Chromatin rearrangements during nucleotide excision repair

Biochimie 81 (1999) 45−52 © Société française de biochimie et biologie moléculaire / Elsevier, Paris

Chromatin rearrangements during nucleotide excision repair Jonathan G. Moggs, Geneviève Almouzni* Dynamique de la Chromatine, Institut Curie, Section de Recherche, UMR 144, 75231 Paris, France (Received 21 June 1998; accepted 30 November 1998) Abstract — The removal of DNA damage from the eukaryotic genome requires DNA repair enzymes to operate within the complex environment of chromatin. We review the evidence for chromatin rearrangements during nucleotide excision repair and discuss the extent and possible molecular mechanisms of these rearrangements, focusing on events at the nucleosome level of chromatin structure. © Société française de biochimie et biologie moléculaire / Elsevier, Paris chromatin rearrangements / nucleotide excision repair

1. Introduction Eukaryotic genomes are exposed to a wide range of DNA damaging agents originating from both endogenous (e.g., hydrolytic and oxidative metabolites) and exogenous (e.g., sunlight, pollution, ionising radiation) sources. Multiple DNA repair mechanisms are present in eukaryotic cells for the processing of DNA damage [1]. Their importance in man is highlighted by the severe clinical phenotypes associated with defects in DNA repair enzymes, including in some cases a predisposition to cancer. The recognition and processing of DNA lesions in the eukaryotic genome requires repair enzymes to operate within the complex environment of chromatin. This review focuses on the chromatin rearrangements (largely at the nucleosome level) which can occur during nucleotide excision repair (NER), a major repair pathway which is highly conserved throughout evolution. Briefly, chromatin is a nucleoprotein complex which consists of basic repeating units known as nucleosomes. The core particle of a nucleosome contains ≈ 1.8 superhelical turns (146 bp) of DNA wrapped around a core histone octamer comprising the histones H2A, H2B, H3 and H4 whose structure has recently been solved at high resolution [2]. Nucleosome core particles are separated from each other by linker DNA whose length can vary between different cell types and species. To these basic components, additional linker histones as well as non-

histone proteins must be incorporated in order to attain a complex chromatin structure whose maintenance is critical to ensure regulated DNA metabolism [3]. NER enzymes can recognise and remove a wide range of DNA lesions including UV photoproducts and bulky chemical adducts. At least 30 polypeptides are required for NER of naked DNA in vitro [4, 5]. The biochemical mechanism of NER involves damage recognition and open complex formation by factors that include XPA and RPA, XPC and TFIIH, dual incision of the damaged DNA strand by the structure-specific endonucleases XPFERCC1 and XPG, repair synthesis mediated by a PCNAdependent DNA polymerase and ligation of the repaired DNA strand (figure 1A). This chapter is aimed at discussing the problem of how NER enzymes can gain access to DNA lesions within chromatin, repair the damaged DNA strand and restore the chromatin structure present prior to the formation of DNA damage. This process contributes to the maintenance of genetic information as a whole ranging from the nucleotide sequence level of DNA up to its chromatin organisation. In order to understand how this genomic maintenance is achieved it is first necessary to consider how DNA damage formation itself can be modulated within chromatin. We will then discuss the extent and potential mechanism(s) of chromatin rearrangements associated with NER along with factors which may facilitate these rearrrangements. 2. DNA damage formation within chromatin

* Correspondence and reprints Abbreviations: NER, nucleotide excision repair; XP, xeroderma pigmentosum; ERCC, excision repair cross complementing; CS, Cockayne’s syndrome; CAF-1, chromatin assembly factor 1; PCNA, proliferating cell nuclear antigen; RPA, replication protein A.

It is important to appreciate the heterogeneous nature of DNA damage formation within the genome when considering the relationship between chromatin structure and NER. This topic has been recently reviewed [6] and we have highlighted a few important points below. Exposure of cells to UV-irradiation is thought to result in the

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Figure 1. Model for chromatin rearrangements during nucleotide excision repair. A. The biochemical mechanism of NER involves damage recognition and open complex formation by factors that include XPA and RPA, XPC and TFIIH, dual incision of the damaged DNA strand by the structure-specific endonucleases XPF-ERCC1 and XPG, repair synthesis mediated by a PCNA-dependent DNA polymerase and ligation of the repaired DNA strand [4, 5]. B. The recognition and repair of DNA lesions is associated with a destabilisation of the pre-existing chromatin structure [23], the extent of which is not yet clear. Nucleosome assembly is initiated prior to DNA repair synthesis [59] and may involve the recruitment of factors including CAF-1 by an incised DNA strand and/or associated protein factors. Nucleosomal arrays are propagated bidirectionally from the repair site [59] either to reset a pre-existing chromatin structure or to establish de novo a repressive chromatin structure.

formation of UV photoproducts (mainly cyclobutane pyrimidine dimers and 6-4 photoproducts) throughout the genome depending mainly on local DNA sequence, although nucleosome organisation can also modulate their distribution. Indeed, analysis of the cyclobutane pyrimidine dimer distribution within a population of nucleosomes containing a mixture of DNA sequences revealed that pyrimidine dimers form preferentially at sites within nucleosomes where the minor groove of the DNA helix faces into solution [7, 8]. Interestingly, a distinct distribution of pyrimidine dimers was observed in a positioned nucleosome containing poly(dA.dT) tracts [9] suggesting that damage formation in specific DNA sequences may differ from the average genomic lesion distribution. In contrast, 6-4 photoproducts (which are repaired more efficiently than pyrimidine dimers) are more randomly distributed within nucleosome cores and appear to form preferentially in linker and nucleosome-free regions of chromatin [10] although their distribution within specific DNA sequences has not yet been characterised. Having considered that nucleosome organisation can modulate the distribution of DNA lesions in the genome, it is equally important to appreciate that the nucleosome organisation of the genome itself is not uniform. Transcriptionally active regions are generally more accessible

than inactive or repressed regions and chromatin organisation can vary between different cell types and states of differentiation. Since lesions are clearly not only distributed within nucleosome-free regions of the genome it is important to consider the problem of how NER enzymes access DNA lesions within a nucleosomal context. 3. Restricted access of NER enzymes to DNA lesions within chromatin At the nucleosomal level, the ability of NER factors to access DNA lesions is likely to be restricted by the wrapping of the DNA helix around core histone octamers. The restricted accessibility of nucleosomal DNA to transacting factors has been extensively documented for transcription factors [11] and this provides a paradigm for the potential mechanisms of nucleosome rearrangements associated with NER. The DNA helix within core nucleosome particles exhibits significant distortions, restricted flexibility and is partially concealed through interactions of its inner surface with the core histones and by the close proximity of superhelical DNA gyres [2]. The compaction of nucleosomes into higher order chromatin structures may further limit the accessibility of DNA lesions. These

Chromatin rearrangements during NER potential restrictions combined with the observation that NER enzymes require a minimum of 100 bp flanking a DNA lesion to efficiently repair naked DNA in vitro [12] suggest that at least one nucleosome needs to be rearranged or disassembled during NER. In support of this notion, NER activity in human cell extracts can be suppressed by chromatin structure [13, 14]. Furthermore, high resolution analysis of pyrimidine dimer repair at specific sites within small yeast minichromosomes, possessing a well characterised nucleosomal organisation, have demonstrated preferential repair of linker DNA and a modulation of repair within a positioned nucleosome in the non-transcribed strand of the URA3 gene [15]. 4. Chromatin rearrangements during NER: the ‘unfolding/refolding’ model Chromatin rearrangements during NER have been largely observed in UV-irradiated cultured mammalian cells using either light microscopy [16, 17] or enzymatic and chemical probes of chromatin structure [18–22]. Although relatively large regions of chromatin appear to become more accessible during NER it is not yet clear how, and to what extent, nucleosome structure is disrupted. Furthermore, analysis of chromatin rearrangements within whole genome prevents the elucidation of the potentially different mechanisms by which NER enzymes access DNA lesions within distinct genomic regions (e.g., transcriptionally active versus inactive DNA). However, observations of transient changes in the nuclease sensitivity of chromatin in UV-irradiated cultured mammalian cells have led to the proposal of a simple working model for how nucleosomes are rearranged during NER of the genome as a whole [23, 24]. The nucleosomal organisation of genomic DNA was assessed using nucleases as probes for chromatin structure. Micrococcal nuclease (MNase) preferentially cleaves nonnucleosomal DNA and can be used to isolate nucleosome core particles. DNase I digestion of nucleosomal DNA results in a 10.4 base repeat pattern reflecting the binding of duplex DNA to the surface of the histone octamer. Newly repaired DNA was rapidly digested by both MNase and DNase I, did not yield a 10.4 base repeat after DNase I digestion and was absent from isolated nucleosome cores. With a half-life of approximately 20 min, these regions became increasingly nuclease resistant, associated with isolated nucleosome cores and produced a 10.4 base pattern after DNase I digestion. These results have been explained by a process involving the initial unfolding or ‘disassembly’ of nucleosomal DNA for processing of UV photoproducts by repair enzymes followed by the refolding or ‘reassembly’ of newly repaired DNA into a nucleosome structure [23] (figure 1b). The potential mechanisms by which these nucleosome rearrangements may operate are discussed successively below.

47 5. Chromatin unfolding/disassembly during NER How do NER enzymes search for and recognise DNA lesions which are potentially distributed throughout the genome? The dynamic nature of chromatin is likely to facilitate the genome-wide search for lesion-induced distortions in the DNA helix. Generalised rearrangements of chromatin structure may facilitate the initial search for lesions whilst more localised rearrangements may only occur during the recognition and repair of lesions. Nucleosome rearrangement mechanisms could include the transient dissociation of DNA strand(s) from nucleosome surface, displacement of some or all of the core histones and/or movement of the nucleosomes with respect to the DNA sequence (figure 2). In the latter case any mechanism which alters nucleosome positioning could be considered. Although a number of parameters are likely to influence nucleosome positioning in vivo [25, 26], nucleosome positioning was found to be dependent on the continuous presence of a DNA binding factor acting as a barrier to nucleosome movement in vitro [27]. In that situation the simple release of the factor resulted in the loss of nucleosome positioning. It is perhaps conceivable that disruption of the binding of such ‘boundary’ factors could increase lesion accessibility without directly interacting with nucleosomes. Certain regions of the genome may become more accessible to NER enzymes through various forms of DNA metabolism including transcription. Actively transcribed pol II genes are preferentially repaired relative to inactive regions of the genome largely due to the faster (transcription-coupled) repair of the transcribed DNA strand [28]. Transcription-coupled repair may be accounted for by the more ‘open’ chromatin configuration of actively transcribed genes [29]. In addition, the presence of factors like TFIIH, directly involved in both transcription and NER, could be critical [30]. The contribution of chromatin remodelling machines associated with gene expression [31–34] should also be considered since they could potentially facilitate NER enzyme access to DNA lesions within transcribed DNA. 5.1. Factors required for chromatin disassembly associated with NER It is not yet clear which proteins facilitate nucleosome rearrangements during the initial stages of NER. These types of rearrangements could be modulated by reversible modifications of histone and non-histone proteins including acetylation [35, 36], poly ADP-ribosylation [37] and factor driven nucleosome remodelling mechanisms [38, 39]. Histone acetylation has been widely implicated in facilitating genome accessibility. Consistent with this idea, pretreatment of human cells with n-butyrate (producing a global increase in the acetylation levels of the chromatin) prior to UV-irradiation increased subsequent

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Figure 2. Possible nucleosome rearrangements which facilitate lesion accessibility during nucleotide excision repair. The relative accessibility of DNA lesions depends in part on whether they are positioned within linker DNA or DNA associated with core histone particles. Furthermore, lesions may be positioned either away from or in close proximity to the histone octamer surface. Possible nucleosome rearrangement mechanisms include: nucleosome sliding (a); post-translational modifications of core histones (b); displacement of core histones (c); altered conformation of core histones (d); unfolding of the DNA helix from the histone octamer surface (e, f). Factors which could facilitate these rearrangements include histone modifying enzymes, core histone binding factors and chromatin remodelling machines (see text for details). In addition to altering the accessibility of DNA lesions within individual nucleosomes, these mechanisms could also potentially lead to a more general increase in lesion accessibility within chromatin by altering internucleosomal contacts and thus higher order chromatin organisation.

NER activity [40–42]. In light of the recent advances in characterising the enzymes regulating histone acetylation (histone acetyl transferases and histone deacetylases) [43] it would be interesting to re-examine their possible roles during NER. Chromatin disassembly during NER may be an intrinsic function of the factors involved in the early reaction steps of NER such as XPA, TFIIH, XPC-HHR23B, XPF-ERCC1, RPA and XPG. Such an intrinsic function has been recently attributed to XPC protein which may play a role in transient nucleosome unfolding during NER [44]. On the other hand, mutant cells within the XP-A, XP-C, XP-D and XP-G complementation groups are deficient in localised, but not generalised alterations of chromatin structure in cultured human cells after UVirradiation [17]. Thus additional proteins may be neces-

sary to allow these NER enzymes access to lesions within chromatin. It has been suggested that the UV-DDB protein (a factor defective in some XP-E cells) might play a role in the repair of chromatin [45] and it is intriguing that the p48 subunit of UV-DDB, as well as the transcriptioncoupled repair factor CSA, exhibit some homology to WD repeat proteins, including the smallest subunit of chromatin assembly factor 1 (CAF-1) [46]. This subunit of CAF-1 (p48) is a member of the p46/p48 family of core histone binding proteins present in multiple complexes involved in histone metabolism [47]. The role of CAF-1 in nucleosome assembly coupled to NER is discussed in the following section. Genetic studies in S. cerevisiae have revealed additional factors that potentially perturb chromatin structure during NER. Rad7 and Rad16 form a complex which can act as an ATP-dependent damage-

Chromatin rearrangements during NER sensor in vitro [48] and mutations in these genes can abolish repair of the non-transcribed DNA strand of genes and inactive regions of the genome [49–52]. Together with the homology of RAD16 to the SNF2-like family of proteins [53], present in a variety of ATP-dependent chromatin remodelling machines [38, 39], these data raise the possibility that the Rad7-Rad16 complex may play a role in the remodelling of chromatin structure to facilitate NER. The interaction of Rad7 with Sir3 [54] suggests a mechanism by which the NER machinery could gain access to silenced chromatin. It is noteworthy that the transcription-coupled repair factor CSB also resembles an ATPase of the SNF2-like family [55]. In summary, we still know very little about the exact mechanism of chromatin rearrangements during the early stages of NER or the molecular machines participating in their regulation. The development of suitable in vitro experimental systems for analysis of NER within chromatin combined with genetic approaches (e.g., cells possessing defined mutations in genes encoding proteins which potentially facilitate chromatin rearrangements during NER) should improve our understanding of these events. 6. Chromatin refolding/reassembly during NER 6.1. NER-coupled nucleosome assembly The reassembly of nucleosomes during NER appears to be a multistep process [56, 57]. Most repair-induced nuclease sensitivity is rapidly lost at early times after repair due to nucleosome refolding. The remaining nuclease sensitivity persists for many hours in a slow phase probably reflecting the maturation of newly formed chromatin including the repositioning of nucleosomes within repaired regions. This latter phase is likely to be critical for the restoration of the precise chromatin structures present prior to the formation of DNA damage. A simplified approach for investigating the biochemical mechanism of nucleosome rearrangements associated with NER has been developed using naked damaged DNA substrates as a model for reaction intermediates in the disrupted chromatin organisation observed during NER in vivo. These substrates may also serve as a model for repair of an open chromatin organisation arising during other DNA transactions. Incubation of such substrates in cellfree extracts derived from either Xenopus eggs, human somatic cells or Drosophila preblastoderm embryos revealed that de novo nucleosome assembly on DNA occurs concomitantly with UV damage-dependent repair synthesis [58, 59]. These observations suggest that a mechanistic coupling may exist between the two processes which is conserved amongst higher eukaryotes. A complementation assay revealed that CAF-1, a histone chaperone required for replication-coupled nucleosome assembly [60, 61], was also required for repair-associated nucleosome assembly [58]. The deletion of CAF-1 subunits from the S.

49 cerevisiae genome led to a moderately UV sensitive phenotype [62] consistent with a role for CAF-1 in chromatin assembly coupled to DNA repair. Furthermore, specific phosphorylated forms of CAF-1 are recruited to chromatin during repair of UV damage in human cells in proportion to the amount of lesions formed [63]. Reversible phosphorylation of CAF-1 subunits may be involved in the regulation of nucleosome assembly coupled to NER. It is interesting to note that the activity of NER enzymes themselves are subject to regulation by reversible phosphorylation [64] which could possibly form part of a co-ordinated DNA damage response. Several important questions arise from the observation that NER is coupled to CAF-1 dependent nucleosome assembly. What is the mechanism of nucleosome reassembly at sites of repair? How many nucleosomes are reassembled per repair event? Biochemical analysis of nucleosome assembly from a single NER site (discussed below) has provided further insights into these intriguing questions. 6.2. Mechanism of nucleosome assembly coupled to NER The selective analysis of nucleosome assembly from a single target site for NER has been facilitated by combining DNA substrates containing an efficiently repaired site-specific 1,3-intrastrand cisplatin-DNA cross-link with Drosophila preblastoderm embryo extracts under reaction conditions which suppress nucleosome assembly on bulk DNA [59, 65]. Nucleosome formation was initiated at the target site for NER and regularly spaced nucleosomal arrays were propagated in both directions away from the repair site. Since NER involves a relatively limited (approximately 30 nucleotides) and unidirectional DNA synthesis reaction, the bidirectional propagation of large nucleosomal arrays suggested that the initiation of nucleosome assembly might occur at an earlier reaction step during NER. In support of this notion, nucleosomal arrays flanking the repair site were assembled in the presence of aphidicolin, an inhibitor of DNA repair synthesis, although the repair site itself was particularly sensitive to MNase digestion possibly reflecting an unligated gap and/or an incompletely formed nucleosome. Thus, although nucleosomes can be assembled prior to the completion of NER, repair patch ligation appears to be required for the restoration of a canonical nucleosome structure at the repair site consistent with observations made during NER of chromatin in vivo [66, 67]. Both damage recognition and dual incision of the damaged DNA strand still occured under these reaction conditions, raising the possibility that the initial recruitment of the chromatin assembly machinery (and by inference CAF-1) is due to intermediate DNA structures and/or associated protein factors during NER reaction steps preceding DNA repair synthesis (figure 1B). Importantly, in the absence of NER activity the 1,3-intrastrand cisplatin cross-link itself does not promote the recruitment of nucleosomes but

50 rather represses nucleosome formation (Moggs and Almouzni, unpublished results). This is consistent with observations that UV-irradiation of naked DNA partially inhibited the formation of a positioned nucleosome [68] probably due to the presence of 6-4 photoproducts [69]. Assembly of nucleosomal arrays has also been observed during the repair of a U.G base pair and a single-strand break (Moggs and Almouzni, manuscript in preparation). Consistent with the possibility that nucleosome assembly can be coupled to some forms of base excision and single-strand break repair, nucleosome rearrangements have been observed in human cells treated with bleomycin and methyl methanesulfonate [70, 71]. It seems likely that reaction intermediates common to these distinct repair pathways are responsible for the recruitment of chromatin assembly machinery and a similar connection may exist between nucleosome assembly and additional forms of DNA repair such as mismatch repair. Strong candidates for the recruiting signal include an incised damaged DNA strand and/or associated protein factors. The bidirectional propagation of nucleosome arrays away from the repair site might be achieved by the tracking of the recruiting signal along the DNA. Such a property is conceivable for the DNA polymerase accessory factor PCNA, although other factors cannot be excluded. PCNA is required for NER [72, 73], some forms of base excision repair [74-76] and possibly single strand break processing. PCNA associates with chromatin undergoing NER [77, 78] and colocalises with both the p60 and p150 subunits of CAF-1 in UV-irradiated cultured human cells [63]. The propagation of regularly spaced nucleosomal arrays in both directions away from a single repair site [59] may have important implications for the functional status of chromatin reassembled during DNA excision repair. Although reassembly of the preexisting chromatin structure is important for the restoration of a functional and precisely regulated genome, the propagation of nucleosomes from a repair site may serve a different function by setting up a special form of chromatin structure which could repress DNA transactions as part of the cellular response to DNA damage. In this respect it is noteworthy that the inheritance of transcriptionally silent heterochromatin involves both PCNA [79, 80] and CAF-1 [62, 81–83] proteins. Although significant progress has been made in elucidating mechanisms by which nucleosome assembly can be coupled to DNA excision repair, it will also be important to investigate how assembled nucleosomal arrays are converted back into the chromatin configuration which was present prior to the formation of DNA damage and perhaps into repressive chromatin structures. Furthermore, there may be distinct nucleosome assembly mechanisms associated with the repair of active genes. In this case nucleosome assembly might be coupled to components of the transcription machinery rather than to repair reaction intermediates.

Moggs and Almouzni 7. Future perspectives This review has focused on the potential roles of chromatin rearrangements during DNA excision repair. In addition, there are indications that chromatin structure plays an important role during several other DNA damage processing mechanisms. These include photolyasemediated repair of pyrimidine dimers [84], repair of DNA double-strand breaks induced by ionising radiation [85, 86] and RAD6-dependent postreplicative repair and damage-induced mutagenesis [87, 88]. A major challenge for the future will be the development of methodology for assessing the precise nature of chromatin rearrangements occurring during DNA repair both at the nucleosomal level and within more complex chromatin structures. Furthermore, it will be important to investigate the possible connections between chromatin rearrangements during DNA repair and the cell cycle. Such chromatin rearrangements might be important for the establishment and/or regulation of cell cycle checkpoints in response to DNA damage. Finally, it is possible that human diseases may arise through defects in chromatin rearrangements during DNA repair. Recent reports have shown that alterations in the enzymes which regulate histone acetylation [89, 90], as well as in a member of the SWI/SNF family of chromatin remodelling machines [91], contribute to the onset of certain cancers. An interesting possibility is that patients with mutations in the CSB and XPE proteins might have defective chromatin rearrangement mechanisms based on the homology of these repair proteins to components of factors which modify chromatin structure. Acknowledgments We thank members of our laboratory for critical reading of the manuscript. This work was supported by an EMBO long-term fellowship (J.G.M.), an ATIPE from the CNRS (G.A.), Institut Curie and ARC.

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