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Repair of DNA lesions in chromosomal DNA
DNA Repair 4 (2005) 919–925
Mini review
Repair of DNA lesions in chromosomal DNA Impact of chromatin structure and Cockayne syndrome proteins Maria Fousteri, Anneke van Hoffen, Hana Vargova, Leon H.F. Mullenders ∗ Department of Toxicogenetics, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Accepted 18 April 2005 Available online 14 June 2005
Abstract Decondensation of chromatin is essential to facilitate access to DNA metabolizing processes such as transcription and DNA repair. Disruption of histone–DNA contacts by histone modification or by ATP dependent chromatin remodelling allows DNA-binding proteins to compete with histones for DNA. The efficiency of global genome nucleotide excision repair (GGR) that removes a variety of helix distorting DNA lesions is known to be affected by chromatin structure most notably demonstrated by the slow repair of heterochromatin. In addition, the efficiency of GGR to repair lesions in transcriptionally active genes requires functional CSA and B proteins. We found that repair of UV-photolesions in both strands of the active adenosine deaminase gene was delayed in CS cells when compared to normal human fibroblasts. We suggest that the lack of transcription recovery characteristic for CS cells exposed to DNA damaging agents, might lead to changes in the chromatin structure of active genes, causing less efficient repair of lesions in these genes when compared to normal cells. © 2005 Published by Elsevier B.V. Keywords: Chromatin remodelling; Nucleotide excision repair; Histone modification; Cockayne syndrome proteins
Contents 1. 2. 3. 4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatin alterations and DNA metabolizing processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Repair of DNA lesions in chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A role for the Cockayne syndrome B protein in GGR? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The condensation of DNA into chromatin is achieved in a way so as this DNA protein complex facilitates DNA metabolizing reactions such as replication, transcription and repair. Yet it is evident from in vitro reactions that the interactions of DNA with histone proteins, i.e. the wrapping of approximately 147 basepairs of DNA around a histone octamer, imposes constraints to DNA dependent processes. Histones ∗
Corresponding author. Tel.: +31 71 5276126; fax: +31 71 5276173. E-mail address: [email protected] (L.H.F. Mullenders).
1568-7864/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.dnarep.2005.04.011
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H1 and H5 can bind to linker DNA thereby making the DNA template relatively inaccessible for regulatory proteins. For example, the presence of linker histones in a template containing two nucleosomes, strongly inhibited in vitro transcription [1]. For DNA repair particularly for nucleotide excision repair (NER), convincing evidence has been presented that the recognition and repair of DNA lesions such as those induced by UV, is seriously hindered at least in vitro by their location in chromatin [2]. NER is able to remove a variety of structurally unrelated DNA lesions and operates via two subpathways, i.e. transcription coupled repair (TCR) and global genome repair
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(GGR). TCR is directly coupled to RNA polymerase II driven transcription resulting in selective repair of lesions in the transcribed strand of active genes whereas GGR is responsible for repair of lesions throughout the genome. In eukaryotic cells repair of DNA damage by NER occurs efficiently throughout the genome and at all stages of the cell cycle. Moreover, there is clear evidence that rate and efficiency of repair might be influenced by chromatin context, the structure of DNA lesions and the repair pathway involved [3]. Nevertheless, the timely and fast repair of DNA damage in vivo reveals that the repair machinery is somehow capable of recognizing small numbers of structural different DNA lesions in their chromatin environment and coping with the DNA protein interactions that inhibit processing of the damage at least in vitro. It is obvious that in vivo alterations of chromatin either by the DNA lesion itself, by specific chromatin remodelers such as histone acetyltransferases [4] or by DNA metabolizing processes such as transcription, are required to facilitate damage recognition and repair. In addition, specific sequences such as matrix- or scaffold attachment regions might influence repair by enhancing the accessibility of the chromatin [5,6] to repair factors. At the nucleosome level chromatin can undergo several types of modification that influence the compactness of the DNA. The amino terminal, lysine-rich tails of the core histones are positioned on the outside of the nucleosome and are targets for acetylation. The binding of acetylated histones H3 and H4 to either H2A or H2B and/or to DNA is less stable, thereby facilitating transcription factor assembly at promoter sites and progression of the transcription machinery along the DNA [7,8]. Acetylation of histones has been shown to stimulate nucleotide excision repair. When human fibroblasts were treated with sodium butyrate (a fatty acid that inhibits histone deacetylase) UV-induced repair synthesis was stimulated two-fold and occurred preferentially in hyperacetylated nucleosomes [9]. The enhanced incorporation was due to increased repair events reflected by a two-fold stimulation of incision activity and a similar increase in the removal of sites sensitive to a UV endonuclease that cuts DNA at cyclobutane pyrimidine dimers-CPDs). Most likely, the more open chromatin structure in hyperacetylated nucleosomes facilitates repair [10]. Similar results were obtained with XP-C cells [11]that perform transcription-coupled repair (TCR) only [12] thereby demonstrating that TCR of UV photolesions is stimulated by acetylation of histones in transcriptionally active genes as well. The similar extent of increase of repair in normal human and XP-C cells suggests that acetylation of histones is a stimulating factor for repair of UV-photolesions and other lesions removed via both TCR and GGR.
2. Chromatin alterations and DNA metabolizing processes Methylation of cytosines in DNA has consequences for transcription and chromatin structure. Inactivation of genes
in cultured mammalian cells is accompanied by methylation of CpG islands [13]. Studies by Boyes and Bird [14] revealed the existence of two proteins, i.e. MeCP1 and MeCP2, that specifically bind to methylated sequences [15,16]. MeCP1 only binds when a stretch of CpG is methylated, and MeCP2 binds to sparsely methylated DNA, but only when a CpG is methylated on both DNA strands. A model has been proposed in which the binding of these proteins sterically interferes with the binding of transcription factors and thereby inhibits transcription initiation [17]. The presence of binding proteins in methylated sequences might interfere with the binding of repair proteins to those sequences and thereby inhibit repair of lesions in these DNA regions. Since basal transcription factor TFIIH is also an essential NER component, the presence of this complex at active genes might favor rapid repair of lesions in those sequences and vice versa, when accumulation of transcription factors is inhibited by methylation of the promoter region, TFIIH will not be directly available for NER at that site. Indications that methylation and associated protein interactions in heterochromatin might influence the efficiency of GGR come from studies that focused on repair of DNA lesions in specific genes, by comparing housekeeping genes with X-chromosomal inactive genes (see below). Whereas core histone acetylation and CpG-methylation directly or indirectly influence transcription initiation, other chromatin modifications are connected with transcription elongation. Ubiquitination of specifically histone H2B is dependent on ongoing transcription [18]. VanHolde et al. [19] proposed that histones H2A and H2B have to be transiently released in order to let the transcription machinery pass by, and that this can be stimulated by ubiquitination of H2B. A similar explanation was given for the observed phosphorylation of histone H1b that is coupled to ongoing transcription [20]. H1b can only be phosphorylated when dissociated from chromatin and this function relates to the preservation of decondensed chromatin in order to enable continued transcription [21]. The open chromatin structure in actively transcribed genes, caused by ubiquitination and phosphorylation of histone proteins, might favor efficient recognition and repair of DNA damage even in the non-transcribed strand. Ubiquitination has been directly implicated in DNA repair by the identification of the yeast RAD6 gene product as an ubiquitin conjugating protein. Moreover, the identification of the repair proteins DDB2 (UV-damaged DNA binding protein 2, involved in GGR) and CSA (Cockayne syndrome gene product A, involved in TCR) as core components of CUL4A E3 Ub ligase complexes [22] provides additional evidence for a role of ubiquitination in NER that may well be critical for the regulation of both subpathways. CUL4A forms a complex together with ROC1/Rbx1, DDB1 and either DDB2 or CSA. CSA- and DDB2-Ub ligase complexes also associate with the COP9 signalosome (CSN), a Nedd8 isopeptidase and a known regulator of Cullin-based Ub ligases. Upon UV irradiation the DDB2-DDB1-CUL4A-ROC1/Rbx1 complex was bound to chromatin and removed from CSN, whereas CSN
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rejoined the complex at a later stage. However, in response to UV damage the CSA-DDB1-CUL4A-ROC1/Rbx1 Ubligase complex was tightly bound to CSN and it was also found to interact with the elongating hyperphosphorylated RNA polymerase II (RNAPIIo) [22]. This indicates that an UV-dependent differential regulation of the two complexes by CSN is required in the two distinct NER pathways. Furthermore, using a mouse temperature sensitive mutant (ts20) of a ubiquitin-activating enzyme (E1), Wani and coworkers [23] showed that inactivation of E1 significantly impaired NER. In the same study, using single cell analysis and local UV irradiation in the presence or absence of proteasome inhibitors they showed reduced recruitment of NER specific factors to damage sites underlying the importance of the ubiquitin-proteasome pathway for efficient NER, probably by facilitating the recruitment of repair factors to the damaged DNA.
3. Repair of DNA lesions in chromatin The efficiency of GGR might be influenced by several factors. DNA helix distortion due to DNA damage is likely the signal for lesion recognition by NER and therefore, lesions that cause severe distortion of the DNA helical structure, will be an attractive substrate for GGR. On the other hand, lesions that do not, or only mildly affect the DNA helical structure are recognized inefficiently. Second, inactive DNA sequences on the X-chromosomes have been shown to be heavily methylated and their chromatin structure is relatively insensitive to DNAse I treatment [24]. In more recent years detailed information has been gained on the types of histone modifications in heterochromatin [25]. It is conceivably that heterochromatic sequences would be relatively inaccessible to enzymes including repair proteins or be refractory to chromatin modifications required for efficient repair. Indeed, several reports have shown that repair of bulky DNA lesions such as CPDs and 6-4PP, deoxyguanosine-C8-aminofluorene and benzo(a)pyrene adducts was far less efficient in these inactive sequences than in non-transcribed strands of active genes or in autosomal inactive genes [12,26–29]. In GGR studies with naked DNA templates indicate that more than 30 proteins work together to consecutively execute the basic steps of NER, i.e. recognition of the DNA lesion, local unwinding of the DNA around the lesion, excision of the oligonucleotide containing the damage and finally repair synthesis and ligation of the gap [30]. Results of recent studies have identified the XPC-hHR23B complex as the principle initiator of GGR [31,30] and an essential role of UV-DDB to recognize DNA damage and to accelerate GGR [32,33]. UV-DDB is a heterodimer of the p48 and p127 protein products of the DDB2 and DDB1 genes, respectively. In vitro binding studies have revealed that the UV-DDB protein complex exhibits a high affinity for UV-induced DNA lesions and a moderate affinity for several other types of DNA lesions. DDB2 shares homology with chromatin reorganiz-
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ing proteins [34] and UV-DDB interacts with the CBP/p300 histone acetyl transferase [35] consistent with a function in chromatin remodeling to allow efficient repair in the vicinity of the lesion. In vivo studies have shown that p48 and p127 relocalize rapidly to sites containing UV-damaged DNA immediately after irradiation in the absence of functional XPC protein [36]. This observation is consistent with a role for UV-DDB in UV damage recognition, even before XPC is involved and a mechanism in which UV-DDB accelerates GGR by binding to the lesion, facilitating the recruitment of XPC and subsequent factors [37]. It is conceivably that the slow repair of DNA damage in heterochromatin could be due to limited damage recognition by hindered accessibility of UV-DDB to DNA lesions when compared to the more accessible euchromatin. It is evident that UV-DDB is able to interact with DNA damage in heterochromatin as UV–induced CPDs (entirely depending for their repair on UV-DDB) are removed from the inactive X-chromosomal gene 754 and that for coagulation factor IX. The interesting possibility that the efficiency of recruitment of UV-DDB to DNA damage differs for different genomic regions, awaits future research. Two other molecular entities associated with UV-DDB may contribute to the repair efficiency by triggering chromatin remodeling. The CBP/p300 histone acetyltransferase might modify multiple targets including substrates other than histones [4,38]. Heterochromatic specific modification of histones such as histone H3 K9 methylation [25] might interfere with acetylation and hence be inhibitory to DNA repair. Another activity is ubiquitination associated with DDB2 [22], since the E3 ubiquitin ligase activity of DDB2 complex is activated upon UV irradiation. This probably facilitates the access of repair components to otherwise inaccessible DNA lesions.
4. A role for the Cockayne syndrome B protein in GGR? Three rare hereditary human disorders have been shown to be associated with defective NER, i.e. xeroderma pigmentosum (XP), Cockayne syndrome (CS) and PIBIDS, the photosensitive form of trichothiodystrophy (TTD). The clinical and cellular features of CS pose challenging questions on the molecular mechanism underlying repair defects and disease. Clinical symptoms of CS are complex including growth retardation, progressive neurological abnormalities and photosensitivity [39]. In spite of normal levels of repair synthesis, CS cells are sensitive to the lethal effects of UV and unable to recover UV-inhibited DNA and RNA synthesis [40]. The lack of RNA synthesis recovery has been related to a specific defect in TCR [41]. In addition to the defect in TCR, repair of lesions in active genes by GGR was affected in CS cells as well. This was manifested by the less efficient repair of CPD in both strands of the ADA gene in CS cells when compared to the non-transcribed strand of the ADA gene in normal cells [41]. A similar observation has been recently made for the
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Fig. 1. Autoradiograms showing removal of 6-4PPs from the active ADA gene and the inactive 754 gene in Cockayne syndrome group A (CS-A) and group B (CS-B) fibroblasts irradiated with 30 J/m2 UV. Removal of 6-4PPs was measured in the EcoR1 restriction fragment of the active ADA gene (both strands of this fragment are transcribed) and the inactive 754 gene using radioactively labelled DNA probes.
repair of CPDs in transcriptional active genes in UVSS [42] a syndrome closely linked to CS by a common gene mutation [43]. The delayed repair of CPDs was not seen in inactive X-chromosomal genes in CS cells, consistent with the observation that global repair of CPDs in CS cells could not be distinguished from that in normal cells. Retarded repair is also observed for 6-4PP photolesions that are removed extremely fast from active genes without any strand-specificity, even in cells exposed to 30 J/m2 [12]. Induction and repair of 64PP was measured in cells belonging to CS complementation group A (CS3BE and CS-SAIF) and group B (CS1AN and 11961). The results were essentially the same for the two cell strains from each group investigated, and pooled data from each pair are presented. From both complementation groups representative autoradiograms are shown in Fig. 1. The inactive 754 gene is repaired with an efficiency similar to that in normal cells. Taken together, the results demonstrate that the CS defect extends beyond the level of TCR and that the rate of repair of lesions that are not subject to TCR is delayed as well. Fig. 2 shows the percent removal of 6-4PPs from the active ADA gene (Fig. 2A) and the inactive 754 gene (Fig. 2B) during the first 8 h after irradiation. It is clear that repair of 6-4PPs in the ADA gene of CS cells belonging to either complementation group A or B is retarded compared to normal cells. However, the delay in repair of 6-4PPs is more pronounced in CS group B cells. The differences in the repair rate of 6-4PP in the active ADA gene between normal and CS cells are most pronounced during the first 2–4 h. Repair of 6-4PPs in the two genes in both normal and CS cells was completed after 24 h (data not shown). It should be noted that both strands of the EcoRI fragment of the ADA gene are transcribed. The delayed repair of DNA damage by global genome repair (GGR) might be a general feature of CS cells. A reduced rate of repair at the ADA gene was also observed when CS cells were treated with NA-AAF and assayed for removal of deoxyguanosine-C8-acetyl-aminofluorene (dG-
Fig. 2. Graphs showing removal of 6-4PPs from the active ADA gene and the inactive 754 gene in Cockayne syndrome group A (CS-A) and group B (CS-B) and normal human fibroblasts irradiated with 30 J/m2 UV. For all three-cell types the data of the different cell strains that were investigated are pooled, since essentially no differences were observed between the cell strains within one group. (A) Repair of 6-4PP in the active ADA gene: (䊉) normal human fibroblasts (VH), () CS-A cells, and () CS-B cells. (B) Repair of 6-4PP in the inactive 754 gene: (䊉) normal human fibroblasts (VH), () CS-A cells, and () CS-B cells. Bars represent standard errors of the mean (S.E.M.).
C8-AAF) adducts [44]. Explanations for the reduced excision of DNA damage from active genes by GGR in CS cells relate to alterations of the chromatin structure of these genes in CS cells exposed to DNA damage and to the nuclear organization of NER. The fact that the phenomenon has been observed in CSA and CSB deficient cells, suggests that the transcription response following DNA damage plays a role. Inhibition of transcription might affect chromatin structure in several ways. In mammalian cells ubiquitinated histone H2B (u-H2B) is highly associated with transcriptionally active chromatin. Davie and Murphy [45] showed that inhibition of transcription in human cells by treatment with actinomycin D strongly reduced the ubiquitination of histone H2B and they suggested that u-H2B would impede nucleosome refolding. It is tentative to speculate that UV-inhibited transcription will give rise to a similar decrease of H2B ubiquitination, thereby
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affecting the chromatin conformation of active genes. Particularly, the lack of transcription recovery in CS cells could lead to a constitutively less open chromatin configuration of RNA polymerase II transcribed genes, reducing the efficiency of repair in these genomic regions. The acetylation status of histones also has been related to transcription and the accessibility of chromatin. Acetylation weakens the DNA binding efficiency of histone proteins and enhances the accessibility of DNA to other proteins. Histone acetyltransferases are targeted to the RNA polymerase II transcription apparatus and thus generate a close coupling of histone acetylation to transcription activity. Prolonged inhibition of transcription in CS cells by UV irradiation, eventually through deficient H2B ubiquitination, might lead to impaired histone acetylation, the alteration of chromatin, the configuration of active genes and subsequently the reduced repair of DNA lesions by GGR. It has been proposed that certain types of oxidative DNA damage, i.e. 8-oxo-guanine [46] might interfere with transcription elongation and that the repair of these damages requires the CSB protein [47]. The latter is consistent with the slight sensitivity of CSB deficient cells to ionizing radiation that induces a multiplicity of damages including 8-oxoguanine. The sensitivity towards oxidative damage induced by ionising radiation or the chemical agent paraquat, is more pronounced in embryo fibroblasts derived from CSB deficient mice [48]. In contrast to CSB−/− fibroblasts, CSA−/− fibroblasts are not hypersensitive to X-rays or paraquat. There are some notions that mutations in the CSB gene not only affect TCR but also other processes. As abovementioned, oxidative damage such as 8-oxo-guanine poses weak blocks to transcription and there is accumulating evidence that transcriptional bypass of these lesions requires CSB or its homologue Rad26 in respectively human cells [46] and S. cerevisiae [49]. In addition, evidence has been presented suggesting that CSB might have an impact on GGR of 8-oxoguanine. This is impaired in human CSB deficient cells [50] and CSB deficient mouse cells accumulate 8-oxo-guanine induced by the photosensitiser RO 19-8022 and visible light [51]. The CSB gene encodes a 168 kDa protein containing a helicase domain that shows strong homology with similar domains in SWI2/SNF2-like proteins. The CSB protein, like most members of this family, shows ATPase and DNA binding activity, but not helicase activity. Interestingly and in contrast to CSA, the CSB protein possesses chromatin remodelling activity and binds to histone tails [52] Although nuclear extracts of CSB deficient cells are impaired in removal of 8oxo guanine from oligonucleotides without proper chromatin structure, it is tempting to speculate that at least some of the observed effects are related to a chromatin remodelling function of CSB. In support of this we found a general reduction of repair synthesis in CSB deficient cells after ionising radiation, indicating a more general defect in the GGR of oxidative damage (unpublished data). It is possible that the CSB protein is required for subtle chromatin modifications to facilitate lesion recognition including those induced by UV. Interest-
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ingly, at least for chemical and UV induced bulky lesions, a deficiency of CSB has no major impact on repair of these lesions in heterochromatin.
Acknowledgements The UvrABC proteins used in this study were obtained from Dr. N. Goosen, Laboratory of Molecular Genetics, Leiden University, The Netherlands. This study was supported by the EU project ‘DNA Repair’ and by the Netherlands Organisation for Health Research and Development (NWO) ‘Nucleotide excision repair in vivo: a paradigm for chromatin-associated processes’ with project nos. CT2003503618 and 912-03-012.
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Mini review
Repair of DNA lesions in chromosomal DNA Impact of chromatin structure and Cockayne syndrome proteins Maria Fousteri, Anneke van Hoffen, Hana Vargova, Leon H.F. Mullenders ∗ Department of Toxicogenetics, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Accepted 18 April 2005 Available online 14 June 2005
Abstract Decondensation of chromatin is essential to facilitate access to DNA metabolizing processes such as transcription and DNA repair. Disruption of histone–DNA contacts by histone modification or by ATP dependent chromatin remodelling allows DNA-binding proteins to compete with histones for DNA. The efficiency of global genome nucleotide excision repair (GGR) that removes a variety of helix distorting DNA lesions is known to be affected by chromatin structure most notably demonstrated by the slow repair of heterochromatin. In addition, the efficiency of GGR to repair lesions in transcriptionally active genes requires functional CSA and B proteins. We found that repair of UV-photolesions in both strands of the active adenosine deaminase gene was delayed in CS cells when compared to normal human fibroblasts. We suggest that the lack of transcription recovery characteristic for CS cells exposed to DNA damaging agents, might lead to changes in the chromatin structure of active genes, causing less efficient repair of lesions in these genes when compared to normal cells. © 2005 Published by Elsevier B.V. Keywords: Chromatin remodelling; Nucleotide excision repair; Histone modification; Cockayne syndrome proteins
Contents 1. 2. 3. 4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatin alterations and DNA metabolizing processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Repair of DNA lesions in chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A role for the Cockayne syndrome B protein in GGR? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The condensation of DNA into chromatin is achieved in a way so as this DNA protein complex facilitates DNA metabolizing reactions such as replication, transcription and repair. Yet it is evident from in vitro reactions that the interactions of DNA with histone proteins, i.e. the wrapping of approximately 147 basepairs of DNA around a histone octamer, imposes constraints to DNA dependent processes. Histones ∗
Corresponding author. Tel.: +31 71 5276126; fax: +31 71 5276173. E-mail address: [email protected] (L.H.F. Mullenders).
1568-7864/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.dnarep.2005.04.011
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H1 and H5 can bind to linker DNA thereby making the DNA template relatively inaccessible for regulatory proteins. For example, the presence of linker histones in a template containing two nucleosomes, strongly inhibited in vitro transcription [1]. For DNA repair particularly for nucleotide excision repair (NER), convincing evidence has been presented that the recognition and repair of DNA lesions such as those induced by UV, is seriously hindered at least in vitro by their location in chromatin [2]. NER is able to remove a variety of structurally unrelated DNA lesions and operates via two subpathways, i.e. transcription coupled repair (TCR) and global genome repair
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(GGR). TCR is directly coupled to RNA polymerase II driven transcription resulting in selective repair of lesions in the transcribed strand of active genes whereas GGR is responsible for repair of lesions throughout the genome. In eukaryotic cells repair of DNA damage by NER occurs efficiently throughout the genome and at all stages of the cell cycle. Moreover, there is clear evidence that rate and efficiency of repair might be influenced by chromatin context, the structure of DNA lesions and the repair pathway involved [3]. Nevertheless, the timely and fast repair of DNA damage in vivo reveals that the repair machinery is somehow capable of recognizing small numbers of structural different DNA lesions in their chromatin environment and coping with the DNA protein interactions that inhibit processing of the damage at least in vitro. It is obvious that in vivo alterations of chromatin either by the DNA lesion itself, by specific chromatin remodelers such as histone acetyltransferases [4] or by DNA metabolizing processes such as transcription, are required to facilitate damage recognition and repair. In addition, specific sequences such as matrix- or scaffold attachment regions might influence repair by enhancing the accessibility of the chromatin [5,6] to repair factors. At the nucleosome level chromatin can undergo several types of modification that influence the compactness of the DNA. The amino terminal, lysine-rich tails of the core histones are positioned on the outside of the nucleosome and are targets for acetylation. The binding of acetylated histones H3 and H4 to either H2A or H2B and/or to DNA is less stable, thereby facilitating transcription factor assembly at promoter sites and progression of the transcription machinery along the DNA [7,8]. Acetylation of histones has been shown to stimulate nucleotide excision repair. When human fibroblasts were treated with sodium butyrate (a fatty acid that inhibits histone deacetylase) UV-induced repair synthesis was stimulated two-fold and occurred preferentially in hyperacetylated nucleosomes [9]. The enhanced incorporation was due to increased repair events reflected by a two-fold stimulation of incision activity and a similar increase in the removal of sites sensitive to a UV endonuclease that cuts DNA at cyclobutane pyrimidine dimers-CPDs). Most likely, the more open chromatin structure in hyperacetylated nucleosomes facilitates repair [10]. Similar results were obtained with XP-C cells [11]that perform transcription-coupled repair (TCR) only [12] thereby demonstrating that TCR of UV photolesions is stimulated by acetylation of histones in transcriptionally active genes as well. The similar extent of increase of repair in normal human and XP-C cells suggests that acetylation of histones is a stimulating factor for repair of UV-photolesions and other lesions removed via both TCR and GGR.
2. Chromatin alterations and DNA metabolizing processes Methylation of cytosines in DNA has consequences for transcription and chromatin structure. Inactivation of genes
in cultured mammalian cells is accompanied by methylation of CpG islands [13]. Studies by Boyes and Bird [14] revealed the existence of two proteins, i.e. MeCP1 and MeCP2, that specifically bind to methylated sequences [15,16]. MeCP1 only binds when a stretch of CpG is methylated, and MeCP2 binds to sparsely methylated DNA, but only when a CpG is methylated on both DNA strands. A model has been proposed in which the binding of these proteins sterically interferes with the binding of transcription factors and thereby inhibits transcription initiation [17]. The presence of binding proteins in methylated sequences might interfere with the binding of repair proteins to those sequences and thereby inhibit repair of lesions in these DNA regions. Since basal transcription factor TFIIH is also an essential NER component, the presence of this complex at active genes might favor rapid repair of lesions in those sequences and vice versa, when accumulation of transcription factors is inhibited by methylation of the promoter region, TFIIH will not be directly available for NER at that site. Indications that methylation and associated protein interactions in heterochromatin might influence the efficiency of GGR come from studies that focused on repair of DNA lesions in specific genes, by comparing housekeeping genes with X-chromosomal inactive genes (see below). Whereas core histone acetylation and CpG-methylation directly or indirectly influence transcription initiation, other chromatin modifications are connected with transcription elongation. Ubiquitination of specifically histone H2B is dependent on ongoing transcription [18]. VanHolde et al. [19] proposed that histones H2A and H2B have to be transiently released in order to let the transcription machinery pass by, and that this can be stimulated by ubiquitination of H2B. A similar explanation was given for the observed phosphorylation of histone H1b that is coupled to ongoing transcription [20]. H1b can only be phosphorylated when dissociated from chromatin and this function relates to the preservation of decondensed chromatin in order to enable continued transcription [21]. The open chromatin structure in actively transcribed genes, caused by ubiquitination and phosphorylation of histone proteins, might favor efficient recognition and repair of DNA damage even in the non-transcribed strand. Ubiquitination has been directly implicated in DNA repair by the identification of the yeast RAD6 gene product as an ubiquitin conjugating protein. Moreover, the identification of the repair proteins DDB2 (UV-damaged DNA binding protein 2, involved in GGR) and CSA (Cockayne syndrome gene product A, involved in TCR) as core components of CUL4A E3 Ub ligase complexes [22] provides additional evidence for a role of ubiquitination in NER that may well be critical for the regulation of both subpathways. CUL4A forms a complex together with ROC1/Rbx1, DDB1 and either DDB2 or CSA. CSA- and DDB2-Ub ligase complexes also associate with the COP9 signalosome (CSN), a Nedd8 isopeptidase and a known regulator of Cullin-based Ub ligases. Upon UV irradiation the DDB2-DDB1-CUL4A-ROC1/Rbx1 complex was bound to chromatin and removed from CSN, whereas CSN
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rejoined the complex at a later stage. However, in response to UV damage the CSA-DDB1-CUL4A-ROC1/Rbx1 Ubligase complex was tightly bound to CSN and it was also found to interact with the elongating hyperphosphorylated RNA polymerase II (RNAPIIo) [22]. This indicates that an UV-dependent differential regulation of the two complexes by CSN is required in the two distinct NER pathways. Furthermore, using a mouse temperature sensitive mutant (ts20) of a ubiquitin-activating enzyme (E1), Wani and coworkers [23] showed that inactivation of E1 significantly impaired NER. In the same study, using single cell analysis and local UV irradiation in the presence or absence of proteasome inhibitors they showed reduced recruitment of NER specific factors to damage sites underlying the importance of the ubiquitin-proteasome pathway for efficient NER, probably by facilitating the recruitment of repair factors to the damaged DNA.
3. Repair of DNA lesions in chromatin The efficiency of GGR might be influenced by several factors. DNA helix distortion due to DNA damage is likely the signal for lesion recognition by NER and therefore, lesions that cause severe distortion of the DNA helical structure, will be an attractive substrate for GGR. On the other hand, lesions that do not, or only mildly affect the DNA helical structure are recognized inefficiently. Second, inactive DNA sequences on the X-chromosomes have been shown to be heavily methylated and their chromatin structure is relatively insensitive to DNAse I treatment [24]. In more recent years detailed information has been gained on the types of histone modifications in heterochromatin [25]. It is conceivably that heterochromatic sequences would be relatively inaccessible to enzymes including repair proteins or be refractory to chromatin modifications required for efficient repair. Indeed, several reports have shown that repair of bulky DNA lesions such as CPDs and 6-4PP, deoxyguanosine-C8-aminofluorene and benzo(a)pyrene adducts was far less efficient in these inactive sequences than in non-transcribed strands of active genes or in autosomal inactive genes [12,26–29]. In GGR studies with naked DNA templates indicate that more than 30 proteins work together to consecutively execute the basic steps of NER, i.e. recognition of the DNA lesion, local unwinding of the DNA around the lesion, excision of the oligonucleotide containing the damage and finally repair synthesis and ligation of the gap [30]. Results of recent studies have identified the XPC-hHR23B complex as the principle initiator of GGR [31,30] and an essential role of UV-DDB to recognize DNA damage and to accelerate GGR [32,33]. UV-DDB is a heterodimer of the p48 and p127 protein products of the DDB2 and DDB1 genes, respectively. In vitro binding studies have revealed that the UV-DDB protein complex exhibits a high affinity for UV-induced DNA lesions and a moderate affinity for several other types of DNA lesions. DDB2 shares homology with chromatin reorganiz-
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ing proteins [34] and UV-DDB interacts with the CBP/p300 histone acetyl transferase [35] consistent with a function in chromatin remodeling to allow efficient repair in the vicinity of the lesion. In vivo studies have shown that p48 and p127 relocalize rapidly to sites containing UV-damaged DNA immediately after irradiation in the absence of functional XPC protein [36]. This observation is consistent with a role for UV-DDB in UV damage recognition, even before XPC is involved and a mechanism in which UV-DDB accelerates GGR by binding to the lesion, facilitating the recruitment of XPC and subsequent factors [37]. It is conceivably that the slow repair of DNA damage in heterochromatin could be due to limited damage recognition by hindered accessibility of UV-DDB to DNA lesions when compared to the more accessible euchromatin. It is evident that UV-DDB is able to interact with DNA damage in heterochromatin as UV–induced CPDs (entirely depending for their repair on UV-DDB) are removed from the inactive X-chromosomal gene 754 and that for coagulation factor IX. The interesting possibility that the efficiency of recruitment of UV-DDB to DNA damage differs for different genomic regions, awaits future research. Two other molecular entities associated with UV-DDB may contribute to the repair efficiency by triggering chromatin remodeling. The CBP/p300 histone acetyltransferase might modify multiple targets including substrates other than histones [4,38]. Heterochromatic specific modification of histones such as histone H3 K9 methylation [25] might interfere with acetylation and hence be inhibitory to DNA repair. Another activity is ubiquitination associated with DDB2 [22], since the E3 ubiquitin ligase activity of DDB2 complex is activated upon UV irradiation. This probably facilitates the access of repair components to otherwise inaccessible DNA lesions.
4. A role for the Cockayne syndrome B protein in GGR? Three rare hereditary human disorders have been shown to be associated with defective NER, i.e. xeroderma pigmentosum (XP), Cockayne syndrome (CS) and PIBIDS, the photosensitive form of trichothiodystrophy (TTD). The clinical and cellular features of CS pose challenging questions on the molecular mechanism underlying repair defects and disease. Clinical symptoms of CS are complex including growth retardation, progressive neurological abnormalities and photosensitivity [39]. In spite of normal levels of repair synthesis, CS cells are sensitive to the lethal effects of UV and unable to recover UV-inhibited DNA and RNA synthesis [40]. The lack of RNA synthesis recovery has been related to a specific defect in TCR [41]. In addition to the defect in TCR, repair of lesions in active genes by GGR was affected in CS cells as well. This was manifested by the less efficient repair of CPD in both strands of the ADA gene in CS cells when compared to the non-transcribed strand of the ADA gene in normal cells [41]. A similar observation has been recently made for the
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Fig. 1. Autoradiograms showing removal of 6-4PPs from the active ADA gene and the inactive 754 gene in Cockayne syndrome group A (CS-A) and group B (CS-B) fibroblasts irradiated with 30 J/m2 UV. Removal of 6-4PPs was measured in the EcoR1 restriction fragment of the active ADA gene (both strands of this fragment are transcribed) and the inactive 754 gene using radioactively labelled DNA probes.
repair of CPDs in transcriptional active genes in UVSS [42] a syndrome closely linked to CS by a common gene mutation [43]. The delayed repair of CPDs was not seen in inactive X-chromosomal genes in CS cells, consistent with the observation that global repair of CPDs in CS cells could not be distinguished from that in normal cells. Retarded repair is also observed for 6-4PP photolesions that are removed extremely fast from active genes without any strand-specificity, even in cells exposed to 30 J/m2 [12]. Induction and repair of 64PP was measured in cells belonging to CS complementation group A (CS3BE and CS-SAIF) and group B (CS1AN and 11961). The results were essentially the same for the two cell strains from each group investigated, and pooled data from each pair are presented. From both complementation groups representative autoradiograms are shown in Fig. 1. The inactive 754 gene is repaired with an efficiency similar to that in normal cells. Taken together, the results demonstrate that the CS defect extends beyond the level of TCR and that the rate of repair of lesions that are not subject to TCR is delayed as well. Fig. 2 shows the percent removal of 6-4PPs from the active ADA gene (Fig. 2A) and the inactive 754 gene (Fig. 2B) during the first 8 h after irradiation. It is clear that repair of 6-4PPs in the ADA gene of CS cells belonging to either complementation group A or B is retarded compared to normal cells. However, the delay in repair of 6-4PPs is more pronounced in CS group B cells. The differences in the repair rate of 6-4PP in the active ADA gene between normal and CS cells are most pronounced during the first 2–4 h. Repair of 6-4PPs in the two genes in both normal and CS cells was completed after 24 h (data not shown). It should be noted that both strands of the EcoRI fragment of the ADA gene are transcribed. The delayed repair of DNA damage by global genome repair (GGR) might be a general feature of CS cells. A reduced rate of repair at the ADA gene was also observed when CS cells were treated with NA-AAF and assayed for removal of deoxyguanosine-C8-acetyl-aminofluorene (dG-
Fig. 2. Graphs showing removal of 6-4PPs from the active ADA gene and the inactive 754 gene in Cockayne syndrome group A (CS-A) and group B (CS-B) and normal human fibroblasts irradiated with 30 J/m2 UV. For all three-cell types the data of the different cell strains that were investigated are pooled, since essentially no differences were observed between the cell strains within one group. (A) Repair of 6-4PP in the active ADA gene: (䊉) normal human fibroblasts (VH), () CS-A cells, and () CS-B cells. (B) Repair of 6-4PP in the inactive 754 gene: (䊉) normal human fibroblasts (VH), () CS-A cells, and () CS-B cells. Bars represent standard errors of the mean (S.E.M.).
C8-AAF) adducts [44]. Explanations for the reduced excision of DNA damage from active genes by GGR in CS cells relate to alterations of the chromatin structure of these genes in CS cells exposed to DNA damage and to the nuclear organization of NER. The fact that the phenomenon has been observed in CSA and CSB deficient cells, suggests that the transcription response following DNA damage plays a role. Inhibition of transcription might affect chromatin structure in several ways. In mammalian cells ubiquitinated histone H2B (u-H2B) is highly associated with transcriptionally active chromatin. Davie and Murphy [45] showed that inhibition of transcription in human cells by treatment with actinomycin D strongly reduced the ubiquitination of histone H2B and they suggested that u-H2B would impede nucleosome refolding. It is tentative to speculate that UV-inhibited transcription will give rise to a similar decrease of H2B ubiquitination, thereby
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affecting the chromatin conformation of active genes. Particularly, the lack of transcription recovery in CS cells could lead to a constitutively less open chromatin configuration of RNA polymerase II transcribed genes, reducing the efficiency of repair in these genomic regions. The acetylation status of histones also has been related to transcription and the accessibility of chromatin. Acetylation weakens the DNA binding efficiency of histone proteins and enhances the accessibility of DNA to other proteins. Histone acetyltransferases are targeted to the RNA polymerase II transcription apparatus and thus generate a close coupling of histone acetylation to transcription activity. Prolonged inhibition of transcription in CS cells by UV irradiation, eventually through deficient H2B ubiquitination, might lead to impaired histone acetylation, the alteration of chromatin, the configuration of active genes and subsequently the reduced repair of DNA lesions by GGR. It has been proposed that certain types of oxidative DNA damage, i.e. 8-oxo-guanine [46] might interfere with transcription elongation and that the repair of these damages requires the CSB protein [47]. The latter is consistent with the slight sensitivity of CSB deficient cells to ionizing radiation that induces a multiplicity of damages including 8-oxoguanine. The sensitivity towards oxidative damage induced by ionising radiation or the chemical agent paraquat, is more pronounced in embryo fibroblasts derived from CSB deficient mice [48]. In contrast to CSB−/− fibroblasts, CSA−/− fibroblasts are not hypersensitive to X-rays or paraquat. There are some notions that mutations in the CSB gene not only affect TCR but also other processes. As abovementioned, oxidative damage such as 8-oxo-guanine poses weak blocks to transcription and there is accumulating evidence that transcriptional bypass of these lesions requires CSB or its homologue Rad26 in respectively human cells [46] and S. cerevisiae [49]. In addition, evidence has been presented suggesting that CSB might have an impact on GGR of 8-oxoguanine. This is impaired in human CSB deficient cells [50] and CSB deficient mouse cells accumulate 8-oxo-guanine induced by the photosensitiser RO 19-8022 and visible light [51]. The CSB gene encodes a 168 kDa protein containing a helicase domain that shows strong homology with similar domains in SWI2/SNF2-like proteins. The CSB protein, like most members of this family, shows ATPase and DNA binding activity, but not helicase activity. Interestingly and in contrast to CSA, the CSB protein possesses chromatin remodelling activity and binds to histone tails [52] Although nuclear extracts of CSB deficient cells are impaired in removal of 8oxo guanine from oligonucleotides without proper chromatin structure, it is tempting to speculate that at least some of the observed effects are related to a chromatin remodelling function of CSB. In support of this we found a general reduction of repair synthesis in CSB deficient cells after ionising radiation, indicating a more general defect in the GGR of oxidative damage (unpublished data). It is possible that the CSB protein is required for subtle chromatin modifications to facilitate lesion recognition including those induced by UV. Interest-
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ingly, at least for chemical and UV induced bulky lesions, a deficiency of CSB has no major impact on repair of these lesions in heterochromatin.
Acknowledgements The UvrABC proteins used in this study were obtained from Dr. N. Goosen, Laboratory of Molecular Genetics, Leiden University, The Netherlands. This study was supported by the EU project ‘DNA Repair’ and by the Netherlands Organisation for Health Research and Development (NWO) ‘Nucleotide excision repair in vivo: a paradigm for chromatin-associated processes’ with project nos. CT2003503618 and 912-03-012.
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