Clustered Sites of DNA Repair Synthesis during Early Nucleotide Excision Repair in Ultraviolet Light-Irradiated Quiescent Human Fibroblasts

Experimental Cell Research 276, 284 –295 (2002) doi:10.1006/excr.2002.5519, available online at http://www.idealibrary.com on

Clustered Sites of DNA Repair Synthesis during Early Nucleotide Excision Repair in Ultraviolet Light-Irradiated Quiescent Human Fibroblasts Maria Svetlova, Lioudmila Solovjeva, Nadezhda Pleskach, Natalia Yartseva, Tatyana Yakovleva, Nikolai Tomilin, 1 and Philip Hanawalt* Laboratory of Chromosome Stability, Institute of Cytology, Russian Academy of Sciences, 194064 St. Petersburg, Russia; and *Department of Biological Sciences, Stanford University, Stanford, California 94305-5020

The ubiquitous process of nucleotide excision repair includes an obligatory step of DNA repair synthesis (DRS) to fill the gapped heteroduplex following excision of a short (⬃30-nucleotide) damaged single-strand fragment. Using 5-iododeoxyuridine to label repair patches during the first 10 – 60 min after UV irradiation of quiescent normal human fibroblasts we have visualized a limited number of discrete foci of DRS. These must reflect clusters of elementary DRS patches, since single patches would not be detected. The DRS foci are attenuated in normal cells treated with ␣-amanitin or in Cockayne syndrome (CS) cells, which are specifically deficient in the pathway of transcription-coupled repair (TCR). It is therefore likely that the clusters of DRS arise in chromatin domains within which RNA polymerase II transcription is compartmentalized. However, we also found significant suppression of DRS foci in xeroderma pigmentosum, complementation group C cells in which global genome repair (GGR) is defective, but TCR is normal. This suggests that the TCR is responsible for the DRS cluster formation in the absence of GGR. The residual foci detected in CS cells indicate that, even at early times following UV irradiation, GGR may open some chromatin domains for processive scanning and consequent DRS independent of transcription. © 2002 Elsevier Science (USA)

Key Words: nucleotide excision repair; transcription; quiescent fibroblasts; 5-iododeoxyuridine incorporation; tyramide– biotin amplification; DNA repair synthesis; nuclear foci; ␣-amanitin.

INTRODUCTION

Nucleotide excision repair (NER) is one of the beststudied mechanisms for maintenance of genome integrity in prokaryotic and eukaryotic cells. Since its dis1 To whom reprint requests should be addressed. Fax: 7 (812) 247-0341. E-mail: [email protected].

0014-4827/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

covery in Escherichia coli [1–3], most of genes controlling NER have now been identified in mammals and the basic biochemical steps have been well defined (for reviews see [4 – 6]). However, the spatial organization of the NER process in the mammalian nucleus remains to be established. The critical early step of NER is recognition of only one or a few damaged nucleotides within the large excess of undamaged DNA, and an early suggestion was that DNA may be scanned for lesions by a processive protein complex sensing distortions in DNA helix [7]. Although there is some evidence for processive NER in E. coli [8] and for specific damage-recognition enzymes [9], recent experiments using the green fluorescent protein (GFP)tagged human NER complex ERCC1/XPF suggest that NER works in a distributive fashion in mammalian cells [10]. Several other important genetic processes—DNA replication, transcription, and RNA splicing—are compartmentalized within the nucleus (for review see [11– 14]). Splicing takes place within interchromatin granule clusters (IGCs or “speckles”) and an IGC complex containing about 75 proteins has been isolated [15]. One subunit (137 kDa) of the IGC complex exhibits a striking sequence similarity over its entire length to the damaged DNA-binding protein p127 [15] that is associated with the hereditary disease xeroderma pigmentosum complementation group E [16]. DNA replication occurs in compact replication factories, within which the replication proteins are concentrated, and these compartments contain multiple, simultaneously active replication forks [17, 18]. Transcription foci, each accommodating 8 –15 clustered RNA polymerase II (RNAP II) transcription units, have also been visualized [13, 19]. Two major subpathways of NER are known: transcription-coupled repair (TCR) and global genome repair (GGR) [4, 5]. TCR is initiated in transcriptionally active chromatin domains by the arrest of RNAP II at lesions which are then preferentially excised from the

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template DNA strand. Patients with Cockayne syndrome (CS) are defective in TCR, generally as a consequence of mutation in the CSB or CSA genes. However, they have a number of clinical symptoms which have been difficult to explain by defective TCR. These include growth failure of soma and, in brain, neurologic degeneration and demyelinating neuropathy [20]. Since the CSB protein is a chromatin-remodeling ATPase of the SWI2/SNF2 family [21, 22], in CSB patients these symptoms might arise because of an altered chromatin structure and reduced RNAP II transcription in the absence of exogenous DNA damage [23]. An alternative view is that these clinical features are a consequence of the defective TCR of several lesions produced by reactive oxygen species, which are normally repaired by the base excision-repair pathway [24, 25]. GGR can be initiated by at least two different DNA lesion-binding complexes—XPC/hHR23B [26] and UVDDB [27, 28]—that deal with lesions throughout the genome, including the nontranscribed strands of expressed genes and silent domains that include compact heterochromatin. It has been suggested that transient nucleosome unfolding occurs during GGR in normal human cells and that this unfolding may require the XPC protein [29]. Interestingly, the p48 subunit of the UV–DDB complex which is essential for efficient GGR of some lesions [28], contains a WD repeat motif, with homology to proteins that reorganize chromatin [16]. Both TCR and GGR require the XPA protein and result in excision of about 30-base oligonucleotides containing the DNA lesion [30] followed by DNA repair synthesis (DRS) to fill the resulting gap with undamaged nucleotides and then ligation [4 – 6]. In the present study, using the tyramide system of amplification of the immunofluorescent signal [31] from incorporated 5-iododeoxyuridine (IdU), we have analyzed the dynamics of DRS in nuclei of UV-irradiated quiescent human primary fibroblasts. Limited numbers of discrete focal DRS sites were detected in nuclei after relatively short (10- to 60-min) pulses of IdU immediately after UV irradiation. Since a single 30-base DRS patch cannot be detected by immunofluorescence, we conclude that each DRS focus must represent a cluster of many elementary DRS patches. UV-induced DRS foci can be seen in the interphase nucleus soon after irradiation and IdU labeling, but then the label appears several days later in metaphase chromosomes. We found that the number of DRS foci is reduced in normal cells treated with the transcription inhibitor ␣-amanitin and in cells containing mutations in the XPC or CSB genes, indicating that both TCR and GGR contribute to the formation of these clusters of elementary DRS patches during NER at early times following UV irradiation.

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MATERIALS AND METHODS Cell cultures, ultraviolet irradiation, and fixation. Four primary human skin fibroblast cell lines were used in this study: VH-10 (normal), GM00038 (normal), CS1AN (GM00739) from patient with Cockayne syndrome complementation group B (CSB), and GM00030 from patient with xeroderma pigmentosum complementation group C (XPC). VH-10 cells kindly provided by Dr. A. Kolman (Stockholm University) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS). Cells of the other three cell lines obtained from the NIGMS Human Genetic Cell Repository (USA) were grown in MEM supplemented with a twofold higher concentration of essential and nonessential amino acids and vitamins and 15% FCS. Cells were grown to confluence on microscope glass slides in petri dishes and then incubated for 3–5 days in the medium with 0.5% FCS to attain the quiescent state. Then the cells were incubated for 30 min in growth medium supplemented with 0.5% FCS and 0.01 mM 5-fluorodeoxyuridine (FdU). After washing with phosphate-buffered saline (PBS) solution cells were UV-irradiated from a germicidal lamp (254 nm) using different doses (as indicated in the text) and then incubated for indicated times in the same growth medium with addition of 0.01 mM 5-iododeoxyuridine. UV dosimetry employed a commercial dosimeter equipped with an appropriate (254-nm) sensor. After washing two times with PBS, slides were treated for 3.5 min with 0.1% Triton X-100 in PBS at room temperature (RT), washed again with PBS, fixed for 10 min in ice-cold 4% formaldehyde solution in PBS, kept in 70% ethanol at 4°C overnight, dehydrated in 96% ethanol, and air-dried. Treatment of cells with ␣-amanitin. VH-10 cells were grown to confluence in DMEM containing 10% FCS and then incubated for 3 days in the serum-depleted (0.5% FCS) medium; 10 ␮g/ml ␣-amanitin (Sigma) was added to the growth medium 6 h before UV irradiation (5 J/m 2), and 30 min before irradiation 0.5% FCS and 0.01 mM FdU were added to the medium. Procedures of irradiation, incubation with IdU, and fixation were as described above. IdU staining procedure with signal amplification by tyramide. For denaturation of DNA in nuclei slides were placed in 4 N HCl for 30 min at RT. After incubation in PBS for 15 min at RT slides were incubated in 1% blocking reagent (Boehringer, Cat. No. 1096 176) in PBS with 0.02% Tween 20 for 30 min at 37°C and rinsed with 0.5% blocking reagent containing 0.02% Tween 20. The subsequent dilutions of antibodies were in the same solution and incubations were at 37°C. Slides were incubated with mouse monoclonal anti-bromodeoxyuridine (BrdU) antibodies (Boehringer, Cat. No. 1170376; 1:100, 1 h) that also react with IdU. Slides were washed with PBS supplemented with 0.2% Tween 20 for 30 min at RT with shaking; then they were incubated with biotinylated sheep-anti-mouse IgG antibodies (Sigma; 1:100, 40 min) and washed for 30 min in PBS with 0.2% Tween 20 at RT. To increase the signal from the incorporated IdU we used the tyramide signal amplification with TSA-Indirect Renaissance Kit (DuPont) according to the protocol provided by the company. Slides were washed for 30 min in TNT buffer (0.1 M Tris–HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween 20) at RT with shaking. All of the subsequent washes between incubations were done in the same way. Slides were blocked in TNB buffer (0.1 M Tris–HCl, pH 7.5, 0.15 M NaCl, 0.5% DuPont Blocking Reagent) for 30 min at RT and then incubated sequentially at RT with horseradish peroxidase-conjugated streptavidin (1:500 in TNB, 30 min), biotinyl tyramide (1:100 in amplification diluent, 10 min), and fluorescein isothiocyanate (FITC)-conjugated avidin (Boehringer; 1:200) and washed. The DNA in nuclei was counterstained by incubating the slides with ethidium bromide (0.02 mg/ml in PBS, 10 min at RT). Then slides were mounted in CITIFLUOR antifading solution (glycerol/PBS solution; UKS Chemical Laboratories, UK).

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FIG. 1. Visualization of focal sites of DNA repair synthesis in UV-irradiated quiescent VH-10 fibroblasts (A) after 10-min pulse with 5-iododeoxyuridine using tyramide– biotin signal amplification and (B) in control unirradiated cells. UV dose was 30 J/m 2. Counterstaining was with ethidium bromide. Bar, 10 ␮m.

Metaphase chromosome plate preparation and combination of DAPI staining and IdU detection. VH-10 cells were grown to confluence in DMEM with 10% FCS in 10-cm petri dishes and then starved for 3 days in DMEM without FCS. The cells were UVirradiated (3 J/m 2) and IdU was incorporated for 30 min as described above. After washing three times with sterile PBS, cells were put in DMEM containing 10% FCS, 24 h later the cells were replated on glass microscopic slides to stimulate cell division, and 22–24 h after replating 0.06 ␮g/ml colcemide was added for 2 h. The slides were washed in warm Hanks’ solution, treated for 15 min with hypotonic solution (0.075 M KCl/1% sodium citrate, 1:1) at 37°C, and fixed in precooled (⫺20°C) methanol/acetic acid mixture (3:1) for 40 min at 4°C. The slides were placed on a flat horizontal ice surface and blown with a cool air flow from a hair dryer to prepare metaphase plates. Slides were treated for 20 min with 45% acetic acid at RT and then air-dried, kept for 10 min in 70% ethanol at RT, rinsed in 96% ethanol, and air-dried. Staining was done with 0.75 ng/ml DAPI in PBS. After washing for 3 min in running tap water slides were air-dried and mounted in antifading solution. Images of metaphase plates with DAPI banding were obtained with a charge-coupled device (CCD) camera attached to the microscope, and the positions of the microscope stage for each plate were recorded. Slides were then processed for IdU staining with tyramide amplification of signal as described above. The images of IdU-stained metaphase plates obtained using the stage position coordinates identical to those for the DAPI-stained images were collected with the CCD camera, and the pairs of images for each metaphase plate were analyzed. Microscopy and image processing. For fluorescent microscopy the Axioskop microscope (Zeiss, Germany) with appropriate filter sets for green (FITC), red (ethidium bromide), and blue (DAPI) fluorescence and Plan-NEOFLUAR 100/1.30 objective was used. Photographic images were recorded into the computer using a Vario Cam CCD camera (Germany) with the help of a KS-100 program. Images were processed and analyzed with the use of Adobe Photoshop and TRIM (State Optical Institute, St. Petersburg) programs. The TRIM program allowed us to perform segmentation of images with a manually preset threshold level of segmentation. Those objects (bright

foci) that exhibited a gray level higher than the manually chosen background level were left in the image, and the rest of the image was changed to black (gray level 0). The number of objects (segments) left after segmentation was counted by TRIM. However, when very closely spaced, clearly visible, distinct dots were not resolved by TRIM, the foci within segmented areas were manually counted and these counts added to the number of segments. In each experimental variant 30 –100 nuclei were analyzed for statistical evaluation.

RESULTS

Distinct Foci of 5-Iododeoxyuridine Incorporation in UV-Irradiated Quiescent Human Fibroblasts The tyramide amplification of the fluorescent signal facilitates detection of UV-induced DNA repair synthesis after very short times of incubation of the UVirradiated cells with IdU. Figure 1 shows a typical image of nuclear foci in UV-irradiated (30 J/m 2) quiescent VH-10 fibroblasts following a 10-min pulse of IdU. Distinct foci are seen in all nuclei of UV-irradiated cells (Fig. 1A) and no IdU incorporation is detected in control, nonirradiated cells (Fig. 1B), indicating that the foci are dependent upon irradiation and are not associated with replicative DNA synthesis in S-phase cells, which represent a very small fraction of the cells in quiescent fibroblast cultures. In fact, IdU incorporation during S-phase of normally proliferating (nonquiescent) cells detected using the tyramide system leads to intensive overall labeling of the nuclei without visible foci (Fig. 2, compare A and B). As indicated under Materials and Methods, for a semiquantitative analy-

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287

FIG. 2. Comparative tyramide labeling of UV-irradiated non-S-phase cell (A) and S-phase cell (B) after 10-min pulse with IdU immediately after irradiation (30 J/m 2). Asynchronous nonquiescent VH-10 cell culture was used. No ethidium bromide counterstaining. Bar, 10 ␮m.

sis of images of nuclei containing IdU foci we used the computer program TRIM. Figure 3 shows results of the treatment of an original unprocessed image of the UVinduced IdU foci in a human nucleus (Fig. 3A) at two levels of segmentation thresholds (low stringency, Fig. 3B, and high stringency, Fig. 3C). It can be seen that some very dim foci in Figs. 3A and 3B disappear after segmentation at high stringency (Fig. 3C). Figure 3 also shows profiles of relative brightness before (D) and after segmentation at low stringency (E) and at high stringency (F), illustrating that even closely spaced dots can be resolved by TRIM and that signal-to-noise ratio in our experiments was high. We believe that the distinct IdU foci detected using tyramide in UV-irradiated quiescent fibroblasts reflect DNA repair synthesis during early NER. Further evidence for this will be presented below. We note here that the UV dose of 30 J/m 2 (254 nm) induces roughly one million photoproducts per nucleus but the number of visible foci is less than 100. If we assume that only 0.1% (1000) of the photoproducts are eliminated by NER in the first 10 min, it may be calculated that each tyramide focus should accommodate at least 10 elementary DRS patches. On the other hand, a single DRS patch of 30 nucleotides cannot be detected using analysis of incorporated BrdU or IdU, since only 5–10% of thymines in DNA can be substituted by halogenated deoxyuridines upon in vivo labeling [32]. Taking into account the thymine content in mammalian DNA (20%) a single DRS patch would contain less than one (0.3– 0.7) IdU residue. Therefore, the distinct DRS foci visualized in the nucleus (Fig. 1) must represent clusters of many individual DRS patches. Assuming that three to five clustered IdU molecules are detectable using the tyramide system, a single visible focus in a nucleus should accommodate at least 6 –10 DRS patches.

Since the number of UV-induced DNA photoproducts increases linearly with UV dose we examined the UV dose dependence of the number of DRS foci generated per nucleus within a very short fixed period (10 min) of IdU labeling immediately after irradiation. It can be seen in Fig. 4 that the number of foci increases within the very low UV dose range (⬍3 J/m 2) but then remains unchanged as the UV dose is increased up to 30 J/m 2. These observations are consistent with the view that there is a limited number of nuclear “compartments” within which clustering of repair events is occurring. Such clustering may be a consequence of the transient association of widely separated unlinked genomic segments at NER sites or they might arise because of preferential repair within specific, large subchromosomal domains. If the second view is correct, the DRS foci should not disappear upon delayed fixation of cells labeled with IdU just after UV. Our analysis of interphase nuclei which were fixed 3, 6, and 40 h after UV irradiation and short IdU labeling indicates that DRS foci remain upon delayed fixation (data not shown). In addition, we examined whether distinct DRS foci can be detected in metaphase chromosomes fixed 48 h after the 30-min IdU labeling and results are presented in the next section. Detection of UV-Induced DRS Foci in Metaphase Chromosomes Quiescent fibroblasts were UV-irradiated (3 J/m 2) and then incubated with IdU for 30 min in the lowserum (0.5%) growth medium and for 24 h in the growth medium without IdU but with the normal (10%) concentration of serum. Cells were then replated and incubated for an additional 22–24 h before treatment with colcemide and fixation in methanol–acetic acid. Metaphases were then banded using DAPI and

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FIG. 3. Image analysis using computer program TRIM. (A) An unprocessed immunofluorescent image of the nucleus with multiple IdU foci; (B and C) the same nucleus after segmentation at low and high stringency, respectively; (D) brightness profile through an IdU-labeled nucleus before segmentation; (E and F) similar profiles after segmentation at low and high stringency (threshold), respectively. No ethidium bromide counterstaining. Bar, 10 ␮m.

photographed, and the IdU was detected using the tyramide system. Figure 5 shows a single metaphase labeled for IdU (A) and with DAPI (B). It can be seen that the DRS foci do not disappear 48 h after the 30-min incubation of cells with IdU, suggesting that

clustering of DRS patches in cells fixed immediately after labeling (Fig. 1) is not a consequence of some transient association of unlinked genomic segments but rather arises in specific subchromosomal domains. DAPI banding also allowed us to identify some chro-

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FIG. 4. Dependence of formation of IdU foci in quiescent VH-10 cells on UV dose. Average number of IdU foci per nucleus counted after segmentation of images at high stringency using computer program TRIM (50 nuclei were analyzed in each variant). Time of IdU labeling was 10 min.

mosome bands containing DRS foci and to generate a cumulative plot of the DRS signal. Prominent peaks of the signal were found over R-bands 1p36, 2p21 (data not shown), 4q21, 5q13, and 5q31 (Fig. 6), some of which are known to be enriched in expressed genes. For example, a 1-Mbp subregion in human band 5q31 contains a cluster of 23 cytokine genes [33] and band 4q21 contains a cluster of CXC chemokine genes [34]. It can be seen in Fig. 5 that many IdU foci are asymmetrically distributed in sister chromatids, consistent with the view that they might have arisen during strand-specific TCR within clusters of similarly oriented transcription units. Analysis of the almost completely sequenced human chromosomes shows that such clusters may exist. However, asymmetric labeling of chromatids may be observed sometimes in the first mitosis after S-phase BrdU labeling (see Fig. 6 in [35]) when it is expected to be 100% symmetric. Therefore, we cannot yet conclude that these asymmetric DRS foci in metaphase chromosomes (Fig. 5) actually arise during strand-specific repair or as a consequence of uneven signal amplification. This will be evaluated more thoroughly in our ongoing studies. It should be noted that the metaphase shown in Fig. 5 is unlikely to represent a rare S-phase cell labeled with IdU in quiescent fibroblast culture just after UV, since (1) in the tyramide system 30-min IdU labeling of S-phase cells leads to a very intensive fluorescent signal over the nucleus and metaphase chromosomes and (2) we were unable to detect weakly IdU-labeled met-

aphases in parallel unirradiated control quiescent cultures, also incubated with IdU for 30 min (data not shown). Formation of DRS Foci in Normal Fibroblasts Is Suppressed by ␣-Amanitin Clusters of elementary DRS patches can arise during transcription-coupled repair within spatially and genetically linked (clustered) transcription units which are known to exist in mammalian nuclei. We examined whether formation of UV-induced DRS foci is sensitive to ␣-amanitin. Overall RNA synthesis is known to be reduced by this drug by a factor of two, and TCR in the DHFR gene is reduced by 2/3 [36]. In our experiments cells were pretreated with ␣-amanitin (10 ␮g/ml) for 6 h before UV irradiation (3 J/m 2) and IdU labeling. It is known that the treatment of mammalian cells with 10 ␮g/ml of ␣-amanitin for 5.5 h before UV irradiation and for an additional 4 h after irradiation does not affect overall repair replication [36], indicating that the concentrations of active NER proteins are not decreased by this drug. It can be seen in Fig. 7 that 6 h pretreatment of cells with ␣-amanitin inhibits the induction of DRS foci by UV irradiation. Analysis of the number of foci after segmentation of images at high- (Fig. 7C) or low- (not shown) stringency thresholds indicates that the number is decreased by 40 –70% after treatment with this drug. This result is consistent with the view that at

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FIG. 5. Detection of the foci of DNA repair synthesis in human metaphase chromosomes. UV-irradiated (3 J/m 2) quiescent VH-10 cells were incubated immediately after irradiation in growth medium containing IdU for 30 min and then processed as described under Materials and Methods. (A) IdU labeling; (B) DAPI banding pattern.

least some of the clusters of DRS patches can be formed during TCR. The residual clusters of DRS patches in ␣-amanitin-treated cells can arise because of an incom-

plete inhibition of TCR by this drug and/or independently of ongoing Pol II transcription during GGR. Induction of DRS Foci in Human Fibroblasts Deficient in TCR or GGR

FIG. 6. Cumulative DRS signal over human chromosomes 4 and 5. The signal was generated from 15 DAPI-banded metaphases similar to those shown in Fig. 5.

TCR is known to be deficient in Cockayne syndrome cells while GGR is inactive in XPC cells. Both modes of NER depend upon the XPA protein and we have shown earlier that DRS foci cannot be induced in XPA-deficient cells [37]. In this study we compared formation of DRS foci in normal, CS, and XPC fibroblasts and the results are presented in Fig. 8 and Table 1. Figures 8A– 8F show typical original unprocessed images of VH-10 (A, D), XPC (B, E), and CS (C, F) nuclei, in positive (A–C) and negative (D–F) variants, and Figs. 8G– 8I show the same Photoshop-processed negative images with strongly increased contrast and decreased brightness. It can be seen in Fig. 8 that the number of visible IdU foci is decreased in both XPC (B, E, H) and CS (C, F, I) cells compared to normal fibroblasts (A, D, G), with the decrease apparently more profound in the TCR-deficient CS cells. It should be noted that the decrease of formation of IdU foci in CS and XPC cells compared to normal fibroblasts was reproducibly observed in several independent experiments. This indi-

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FIG. 7. Inhibition of formation of the UV-induced DRS foci in VH-10 cells treated with ␣-amanitin. (A) Example of control untreated nucleus after UV irradiation (3 J/m 2) and 30-min IdU labeling; (B) typical nucleus from VH-10 culture pretreated for 6 h with 10 mg/ml of ␣-amanitin before UV irradiation and IdU labeling; (C) results of analysis of 30 –50 cells in each variant after segmentations of images at high stringency using TRIM. Mean values ⫹ 3 SE are shown. Bar, 10 ␮m.

cates that (1) both TCR and GGR contribute to the clustering of DRS patches in the nucleus and (2) a fraction of the clusters can apparently be formed in the absence of GGR (in XPC cells) and in the absence of TCR (in CS cells). Since CS cells usually show normal levels of overall DNA repair synthesis, strong reduction of the number of DRS foci in these cells (Fig. 8C) indicates that the clustered DRS patches represent a minor fraction of overall DRS. Overall DRS is mostly produced by randomly distributed single patches which are not detectable by immunofluorescence. This interpretation is also consistent with saturation of the number of DRS foci at lower UV doses (⬃3 J/m 2; see Fig. 4) compared to overall DRS which saturates at UV doses greater than 10 J/m 2. We attempted to quantitate the observed differences between cell lines using the computer program TRIM. Segmentation of images have been carried out using

two different threshold values (30 and 46), and the results are shown in Table 1. The stringency of segmentation weakly affected the number of foci observed in control VH-10 and GM38 cells, indicating little slideto-slide and line-to-line variations of immunofluorescence under our conditions, but it significantly affected results with CS and XPC cells, indicating the presence of large but variable numbers of dim foci in these NER-deficient cells (see also Figs. 8H and 8I). The decreased staining of many IdU foci in CS cells (which were processed in some experiments in parallel with VH-10 slides) is unlikely to be associated with a decreased intensity of overall DRS, since UV-sensitive CS cells do not have such a defect, but the initial rate of GGR may be decreased in these cells because of an altered chromatin structure [23]. XPC cells have a partial defect of overall DRS (usually ⬃30% of unscheduled DNA synthesis compared to normal cells)

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FIG. 8. Visualization of the UV-induced DRS foci in NER-deficient XPC and CS cells. (A, B, C) Original positive unprocessed images of nuclei from UV-irradiated (6 J/m 2) cultures of VH-10 (A), XPC (B), and CS (C) cells incubated for 30 min with IdU and then fixed; (D, E, F) negative images of the same nuclei as in (A, B, C); (G, H, I) the same set of images as in (D, E, F) processed using Photoshop, with strongly decreased brightness and increased contrast. No counterstaining with ethidium bromide. Bar, 10 ␮m.

and decreased intensity of labeling of DRS sites in these cells may be caused by defective repair of nontranscribed strands during TCR. In any case, counting of the DRS foci using TRIM confirms our conclusions that both TCR and GGR contribute to the clustered DRS patches during early NER and that formation of the foci is possible in the absence of either TCR or GGR. Involvement of TCR is consistent with the ␣-amanitin-induced inhibition of DRS foci in VH-10 cells (Fig. 7). Residual fractions of DRS foci in XPC and CS cells (Table 1) suggest that the contribution from TCR is quantitatively more important than that from GGR. However, it remains uncer-

tain whether there is an overlap between TCR-dependent and GGR-dependent foci in NER-proficient cells or whether both NER modes contribute to DRS foci additively. It appears significant that after segmentation at low stringency the sum of residual numbers of DRS foci in XPC and CS cells (34.6 ⫹ 25.1 ⫽ 59.7 and 65.5 ⫹ 28.0 ⫽ 94.5) is not very different from the number of the foci in normal cells (66.9 and 76.5, respectively). This may be interpreted as an indication that in NER-proficient cells TCR and GGR contribute to formation of clusters of DRS patches additively, but a definitive answer to this question will be sought in future studies.

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TABLE 1 Induction of DRS Foci in Normal, TCR-, and GGR-Deficient Human Cells Average number of DRS foci a per nucleus in the indicated cell line UV dose (J/m 2)

Time of incubation with IdU (min)

Segmentation threshold

VH-10 b

XPC

CS

6 6 6 6 0 0

30 30 60 60 60 60

46 30 46 30 46 30

58.1 ⫾ 13.1 (100%) 66.9 ⫾ 10.2 (100%) 68.1 ⫾ 13.3 (100%) 76.5 ⫾ 10.0 (100%) 1.8 ⫾ 1.0 2.1 ⫾ 1.4

12.1 ⫾ 3.5 (21%) 34.6 ⫾ 8.6 (51.7%) 27.0 ⫾ 7.0 (39.6%) 65.5 ⫾ 12.0 (85.6%) 1.3 ⫾ 1.3 7.4 ⫾ 4.4

4.3 ⫾ 2.5 (7.4%) 25.1 ⫾ 10.5 (37.5%) 3.6 ⫾ 2.6 (5.3%) 28.0 ⫾ 11.0 (36.6%) 0 0

a Foci in 50 –100 nuclei of each variant were counted after segmentation under indicated thresholds using computer program TRIM and average values are shown plus/minus three standard errors. b Formation of DRS foci was also examined in another NER-proficient human primary fibroblast cell line, GM38, and after UV-irradiation with the dose 6 J/m 2 and stringent segmentation threshold (46) average numbers of the foci per nucleus were found to be 61.5 ⫾ 10.9 (30-min IdU labeling) and 67.4 ⫾ 11.6 (60-min IdU labeling) which is very close to the numbers obtained with VH-10 cells. This indicates that slide-to-slide and line-to-line variations in the numbers of DRS foci are smaller than differences observed between NER-proficient and NER-deficient cells.

DISCUSSION

In this study focal sites of the UV-induced DNA repair synthesis were visualized in the nucleus after short (10- to 60-min) incubation of irradiated cells with IdU. We believe that these foci represent clusters of elementary 30-nucleotide DRS patches known from biochemical experiments in vitro and, therefore, detection of the foci is indicative of spatial clustering of NER events in human cells. Because of sensitivity to ␣-amanitin and partial NER deficiency in CS cells many DRS foci are probably formed in association with Pol II transcription and TCR. In agreement with this view we found (Table 1) that significant fractions of the foci are formed in GGR-deficient XPC cells in which only TCR is possible. This indicates that the clusters of DRS patches can arise during TCR in the absence of GGR, which is apparently possible within clusters of adjacent (but independent) transcription units each containing a single transcript [13]. TCR is known to be specifically dependent on CSB and CSA proteins, the first of which has been recently identified as an ATP-dependent chromatin-remodeling factor [21], and the report of reduced Pol II transcription in unirradiated CSB cells [23] would be consistent with an altered state of chromatin in these cells in the absence of DNA damage. An altered state of chromatin in CS cells may not significantly affect the overall efficiency of NER (i.e., overall unscheduled DNA synthesis is not decreased in Cockayne cells) but it may decrease the initial rate of DRS in these cells. Clustering of NER events during TCR within transcriptionally active chromatin is not very surprising taking into account apparent clustering of transcription units [13, 19]. However, it is important that many DRS foci are formed in CS cells and in normal fibro-

blasts in the presence of ␣-amanitin. These foci cannot be associated with TCR but could only arise because of GGR. Therefore, NER events may be clustered during GGR in the absence of TCR, which is consistent with a model for processive scanning of large chromatin domains. The current view of global nuclear organization [11, 12] anticipates dynamic interactions between spongelike chromosome territories (CTs) occupied by chromatin and protein complexes assembled in interchromatin compartments (ICs). Although many RNA splicing, transcription, and DNA repair proteins are mobile in the nucleus and can freely diffuse through ICs and CTs [10, 38 – 40] they can also form stable and functional long-living assemblies such as speckles or interchromatin granule clusters [14, 15]. It should be anticipated that histoneforming nucleosome core particles are immobile [39, 41] as is chromatin itself [42, 43], which indicates that active transcription complexes assembled at the border between ICs and CTs should move along the periphery of CTs to produce RNA. Some relatively rapid (within minutes) exchange of a small fraction (⬃3%) of the histone H2B may be associated with transcription-driven transient displacement of this histone from nucleosomes, but the histones H3 and H4 show little exchange [41]. Imaging with GFP-tagged histone H2B in the nucleus of HeLa cells [41, 44, 45] revealed a very uneven distribution of this histone and its preferential association with compact heterochromatin at the nuclear and nucleolar periphery. H2B-poor domains located within the internal nonnucleolar compartments are enriched in transcription foci [45] representing clusters of transcription units [13, 19]. It is likely that in UV-irradiated NER-proficient cells a major fraction of DRS foci

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may be formed during TCR within these H2B-poor domains. However, some stable and processive GGR complexes probably can be formed within the compact H2B-rich heterochromatin also, resulting in clustered NER events in the absence of TCR. This view is consistent with our previous report of the transient UVinduced insolubilization of NER protein XPA in Cockayne syndrome cells [46]. Such GGR complexes can be assembled sequentially [47] at some repair sites in UV-irradiated cells and then a single complex can processively eliminate several photochemical lesions within one large heterochromatin domain, leading to a cluster of DRS patches in the absence of TCR. Alternatively, several distributive GGR complexes can be assembled simultaneously in one large heterochromatin domain but in this case the cause of clustering of repair events remains unclear. GGR in vitro requires an accessory protein of DNA polymerases ␦ and ␧ proliferating cell nuclear antigen (PCNA) [48]. During normal DNA replication PCNA becomes insoluble in Triton X-100 at replication sites in S-phase cells [49] and similar PCNA insolubilization has been detected in UV-irradiated non-S-phase cells [50 –52], consistent with the view that this protein integrates into immobile complexes during DNA replication or DNA repair. In living cells GFP-tagged PCNA associated with normal replication sites may be stably anchored to an immobile matrix [53] but possible transient changes in diffusional mobility of PCNA in UVirradiated non-S-phase cells has not yet been investigated. Future studies of dynamic changes of repair proteins and chromatin after DNA damage will help to elucidate the precise organization of the DNA repair in the nucleus.

8.

9.

10.

11. 12.

13. 14.

15.

16.

17.

18.

19.

20. This work was supported by NIH Fogarty International Center FIRCA Grant 5R03TW001385-02 to P.C.H. and M.P.S. The laboratory of N.V.T. at the Institute of Cytology RAS in St. Petersburg is supported by grants from the Russian Fund for Basic Research.

21.

REFERENCES 1.

2.

3.

4. 5. 6. 7.

Setlow, R. B., and Carrier, W. L. (1964). The disappearance of thymine dimers from DNA: An error-correcting mechanism. Proc. Natl. Acad. Sci. USA 51, 226 –231. Boyce, R. P., and Howard-Flanders, P. (1964). Release of ultraviolet light-induced thymine dimers from DNA in E. coli K12. Proc. Natl. Acad. Sci. USA 51, 293–300. Pettijohn, D., and Hanawalt, P. C. (1964). Evidence for repair replication of ultraviolet damaged DNA in bacteria. J. Mol. Biol. 9, 395– 410. Hanawalt, P. C. (2001). Controlling the efficiency of excision repair. Mutat. Res. 485, 3–13. Hoeijmakers, J. H. (2001). Genome maintenance mechanisms for preventing cancer. Nature 411, 366 –374. Wood, R. D. (1999). DNA damage recognition during nucleotide excision repair in mammalian cells. Biochimie 81, 39 – 44. Hanawalt, P. C., and Haynes, R. H. (1967). The repair of DNA. Sci. Am. 216, 36 – 43.

22.

23.

24. 25.

26.

Grossman, L., and Thiagalingam, S. (1993). Nucleotide excision repair, a tracking mechanism in search of damage. J. Biol. Chem. 268, 16871–16874. Lloyd, R. S., Hanawalt, P. C., and Dodson, M. L. (1980). Processive action of T4 endonuclease V on irradiated DNA. Nucleic Acids Res. 8, 5113–5127. Houtsmuller, A. B., Rademakers, S., Nigg, A. L., Hoogstraten, D., Hoeijmakers, J. H., and Vermeulen, W. (1999). Action of DNA repair endonuclease ERCC1/XPF in living cells. Science 284, 958 –961. Lamond, A. I., and Earnshaw, W. C. (1998). Structure and function in the nucleus. Science 280, 547–553. Cremer, T., and Cremer, C. (2001). Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat. Rev. Genet. 2, 292–301. Cook, P. R. (1999). The organization of replication and transcription. Science 284, 1790 –1795. Mintz, P. J., and Spector, D. L. (2000). Compartmentalization of RNA processing factors within nuclear speckles. J. Struct. Biol. 129, 241–251. Mintz, P. J., Patterson, S. D., Neuwald, A. F., Spahr, C. S., and Spector, D. L. (1999). Purification and biochemical characterization of interchromatin granule clusters. EMBO J. 18, 4308 – 4320. Hwang, B. J., Toering, S., Francke, U., and Chu, G. (1998). p48 activates a UV-damaged-DNA binding factor and is defective in xeroderma pigmentosum group E cells that lack binding activity. Mol. Cell. Biol. 18, 4391– 4399. Nakamura, H., Morita, T., and Sato, C. (1986). Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus. Exp. Cell Res. 165, 291–297. Hozak, P., Hassan, A. B., Jackson, D. A., and Cook, P. R. (1993). Visualization of replication factories attached to nucleoskeleton. Cell 73, 361–373. Jackson, D. A., Iborra, F. J., Manders, E. M., and Cook, P. R. (1998). Numbers and organization of RNA polymerases, nascent transcripts, and transcription units in HeLa nuclei. Mol. Biol. Cell 9, 1523–1536. Rapin, I., Lindenbaum, Y., Dickson, D. W., Kraemer, K. H., and Robbins, J. H. (2000). Cockayne syndrome and xeroderma pigmentosum. Neurology 55, 1442–1449. Citterio, E., Van Den Boom, V., Schnitzler, G., Kanaar, R., Bonte, E., Kingston, R. E., Hoeijmakers, J. H., and Vermeulen, W. (2000). ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcription-coupling factor. Mol. Cell. Biol. 20, 7643–7653. Eisen, J. A., Sweder, K. S., and Hanawalt, P. C. (1995). Evolution of the SNF2 family of proteins: Subfamilies with distinct sequences and functions. Nucleic Acids Res. 23, 2715–2723. Balajee, A. S., May, A., Dianov, G. L., Friedberg, E. C., and Bohr, V. A. (1997). Reduced RNA polymerase II transcription in intact and permeabilized Cockayne syndrome group B cells. Proc. Natl. Acad. Sci. USA 94, 4306 – 4311. Hanawalt, P. C. (2000). DNA repair: The bases for Cockayne syndrome. Nature 405, 415– 416. LePage, F., Kwoh, E. E., Avrutskaya, A., Gentil, A., Leadon, S. A., Sarasin, A., and Cooper, P. K. (2000). Transcriptioncoupled repair of 8-oxoGuanine: Requirement for XPG, TFIIH, and CSB and implications for Cockayne syndrome. Cell 101, 159 –171. Sugasawa, N., Masutani, C., Iwai, S., van der Spek, P. J., Eker, A. P., Hanaoka, F., Bootsma, D., and Hoeijmakers, J. H. (1998). Xeroderma pigmentosum group C protein complex is the initi-

CLUSTERING OF NUCLEOTIDE EXCISION REPAIR EVENTS ator of global genome nucleotide excision repair. Mol. Cell 2, 223–232. 27.

Hwang, B. J., Ford, J. M., Hanawalt, P. C., and Chu, G. (1999). Expression of the p48 xeroderma pigmentosum gene is p53dependent and is involved in global genomic repair. Proc. Natl. Acad. Sci. USA 96, 424 – 428.

28.

Tang, J. Y., Hwang, B. J., Ford, J. M., Hanawalt, P. C., and Chu, G. (2000). Xeroderma pigmentosum p48 gene enhances global genomic repair and suppresses UV-induced mutagenesis. Mol. Cell 5, 737–744.

29.

Baxter, B. K., and Smerdon, M. J. (1998). Nucleosome unfolding during DNA repair in normal and xeroderma pigmentosum (group C) human cells. J. Biol. Chem. 273, 17517–17524.

30.

Bowman, K. K., Smith, C. A., and Hanawalt, P. C. (1997). Excision repair patch lengths are similar for transcriptioncoupled repair and global genome repair in UV-irradiated human cells. Mutat. Res. 385, 95–105.

40.

41.

42.

43.

44.

31.

Raap, A. K., van de Corput, M. P., Vervenne, R. A., van Gijlswijk, R. P., Tanke, H. J., and Wiegant, J. (1995). Ultrasensitive FISH using peroxidase-mediated deposition of biotinor fluorochrome tyramides. Hum. Mol. Genet. 4, 529 –534.

32.

Raderschall, E., Golub, E. I., and Haaf, T. (1999). Nuclear foci of mammalian recombination proteins are located at singlestranded DNA regions formed after DNA damage. Proc. Natl. Acad. Sci. USA 96, 1921–1926.

47.

33.

Frazer, K. A., Ueda, Y., Zhu, Y., Gifford, V. R., Garofalo, M. R., Mohandas, N., Martin, C. H., Palazzolo, M. J., Cheng, J. F., and Rubin E. M. (1997). Computational and biological analysis of 680 kb of DNA sequence from the human 5q31 cytokine gene cluster region. Genome Res. 7, 495–512.

48.

34.

O’Donovan, N., Galvin, M., and Morgan, J. G. (1999). Physical mapping of the CXC chemokine locus on human chromosome 4. Cytogenet. Cell Genet. 84, 39 – 42.

35.

Ma, H., Samarabandu, J., Devdhar, R. S., Acharya, R., Cheng, P. C., Meng, C., and Berezney, R. (1998). Spatial and temporal dynamics of DNA replication sites in mammalian cells. J. Cell Biol. 143, 1415–1425.

36.

Christians, F. C., and Hanawalt, P. C. (1992). Inhibition of transcription and strand-specific DNA repair by ␣-amanitin in Chinese hamster ovary cells. Mutat. Res. 274, 93–101.

37.

Svetlova, M. P., Solovjeva, L. V., Pleskach, N. A., and Tomilin, N. V. (1999). Focal sites of DNA repair synthesis in human chromosomes. Biochem. Biophys. Res. Commun. 257, 378 –383.

51.

38.

Kruhlak, M. J., Lever, M. A., Fischle, W., Verdin, E., BazettJones, D. P., and Hendzel, M. J. (2000). Reduced mobility of the alternate splicing factor (ASF) through the nucleoplasm and steady state speckle compartments. J. Cell Biol. 150, 41–51.

52.

39.

Phair, R. D., and Misteli T. (2000). High mobility of proteins in the mammalian cell nucleus. Nature 404, 604 – 609.

Received October 9, 2001 Revised version received February 22, 2002 Published online May 2, 2002

45.

46.

49.

50.

53.

295

McNally, J. G., Muller, W. G., Walker, D., Wolford, R., and Hager, G. L. (2000). The glucocorticoid receptor: Rapid exchange with regulatory sites in living cells. Science 287, 1262– 1265. Kimura, H., and Cook, P. R. (2001). Kinetics of core histones in living human cells: Little exchange of H3 and H4 and some rapid exchange of H2B. J. Cell Biol. 153, 1341–1353. Abney, J. R., Cutler, B., Fillbach, M. L., Axelrod, D., and Scalettar, B. A. (1997). Chromatin dynamics in interphase nuclei and its implications for nuclear structure. J. Cell Biol. 137, 1459 –1468. Marshall, W. F., Straight, A., Marko, J. F., Swedlow, J., Dernburg, A., Belmont, A., Murray, A. W., Agard, D. A., and Sedat, J. W. (1997). Interphase chromosomes undergo constrained diffusional motion in living cells. Curr. Biol. 7, 930 –939. Kanda, T., Sullivan, K. F., and Wahl, G. M. (1998). Histone– GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr. Biol. 8, 377–385. Verschure, P. J., van Der Kraan, I., Manders, E. M., and van Driel, R. (1999). Spatial relationship between transcription sites and chromosome territories. J. Cell Biol. 147, 13–24. Svetlova, M., Nikiforov, A., Solovjeva, L., Pleskach, N., Tomilin, N., and Hanawalt, P. C. (1999). Reduced extractability of the XPA DNA repair protein in ultraviolet light-irradiated mammalian cells. FEBS Lett. 463, 49 –52. Volker, M., Mone, M. J., Karmakar, P., van Hoffen, A., Schul, W., Vermeulen, W., Hoeijmakers, J. H., van Driel, R., van Zeeland, A. A., and Mullenders, L. H. (2001). Sequential assembly of the nucleotide excision repair factors in vivo. Mol. Cell 8, 213–224. Aboussekhra, A., Biggerstaff, M., Shivji, M. K. K., Vilpo, J. A., Moncollin, V., Podust, V. N., Protic, M., Hubscher, U., Egly, J.-M., and Wood, R. D. (1995). Mammalian DNA nucleotide excision repair reconstituted with purified components. Cell 80, 859 – 868. Bravo, R., and MacDonald-Bravo, H. (1987). Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: Association with DNA replication sites. J. Cell Biol. 105, 1549 –1554. Toschi, L., and Bravo, R. (1988). Changes in cyclin/proliferating cell nuclear antigen distribution during DNA repair synthesis. J. Cell Biol. 107, 1623–1628. Aboussekhra, A., and Wood, R. D. (1995). Detection of nucleotide excision repair incisions in human fibroblasts by immunostaining for PCNA. Exp. Cell Res. 221, 326 –332. Li, R., Hannon, G. J., Beach, D., and Stillman, B. (1995). Subcellular distribution of p21 and PCNA in normal and repairdeficient cells following DNA damage. Curr. Biol. 6, 189 –199. Leonhardt, H., Rahn, H. P., Weinzierl, P., Sporbert, A., Cremer, T., Zink, D., and Cardoso, M. C. (2000). Dynamics of DNA replication factories in living cells. J. Cell Biol. 149, 271–280.