossref PubMed Scopus (42) Google Scholar). Nuclei were isolated as described (24Allfrey V.G. Chen T.A. Methods Cell Biol. 1991; 35: 315-335Crossref PubMed Scopus (19) Google Scholar) with the following modifications. Harvested cells were pelleted and resuspended in a buffer containing 10 mm Tris, pH 8.0, 1.0 mm CaCl2, 8.6% sucrose, and 5.0 mm sodium butyrate (to inhibit histone deacetylation). Following centrifugation, cells were resuspended in a lysis buffer containing 10 mm Tris, pH 8.0, 1.0 mm CaCl2, 8.6% sucrose, 5.0 mm sodium butyrate, 0.25 mmphenylmethylsulfonyl fluoride (PMSF), 0.25 mm1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), and 0.5% (w/v) Triton X-100. Nuclei, at an approximate concentration of 1 × 107 nuclei/ml, were treated with a mix of three restriction enzymes (0.5 units/μl each of HaeIII, RsaI, andTaqI) for 1 h at 37 °C, with vigorous shaking to digest the chromatin. The nuclei digestion buffer contained 10 mm Tris-HCl, pH 7.4, 25 mm NaCl, 25 mm KCl, 3.0 mm MgCl2, 1.0 mm β-mercaptoethanol, 5.0 mm sodium butyrate, 0.25 mm PMSF, 0.25 mm EPNP. Chromatin fragments were released, with the addition of 50 mm EDTA, from the nuclei into the supernatant, which was collected after centrifugation at 10,000 rpm for 10 min. at 4 °C. This soluble fraction will be referred to as the “S fraction.” Magnetic agarose beads were coupled to a phenylmercury group as described by Chen-Cleland et al. (29Chen-Cleland T.A. Boffa L.C. Carpaneto E.M. Mariani M.R. Valentin E. Mendez E. Allfrey V.G. J. Biol. Chem. 1993; 268: 23409-23416Abstract Full Text PDF PubMed Google Scholar). Alternatively, some beads were made with benzoic acid replacing mercuric benzoate, resulting in beads containing just a benzene ring (and no HgCl). These beads were used in control experiments for nonspecific binding (see below). The soluble chromatin (or S fraction) collected after restriction digestion of nuclei for each sample was loaded onto organomercurial columns or mixed with organomercurial magnetic beads (as described in Refs. 24Allfrey V.G. Chen T.A. Methods Cell Biol. 1991; 35: 315-335Crossref PubMed Scopus (19) Google Scholar and 29Chen-Cleland T.A. Boffa L.C. Carpaneto E.M. Mariani M.R. Valentin E. Mendez E. Allfrey V.G. J. Biol. Chem. 1993; 268: 23409-23416Abstract Full Text PDF PubMed Google Scholar) to selectively bind unfolded nucleosomes. The first column buffer, buffer 1 (10 mm Tris-HCl, pH 7.4, 25 mm NaCl, 25 mm KCl, 2% sucrose, 5.0 mm EDTA, 5.0 mm sodium butyrate, 0.1 mm PMSF, 0.1 mm EPNP), washed out the flow-through fraction and rinsed the column (or beads) of nonbinding material (Fig. 1). The collected fractions were called “peak 1.” The organomercurial matrix was then washed with buffer 2 (buffer 1 plus 0.5 m NaCl), which releases chromatin bound by ionic interactions alone (Fig. 1). Finally, the column (or beads) was washed with buffer 3 (buffer 1 plus 0.5m NaCl and 10 mm dithiothreitol (DTT)). The DTT reduces sulfide linkages, releasing the thiol-reactive chromatin from the matrix (Fig. 1). In the repair analysis, the three classes of nucleosomes separated by the chromatography (class 1, non-reactive; class 2, salt labile; and class 3, thiol-reactive) were stripped of protein by proteinase K digestion (500 μg/ml at 42 °C 1 h), and the level of 3H dpm and 14C dpm was determined by scintillation counting of triplicate samples (Packard 1900CA, Downer's Grove, IL). To clarify the presentation of data, TableI shows scintillation counts (dpm) for a typical experiment. The ratio of 3H dpm (repaired DNA) to 14C dpm (total DNA) gives the relative amount of repair synthesis (per unit of DNA) for a given experiment. Since the levels (specific radioactivity) of3H and 14C incorporated vary for each experiment, we normalized the ratio for the thiol-bound fraction (fraction 3) to the ratio for the flow-through fraction (fraction 1) in each experiment (Table I). Also, the average3H/14C ratio in unirradiated cells was 4 ± 2.7% (mean ± 1 S.D.) that of the irradiated cells in each case.Table IData from repair-labeled chromatin fractionated by organomercurial chromatographyChase timeFraction3H14C3H/14CFraction 3/Fraction 1hdpm0S818220464.00126,32712,2202.15228649223.11319,17046474.131.9224S19,82061273.231128,76540,6783.17216,74051523.25326909392.860.91Data shown are from a typical experiment to explain our calculations. Confluent human fibroblasts were uniformly labeled in DNA during growth with [14C]dThd, labeled for 30 min in NER patches with [3H]dThd after 12 J/m2 UV irradiation, and exposed to chase periods of 0 or 24 h, as described under “Experimental Procedures.” Soluble chromatin (S) was applied to an organomercurial-agarose column and fractionated as described in the legend to Fig. 1, and the amount of DNA in each fraction was determined by absorbance at 260 nm. A portion of each fraction, which was increased for fractions 2 and 3, was analyzed by scintillation counting. A ratio of 3H/14C represents the amount of repair synthesis per unit of DNA. To compare results from different experiments, the thiol-bound fraction (3Smerdon M.J. Lambert M.W. Laval J. DNA Repair Mechanisms and Their Biological Implications in Mammalian Cells. Plenum Publishing Corp., 1989: 271-294Crossref Google Scholar) was normalized to the flow-through fraction (1van Holde K.E. Chromatin. Springer-Verlag New York Inc., New York1989Crossref Google Scholar) for Figs. 6 and 7 (i.e. values of 1.92 and 0.91 for the data shown). Open table in a new tab Data shown are from a typical experiment to explain our calculations. Confluent human fibroblasts were uniformly labeled in DNA during growth with [14C]dThd, labeled for 30 min in NER patches with [3H]dThd after 12 J/m2 UV irradiation, and exposed to chase periods of 0 or 24 h, as described under “Experimental Procedures.” Soluble chromatin (S) was applied to an organomercurial-agarose column and fractionated as described in the legend to Fig. 1, and the amount of DNA in each fraction was determined by absorbance at 260 nm. A portion of each fraction, which was increased for fractions 2 and 3, was analyzed by scintillation counting. A ratio of 3H/14C represents the amount of repair synthesis per unit of DNA. To compare results from different experiments, the thiol-bound fraction (3Smerdon M.J. Lambert M.W. Laval J. DNA Repair Mechanisms and Their Biological Implications in Mammalian Cells. Plenum Publishing Corp., 1989: 271-294Crossref Google Scholar) was normalized to the flow-through fraction (1van Holde K.E. Chromatin. Springer-Verlag New York Inc., New York1989Crossref Google Scholar) for Figs. 6 and 7 (i.e. values of 1.92 and 0.91 for the data shown). Normal human fibroblasts were prelabeled during the growth phase with [14C]dThd to provide a “bulk” DNA label for repair experiments and allow normalization of repair synthesis levels in each fraction from the organomercurial column (i.e. repaired DNA/total DNA in a particular fraction). A typical profile of the complete fractionation on the mercury affinity agarose column is shown in Fig. 2. Nuclei were prepared and digested with a mixture of three restriction endonucleases (TaqI, HaeIII, andRsaI), as described under “Experimental Procedures.” The released (soluble) chromatin, or S fraction, represents 18 ± 5% of the total DNA content, as measured by scintillation counting of the14C content in the pellet and supernatant (data not shown). This value is similar to that obtained by Allfrey and co-workers for human HeLa cells (29Chen-Cleland T.A. Boffa L.C. Carpaneto E.M. Mariani M.R. Valentin E. Mendez E. Allfrey V.G. J. Biol. Chem. 1993; 268: 23409-23416Abstract Full Text PDF PubMed Google Scholar). Thus, only a minor fraction of the total chromatin in cells is released by this procedure. This number (∼18%) is higher than estimated percentages of actual transcriptionally active genes in mature mammalian cells (30Lewin B. Genes VI. Oxford University Press, New York1997: 650-661Google Scholar); therefore, the S fraction must contain a large fraction of inactive (yet open) chromatin regions accessible to restriction endonucleases. The soluble fraction of the digested chromatin (∼18 ± 5% of total) was loaded onto the organomercurial column, which was equilibrated with column buffer 1, and the unbound chromatin was collected as peak 1 (Fig. 2, fractions 5–10). The second wash contained 0.5 m NaCl and was used to release chromatin fragments bound solely by ionic interactions (Fig. 2, fractions 35–39). This peak was collected and is referred to as fraction 2. The column was then washed with a buffer containing 10 mm DTT to release thiol-reactive chromatin, and this peak collected as fraction 3 (Fig. 2, fractions 55–65; inset). Based on 14C dpm values from 10 different experiments, 94 ± 0.3% of the soluble chromatin was recovered in fraction 1, and 3.8 ± 1.2% was collected in fraction 2. The thiol-reactive chromatin released in fraction 3 represents only 1.8 ± 0.4% of the soluble chromatin, or about 0.4% of total chromatin (see below). In the transcription studies on human 3T3 cells, Allfrey and co-workers (24Allfrey V.G. Chen T.A. Methods Cell Biol. 1991; 35: 315-335Crossref PubMed Scopus (19) Google Scholar, 29Chen-Cleland T.A. Boffa L.C. Carpaneto E.M. Mariani M.R. Valentin E. Mendez E. Allfrey V.G. J. Biol. Chem. 1993; 268: 23409-23416Abstract Full Text PDF PubMed Google Scholar) found that ∼10% of the soluble chromatin was recovered in fraction 2 and ∼7% in fraction 3. These differences from our results may reflect differences in solubility of the fragment sizes produced. These authors used different restriction enzymes (6-bp cutters) than our combination of 4-bp cutters and employed a different method for DNA quantitation (Hoescht dye) than we used (14C labeling). However, only a small percentage (<5%) of the human genome is expected to be actively transcribing (and completely disrupted) at any given time in mature human fibroblasts (30Lewin B. Genes VI. Oxford University Press, New York1997: 650-661Google Scholar); therefore, only a small portion of the soluble fraction should be involved in repair. To test whether the mercury affinity matrix may occlude or bind chromatin fragments nonspecifically, magnetic beads were generated containing only the phenyl group, and lacking HgCl (see “Experimental Procedures”). The 14C-labeled chromatin was then fractionated with these beads as for the repair experiments. Fraction 1, representing the nucleosomes that did not bind to the column, contained ∼97% of the total soluble chromatin (14C-labeled DNA), while the wash with buffer 2 (0.5m NaCl) released ∼3% of the chromatin. Only 0.05% of the 14C-labeled chromatin was detected in fraction 3 (10 mm DTT wash), or <3% of the thiol-bound chromatin when the HgCl group is present. Thus, whereas the column matrix itself interacts ionically with a small fraction of the chromatin fragments, there is little chromatin retained in the presence of 0.5 mNaCl. The fraction of chromatin fragments expected to contain repair patches can be estimated as follows. About 18% of the total genome is liberated from the nucleus by the restriction enzyme digest (see above), and about 2% of these fragments are captured by the organomercurial matrix and released as fraction 3. Therefore, about 0.4% (2% × 0.18) of the total chromatin is eluted in fraction 3. 2Clearly, the insoluble (or pellet) fraction, representing ∼80% of total chromatin, may also contain thiol-reactive proteins at (or near) nascent repair sites, and it is expected that this value of 0.4% is less than the actual fraction of thiol-reactive fragments in total chromatin. There is an average of one cyclobutane pyrimidine dimer per 6–8 kilobase pairs at 12 J/m2 in these cells (31Gale J.M. Smerdon M.J. Biochemistry. 1988; 27: 949-958Crossref Scopus (17) Google Scholar, 32Ramanathan B. Smerdon M.J. J. Biol. Chem. 1989; 264: 11026-11034Abstract Full Text PDF PubMed Google Scholar), and the average size of DNA fragments, following the restriction enzy" @default.
• W2125778284 created "2016-06-24" @default.
• W2125778284 creator A5022310342 @default.
• W2125778284 creator A5080261955 @default.
• W2125778284 date "1998-07-01" @default.
• W2125778284 modified "2023-09-29" @default.
• W2125778284 title "Nucleosome Unfolding during DNA Repair in Normal and Xeroderma Pigmentosum (Group C) Human Cells" @default.
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