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Previous Article | Next Article 
Molecular and Cellular Biology, April 2007, p. 2538-2547, Vol. 27, No. 7
0270-7306/07/$08.00+0 doi:10.1128/MCB.01288-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Graduate School of Frontier Biosciences, Osaka University,1 Solution Oriented Research for Science and Technology of Japan Science and Technology Agency, Yamadaoka 1-3, Suita, Osaka 565-0871, Japan2
Received 14 July 2006/ Returned for modification 6 September 2006/ Accepted 8 January 2007
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ABSTRACT |
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INTRODUCTION |
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Cockayne syndrome (CS) is a rare autosomal recessive disorder, which shows a diversity of clinical symptoms (2, 8). A comprehensive review of 140 cases of CS (27) indicated that growth failure is a basic clinical feature and generally begins within the first year of life. In addition, individuals with CS develop neurological dysfunctions, such as mental retardation and microcephaly. Associated clinical features are ophthalmologic abnormalities, such as cataracts and optic atrophy; sensorineural hearing loss; and dental caries. Affected individuals also manifest photosensitivity of the skin but have no predisposition to sunlight-induced skin cancer, in contrast to xeroderma pigmentosum (XP) patients (2, 27).
Cells from CS patients are hypersensitive to UV light and show reduced recovery of DNA and RNA synthesis after exposure to UV light. The cellular abnormalities in CS have been attributed to a specific defect in TCR (41, 43). Complementation analysis using the recovery of DNA or RNA synthesis after UV irradiation as a marker has defined two genetic complementation groups in CS: CS-A and CS-B (19, 36). In addition, XP group B (XP-B) patients and certain patients with XP-D or XP-G show features of CS in addition to symptoms of XP (XP-B/CS, XP-D/CS, and XP-G/CS) (2, 8).
The CSA and CSB genes have been cloned (13, 39). The CSA gene encodes a 44-kDa protein with five WD-40 repeats that appears to have the potential to interact with other proteins. It has been shown that the CSA protein interacts with XAB2, CSB, and the p44 subunit of TFIIH (13, 26). We have recently reported that CSA forms a ubiquitin ligase complex containing DDB1, cullin 4A, and Roc1 and that the complex interacts with the COP9 signalosome (10). In addition, this CSA complex binds to the hyperphosphorylated form of RNA polymerase II in the chromatin fraction after UV irradiation. The CSB protein is a member of the SWI2/SNF2 family and has DNA-dependent ATPase and ATP-dependent chromatin-remodeling activities (4, 5, 31). It has been reported that CSB interacts with RNA polymerase II elongation complex in vitro and in vivo (37, 40, 42) and stimulates elongation by RNA polymerase II in vitro (32). However, CSB did not remove a stalled RNA polymerase II elongation complex from the DNA template (31). On the other hand, CSB was found to be involved in transcription driven by RNA polymerases I and III (3, 49).
The nuclear matrix is thought to play an important role in nuclear metabolism. The hyperphosphorylated form of RNA polymerase II is known to associate with the nuclear matrix (24, 28, 47). As for repair, several reports suggest that NER is associated with the nuclear matrix (12, 18, 21). It has been reported that UV-induced repair patches were enriched in the nuclear matrix. Interestingly, this phenomenon was enhanced in TCR-proficient XP-C cells and abolished in TCR-deficient CSB cells (16, 25). These findings indicate that TCR takes place in the nuclear matrix. We have shown that CSA protein is translocated to the nuclear matrix after UV irradiation and that the translocation requires CSB, which is involved in TCR, but not XPC, which acts as a damage sensor in the GGR-specific process, or XPA, which functions in the core NER reactions (15). These results suggested that the UV-induced translocation of CSA is relevant to TCR. In this study, we established a cell-free system for examining the UV-induced translocation of CSA and found that the process is dependent on functional TFIIH, as well as CSB, and on chromatin structure and transcription elongation.
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MATERIALS AND METHODS |
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Antibodies. Antihemagglutinin (anti-HA) antibody (3F10) was purchased from Roche Diagnostics. Anti-green fluorescent protein (anti-GFP) antibody (JL-8) was from Clontech. Peroxidase-conjugated anti-rat immunoglobulin G and Alexa Fluor 488-conjugated anti-rat antibody were from Amersham Biosciences and Molecular Probes, respectively.
Plasmid constructs for CSA expression. CSA cDNA without a stop codon was amplified by PCR from pBS-CSA (26) with the following primers: 5'-ATTCTCGAGCACCATGCTGGGGTTTTTGTCC-3' (forward; CSA cDNA flanked by a Kozak sequence and an XhoI site) and 5'-CTGCTCTAGATCCTTCTTCATCACTGCTGC-3' (reverse; CSA cDNA flanked by an XbaI site). CSA cDNA regions are underlined. The PCR product was cloned into pBluescript (pBS-CSA-C). Sequencing of the plasmid ruled out the presence of PCR-derived mistakes. The XhoI-NotI fragment from the above-described plasmid was cloned in frame and upstream of the sequence encoding a FLAG epitope, followed by an HA epitope in pOZ-C (provided by Y. Nakatani), resulting in pOZ-C-CSA. For the generation of a tagged CSA expression construct, the BglII-AvrII fragment containing the full-length CSA-coding region with the epitope tags from pOZ-C-CSA was ligated between the BamHI and XbaI sites in pcDNA3 (Invitrogen), yielding pcDNA3-C-CSA.
Point mutations identified in CS-A cell lines (CS2SE and CS2IAF) were introduced into pBS-CSA-C using a QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The cDNAs encoding truncated CSA were amplified by PCR with appropriate primers and cloned into pBluescript. DNA sequencing excluded additional mutations introduced elsewhere in the mutant cDNA. The part of the cDNA containing the mutation was isolated by digestion with XhoI-BamHI or BamHI-NotI and exchanged with the corresponding sequences in pcDNA3-C-CSA.
Isolation of stable transfectants. CS3BESV cells were transfected with the CSA expression constructs using Effectene transfection reagent (QIAGEN) according to the manufacturer's recommendations. Stable transfectants were selected in the presence of G418 (500 µg/ml). By immunoblot analysis using anti-HA antibody, the expression of CSA in each transfectant was compared to that in dtCSA/CS3BESV cells as a control for normal expression levels (15). Isolation of CS3BESV cells stably expressing CSA-GFP has been described previously (35).
Translocation of CSA protein in the cell-free system. CS3BESV cells were UV irradiated at 20 J/m2 or not irradiated and incubated for 1 h. The cells were harvested by trypsinization, washed with phosphate-buffered saline (PBS), and then extracted in cytoskeleton (CSK)-Triton buffer [10 mM PIPES {piperazine-N,N'-bis(2-ethanesulfonic acid)}, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.5% (vol/vol) Triton X-100, 1 mM dithiothreitol, 1 mM EGTA, and complete protease inhibitor cocktail (Roche)] at 4°C for 10 min. The pellet was separated from soluble proteins by centrifugation at 4,000 x g for 3 min, washed, and resuspended in the same buffer. This suspension was used as the CSK-ppt fraction. CSA-FLAG-HA/CS3BESV cells were treated with the same buffer described above, and the supernatant (CSK-sup fraction) was recovered by centrifugation. The CSK-ppt fraction was incubated with the CSK-sup fraction containing CSA-FLAG-HA at 4°C for 1 h with occasional gentle tapping. After being separated by centrifugation as described above, the pellet was washed twice with the same buffer, treated with DNase I (2.8 units/µl; Takara) at 30°C for 10 min, and then washed three times with the same buffer. The proteins in the remaining pellet (the DNase I-insoluble fraction) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. CSA in the DNase I- insoluble fraction was detected with anti-HA antibody using enhanced chemiluminescence plus Western blotting detection reagents (Amersham Biosciences).
When the translocation of CSA was measured simultaneously with the synthesis of RNA, the cells were harvested immediately after UV irradiation in the presence of 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole. The CSK-ppt fractions were prepared and resuspended in glycerol storage buffer (50 mM Tris-HCl, pH 8.3, 40% glycerol, 2 mM MgCl2, and 0.1 mM EDTA). The suspension was mixed with an equal volume of 2x reaction buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 300 mM KCl, and 5 mM dithiothreitol) containing 1 mM nucleoside triphosphates (NTPs) and purified CSA complex and incubated at 30°C for 1 h.
Survival and recovery of RNA synthesis after UV irradiation.
The survival of the transfectants was assessed based on colony-forming ability. Exponentially growing cells were plated at 5 x 102 to 10 x 102 cells per 100-mm dish and exposed to UV light at various dosages 
14 h after being plated. The cells were then cultured for 7 to 10 days, fixed with 3% formaldehyde, and stained with 0.1% crystal violet. Colonies were counted using a binocular microscope.
To measure the recovery of RNA synthesis after UV irradiation, cells were seeded in 35-mm dishes at 2 x 105 cells/dish 1 day before being irradiated. The cells were washed with PBS, either irradiated at 10 J/m2 or not irradiated, and incubated in fresh medium. At various times after irradiation, the cells were labeled with 370 kBq/ml of [3H]uridine for 30 min. The labeling was terminated by adding NaN3 at a final concentration of 200 µg/ml to the culture. After being washed with PBS, the cells were lysed with 0.8% SDS at room temperature for 30 min, and then an equal volume of 10% trichloroacetic acid containing 0.1 M sodium pyrophosphate was added to the lysate. Acid-insoluble materials were collected on a Whatman GF/C glass fiber filter, and the radioactivity was measured with a liquid scintillation counter.
Analysis of translocation of CSA protein using in situ visualization and cellular fractionation. Immunofluorescence microscopy was performed as described previously (15). For the fractionation method, cellular proteins were fractionated as follows. Cells were extracted in CSK-Triton buffer at 4°C for 10 min. The insoluble fractions were separated from soluble proteins (fraction 1) by centrifugation at 4,000 x g for 3 min. The pellet was washed twice with a solution containing 250 mM sucrose and 5 mM MgCl2 (fraction 2) and resuspended with 25 mM Tris-HCl, pH 7.4, 250 mM sucrose, 5 mM MgCl2, and 1 mM phenymethylsulfonyl fluoride. Chromatin was solubilized by digesting DNA with 1 mg/ml of DNase I (Roche Diagnostics; grade II) at 30°C for 1 h. The sample was centrifuged at 4,000 x g for 3 min (fraction 3). The pellet was washed three times with a low-salt buffer (10 mM Tris-HCl, pH 7.4, 0.2 mM MgCl2, 1 mM phenymethylsulfonyl fluoride) (fraction 4), extracted consecutively with the low-salt buffer containing increasingly higher concentrations of NaCl (0.3, 0.5, and 2.0 M) for 15 min, and centrifuged at 18,000 x g for 15 min (fractions 5, 6, and 7, respectively). The high-salt pellet was finally extracted with the low-salt buffer containing 1% (vol/vol) Triton X-100 for 15 min and centrifuged at 18,000 x g for 15 min (fraction 8). The remaining pellet was washed twice with the low-salt buffer and solubilized in SDS-PAGE loading buffer (fraction 9).
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RESULTS |
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Next, the post-UV incubation time-dependent translocation of CSA to the nuclear matrix was examined. CSK-ppt fractions prepared at various time points after UV irradiation (20 J/m2) were incubated with CSK-sup fractions prepared from the CS3BESV cells expressing HA-tagged CSA (Fig. 1D). CSA was retained in the DNase I-insoluble fractions derived from cells immediately after UV irradiation. More CSA was retained in the DNase I-insoluble fractions from cells incubated for a longer time (0.5 to 2 h) after UV irradiation. These results were consistent with previous results obtained by the conventional cellular-fractionation analysis (15). It took more than 60 min at 4°C for maximum retention of HA-tagged CSA in the DNase I-insoluble fractions (data not shown).
We examined the factor(s) that can substitute for the CSK-sup fraction. Whole-cell extracts (20, 48), extracts fractionated with phosphocellulose (CF-I) (34, 42), and even a purified CSA complex (10) were competent as a CSK-sup fraction for the UV-induced translocation of CSA (Fig. 1E and data not shown).
We have shown that the UV-induced translocation of CSA to the nuclear matrix depends on CSB, not on XPA or XPC (15). We examined whether CSB is required for translocation in this cell-free system as well. When the CSK-ppt fraction prepared from UV-irradiated CS-B (CS1ANSV) cells was used, little CSA was retained in the DNase I-insoluble fractions (Fig. 1F, lane 3). In contrast, a significant amount of CSA was retained in the DNase I-insoluble fractions when the CSK-ppt fractions prepared from UV-irradiated CS1ANSV cells stably expressing double-tagged CSB (dtCSB/CS1ANSV) (42), as well as XP-C and XP-A cells (lanes 5, 8, and 11), were used.
Taken together, the results validated the fact that this cell-free system faithfully reproduces the phenomenon of translocation of CSA to the nuclear matrix in UV-irradiated cells.
CSA in the CSK-ppt fraction is exchangeable with exogenous CSA. When the CSK-ppt fraction prepared from UV-irradiated normal (WI38VA13) cells was incubated with the CSK-sup fraction containing HA-tagged CSA at 4°C, exogenous CSA was retained in the DNase I-insoluble fraction (Fig. 2A). The retention in the DNase I-insoluble fraction from normal cells was less than that from UV-irradiated CS-A cells (compare lanes 1 and 2). When the CSK-ppt and CSK-sup fractions were incubated at 30°C, the retention was more pronounced. These results suggest that CSA in the CSK-ppt fraction is replaced with exogenous CSA during incubation in this cell-free system. To verify the exchange of CSA, the CSK-ppt fraction prepared from UV-irradiated CSA-HA/CS-A cells and the CSK-sup fraction containing GFP-tagged CSA were used (Fig. 2B). CSA-GFP was competent for translocation in this system (Fig. 2B, lane 6). The amount of CSA-HA retained in the DNase I-insoluble fraction decreased when the CSK-ppt fraction was incubated with the CSK-sup fraction containing CSA-GFP. The decrease of CSA-HA was more pronounced when both fractions were incubated at 30°C (compare lanes 4 and 5 with 2). In contrast, the amount of CSA-GFP retained in the DNase I-insoluble fraction was increased at 30°C (lane 4). These results support the above hypothesis that CSA in the CSK-ppt fraction is exchangeable.
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322-396, 282-374, 348-374, and Q106P are mutant forms of CSA derived from CS patients. The CSA patient CS2IAF is homozygous for the Tyr 322-to-stop mutation (322-396) (13, 22). Two deletions removing either exon 11 (348-374) or exons 10 and 11 (282-374) were identified in CSA cDNA isolated from CS-A patients CS5BR and CS6BR and are presumably derived from a homozygous mutation at a splice donor site (13). An amino acid substitution at Gln 106 to Pro (Q106P) was identified in the CS-A patient CS2SE (29). The transfectants expressing each of these mutant CSAs were for convenience designated 322-396 cells, 282-374 cells, 348-374 cells, and Q106P cells, respectively. In addition, we established transfectants expressing mutant CSA with an in-frame deletion of exons 2 and 3 (27-92 cells), with a truncated N terminus (2-4 and 2-7 cells), and with a truncated C terminus (365-396 and 385-396 cells). All the WD-40 repeat motifs are preserved in these terminally truncated mutants. We chose the transfectants that expressed a normal amount of CSA protein.
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322-396 and 282-374 cells were very sensitive to UV irradiation, as were the parental CS3BESV cells. Q106P cells were also extremely sensitive to UV irradiation (29). 348-374, 27-92, 2-7, and 385-396 cells were slightly more resistant than the parental CS3BESV cells. 2-4 and 365-396 cells showed less UV hypersensitivity than 348-374, 27-92, 2-7, and 385-396 cells. Thus, all the transfectants expressing mutant CSA showed more or less UV hypersensitivity. Next, the recovery of RNA synthesis after UV irradiation, which is an index of TCR, was measured. RNA synthesis in WI38VA13 and CS3BESV cells at 2 h after UV irradiation was about 35% and 20% of that in nonirradiated cells, respectively (Fig. 3C). The RNA synthesis in WI38VA13 cells and the transfectant expressing wild-type CSA at 16 and 24 h after UV irradiation had recovered to about 90% of the level in nonirradiated cells, but in the parental CS3BESV cells, no such recovery was observed. None of the transfectants expressing mutant CSA showed a recovery of RNA synthesis at 16 and 24 h after UV irradiation. Thus, all the CSA mutants were deficient in TCR.
We then examined whether the mutant CSA proteins were translocated to the nuclear matrix after UV irradiation by using the cell-free method. CSK-sup fractions were prepared from CS3BESV cells expressing various types of mutant CSA (Fig. 4). When these fractions were incubated with the CSK-ppt fractions from nonirradiated CS3BESV cells, no mutant CSA proteins were retained in the DNase I-insoluble fractions. When the CSK-sup fractions prepared from 365-396 and 385-396 cells, as well as wild-type cells, were incubated with the CSK-ppt fractions derived from UV-irradiated CS3BESV cells, CSA proteins were detected in the DNase I-insoluble fractions. When the CSK-sup fraction was prepared from 2-4 cells, a small amount of CSA was translocated to the DNase I-insoluble fraction. However, no CSA protein was retained in the DNase I-insoluble fractions when the CSK-sup fractions derived from 2-7, 348-374, 282-374, 27-92, Q106P, and 322-396 cells were used.
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282-374, 348-374, Q106P, and 365-396) were also detected in the cytoplasm. On the other hand, wild-type CSA protein was retained in the nuclei even after the pretreatment with CSK-Triton buffer, as described previously (15), but none of the mutant CSA proteins were. Neither the wild-type nor any of the mutant CSA proteins was retained in the nuclei when the cells were not irradiated with UV (data not shown). Next, the translocation of the mutant CSA proteins to the nuclear matrix was monitored by immunoblotting of fractionated cellular proteins (Fig. 5B). In the nonirradiated cells, CSA was mainly detected in the fractions extracted with CSK-Triton buffer (fractions 1 and 2). As described previously (15), and consistent with the results of the immunofluorescence analysis, wild-type CSA was detected in the nuclear-matrix fraction (fraction 9) after UV irradiation. Three truncated CSA proteins, 365-396, 385-396, and 2-4, were also translocated to the nuclear-matrix fraction after UV irradiation, although the amounts translocated to the nuclear matrix appeared to be less than that of wild-type CSA protein. The other mutant CSA proteins were not translocated to the nuclear-matrix fraction after UV irradiation. The difference between the results by immunofluorescence staining and immunoblotting could be caused by the difference in the sensitivities of these two detection methods.
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Involvement of TFIIH in the UV-induced translocation of CSA. TFIIH consists of 10 subunits and is involved in TCR, as well as GGR and transcription. Hereditary mutations in the subunits, XPB, XPD, and TTDA, are associated with three UV-sensitive disorders: XP, XP combined with CS, and trichothiodystrophy (TTD) (2, 9). We examined whether TFIIH participates in the CSA translocation by using the cell-free system. We prepared CSK-ppt fractions from various cell lines mutated in XPD, XPB, or TTDA and tested their competence for the UV-induced CSA translocation (Fig. 6). When CSK-ppt fractions were prepared from UV-irradiated XPD- or XPB-deficient cells (XP6BE [XP-D], XPCS2 [XP-D/CS], TTD1RO [XP-D/TTD], and XPCS2BA [XP-B/CS]), a small amount of CSA was retained in the DNase I-insoluble fractions, while a significant amount of CSA was retained when the CSK-ppt fraction from XPD- or XPB-corrected cells (XPD/XP6BE, XPD/XPCS2, XPD/TTD1RO [46], and XPB/XPCS2BA [14], respectively) was used. On the other hand, there were no significant differences in the retention of CSA between TTDA-deficient (TTD1BR) and corrected (TTDA/TTD1BR [9]) cells. It was reported that the steady-state level of TFIIH in TTD1BR cells was reduced but the main enzymatic activities of TFIIH in TTD1BR cells were globally intact (45). These results suggest that functional TFIIH is involved in the UV-induced translocation of CSA to the nuclear matrix and that the CSA translocation is not correlated with phenotypes caused by mutations in the subunits of TFIIH.
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DISCUSSION |
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We have shown that CSA forms a ubiquitin ligase complex with DDB1, cullin 4A, Roc 1, and the COP9 signalosome (10). However, our cell-free system with a purified CSA complex does not support a ubiquitination reaction. In the absence of ATP, the translocation was detected (Fig. 1E). These results suggest that the ubiquitin ligase activity of the CSA complex is not involved in the translocation.
In order to identify which regions in CSA are required for the UV-induced translocation to the nuclear matrix, we established CS-A (CS3BESV) cells stably expressing various types of mutant CSA (Fig. 3A). The UV-induced translocation to the nuclear matrix was examined with both the cell-free system and conventional fractionation and immunofluorescence methods. As shown in Fig. 3 to 5, the CSA mutants derived from CS-A patients (282-374, 348-374, 322-396, and Q106P) were not translocated to the nuclear matrix. Moreover, the transfectants expressing these mutants showed UV hypersensitivity and were deficient in the recovery of RNA synthesis after UV irradiation (Fig. 3B and C). CSA protein has five WD-40 repeat motifs and is thought to form a ß-propeller structure. All the mutants derived from CS-A patients used here lacked the last WD-40 repeat motif or had a substitution of amino acid residues in the second motif. It is conceivable that these mutants could not form the proper structure and so were deficient in the UV-induced translocation to the nuclear matrix and TCR function. In addition, the CSA mutant 27-92 lacks the first WD-40 repeat motif and the phenotypes of 27-92 cells were similar to those of the CS-A patients' cells, as expected. On the other hand, the four CSA mutants with terminal deletions (2-4, 2-7, 365-396, and 385-396) retained all the WD-40 repeat motifs and are assumed to form a ß-propeller structure. In fact, the 2-4, 365-396, and 385-396 CSA mutants were translocated to the nuclear matrix to some extent, and these mutant cells showed partial resistance to UV, although the 2-7 cells were deficient in UV-induced translocation. These results suggest that the WD-40 repeat motifs and some N-terminal regions are indispensable for the UV-induced translocation of CSA and TCR while some C-terminal regions are dispensable for the translocation to the nuclear matrix but required for full TCR activity.
The C-terminal region (amino acid residues 365 to 396) of CSA is highly conserved and has clusters of acidic amino acid residues. 365-396 lacks the entire C-terminal region, while 385-396 retains half of it. However, 365-396 cells were less UV-sensitive than 385-396 cells. These results suggest that incomplete deletion of the C-terminal region has negative effects on the viability of cells after UV irradiation, although there were no significant differences in the recovery of RNA synthesis after UV irradiation between 365-396 and 385-396 cells.
We showed, using a cell-free system, that TFIIH, as well as CSB, is involved in the UV-induced translocation of CSA. In NER, XPB and XPD helicases in TFIIH cooperate in the unwinding of duplex DNA around lesions to allow the recruitment of the NER factors XPA, RPA, XPG, and XPF-ERCC1 to the damaged site. In this study, we tested three cell lines with mutations in the XPD gene. All the mutations found in the cell lines were shown to impair XPD helicase activity (7). Impairment of TFIIH function in the XPB-deficient cell line has also been reported (44). In contrast, functional TFIIH was shown to exist in the TTDA-deficient cell line, although the steady-state level of TFIIH was reduced (45). This could be a reason why the UV-induced translocation of CSA was not impaired in the TTDA-deficient cell line. We do not yet know the molecular mechanism of TFIIH function in the CSA translocation process. It has been reported that TFIIH interacts with the RNA polymerase II-CSB-DNA-RNA complex (38) and remodels the stalled RNA polymerase II in a manner dependent on the hydrolysis of ATP (30). These results suggest that TFIIH is involved in the UV-induced translocation of CSA to the nuclear matrix by remodeling the stalled RNA polymerase II in collaboration with CSB.
We showed that the CSK-ppt fraction was competent for RNA synthesis even if it was prepared from UV-irradiated cells and that the UV-induced translocation of CSA to the DNase I-insoluble fraction was dependent on RNA synthesis in the CSK-ppt fraction. It has been shown that the synthesis of RNA by RNA polymerase II is completely blocked at UV-damaged sites (6, 23, 33). These results suggest that the blockage of elongating RNA polymerase II is necessary for the translocation of CSA.
We also showed that the pretreatment of the CSK-ppt fraction with either DNase I or a high-salt buffer reduced the UV-induced translocation of CSA, indicating that the disruption of the chromatin structure resulted in the decrease in translocation and, therefore, that alteration of the chromatin structure at the RNA polymerase II stall sites is a prerequisite for the UV-induced translocation of CSA. It has been reported that CSB interacts with RNA polymerase II engaged in ternary complexes containing DNA and RNA in vitro (30, 37) and that the interaction between CSB and the transcription elongation machinery is stabilized by the presence of DNA damage in vivo (40). It has also been reported that CSB wraps DNA around its surface, dependent on ATP binding (1), and has ATP-dependent chromatin-remodeling activity in vitro (5). Taken together, these results suggest that CSB and TFIIH cooperate to alter the chromatin structure at the DNA-damaged site where the elongating RNA polymerase II is stalled so that CSA can be recruited to the RNA polymerase II stall site in the nuclear matrix.
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ACKNOWLEDGMENTS |
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This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), by the Solution Oriented Research for Science and Technology program of the Japan Science and Technology Agency, and also by the Takeda Science Foundation.
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FOOTNOTES |
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Published ahead of print on 22 January 2007.
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-32P]UTP into the acid-insoluble materials. (D) CSK-ppt fractions were prepared immediately after UV irradiation or nonirradiation and incubated with purified CSA complex in the presence (lanes 1 and 2) or absence (lanes 3 and 4) of NTPs at 30°C for 1 h. Ten percent of the CSA complex was loaded in lane 1.