Báo cáo Y học: Nucleotide excision repair and chromatin remodeling pptx

MINIREVIEW Nucleotide excision repair and chromatin remodeling Kiyoe Ura 1 and Jeffrey J. Hayes 2 1 Division of Gene Therapy Science, Osaka University School of Medicine, Suita, Osaka, Japan; 2 Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA The organization of DNA within eukaryotic cell nuclei poses special problems and opportunities for the cell. For example, assembly of DNA into chromatin is thought to be a principle mechanism by which adventitious general transcription is repressed. However, access to genomic DNA for events such as DNA repair must be facilitated by energy-intensive processes that either directly alter chro- matin structure or impart post-translational modifications, leading to increased DNA accessibility. The assembly of DNA into chromatin affects both the incidence of damage to DNA and repair of that damage. Correction of most damage to DNA caused by UV irradiation occurs via the nucleotide excision repair (NER) process. NER requires extensive involvement of large multiprotein complexes with relatively large stretches of DNA. Here, we review recent evidence suggesting that at least some steps of NER require ATP-dependent chromatin remodeling activities while perhaps others do not. INTRODUCTION In vivo, eukaryotic DNA is packaged with histones and other accessory proteins into chromatin. The assembly of nucleosomes, the basic unit of chromatin, changes the structure of DNA and restricts access of DNA binding factors to their recognition sites [1]. In particular DNA within the nucleosome is highly bent, with  150 bp of DNA wrapped in  1 3 4 loops around a central ÔspoolÕ consisting of the core histone proteins [2,3]. Although nucleosomal DNA is quite accessible to small molecules, the DNA binding activity of larger molecules and complexes that require interaction with multiple base pairs is typically severely restricted within the nucleosome [4]. However, nucleosomes are dynamic structures and undergo transi- tions to states in which portions of nucleosomal DNA are as accessible as naked DNA [5]. Details of these transitions have been described and indicate that the core histones behave merely as competitors for binding to DNA, effectively reducing the association constants for DNA- binding proteins by factors of 10 3 )10 6 , dependent on sequence and location within the nucleosome [5,6]. In addition, it is important to note that strings of nucleosomes exist in vivo compacted into Ôchromatin fibersÕ  30 nm in diameter, which are in turn assembled into higher-order structures [2]. These structures contribute additional, severe limitations to the accessibility of DNA, beyond that provided by nucleosomes [1,2]. Clearly, the effects of packaging DNA into nucleosomes must be considered in investigations of all processes that use nuclear DNA as a substrate, including transcription, replication, recombination and DNA repair. Several strat- egies are employed by eukaryotic cell nuclei to modulate the accessibility of DNA within chromatin, including post-translational modification of the histones and ATP- dependent chromatin remodeling machines [7–10]. These play important roles in regulation of transcription and other DNA-dependent nuclear processes and typically involve targeted modifications of distinct regions in chromatin. In contrast, although the assembly of DNA into chromatin does affect the incidence of formation of some DNA lesions, DNA damage is widespread throughout the genome. Thus, NER reaction is required everywhere in the genome, irrespective of chromatin structure or the gene expression profile of a particular cell. NUCLEOTIDE EXCISION REPAIR DNA is frequently damaged by a variety of environmental and endogenous agents produced as products or byproducts of physiological reactions. Damage to DNA causes struc- tural defects that can impede or block transcription or replication and potentially result in mutations [11]. For example, all living organisms have suffered the genotoxic effects of solar UV radiation since the beginning of the evolution of life. It has been estimated that under the strong sunlight typically encountered on a beach, an exposed cell in the human epidermis develops about 40 000 damaged sites in one hour, primarily from absorption of UV radiation by DNA ( 200–320 nm). UV light induces two major classes of mutagenic DNA lesions: cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6–4) pyrimidone photoproducts (6–4PPs), which induce a DNA bend or kink of 7–9° and 44°, respectively [11,12]. Correspondence to J. J. Hayes, Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA. Fax: + 1 716 271 2683, Tel.: + 1 716 273 4887, E-mail: JJHS@uhura.cc.rochester.edu Abbreviations: NER, nucleotide excision repair; XP, xeroderma pigmentosum; RPA, replication protein A; RFC, replication factor C; CPDs, cyclobutane pyrimidine dimers; 6-4PPs, pyrimidine (6-4) pyrimidone photoproducts; CHD, chromain ATPase; ACF, ATP- utilizing chromatin assembly and remodeling factor; GG-NER, global genome repair; TC-NER, transcription-coupled repair. Dedication: This Minireview Series is dedicated to Dr Alan Wolffe, deceased 26 May 2001. (Received 8 October 2001, revised 21 February 2002, accepted 28 February 2002) Eur. J. Biochem. 269, 2288–2293 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02888.x To insure survival in this background, cells have developed multiple strategies for dealing with DNA damage including the direct correction or ÔrepairÕ of DNA changes. Both bacterial and eukaryotic cells have several dedicated repair systems that maintain the integrity of their genomic information. Among these, nucleotide excision repair (NER) is one of the best- studied pathways of DNA repair. NER is capable of eliminating a broad range of structurally unrelated bulky lesions from DNA, including those from UV-induced damage and some chemical damage [11]. Indeed, defects in components of the NER are responsible for genetic diseases exemplified by sensitivity to UV radiation and predisposition to skin cancer such as xeroderma pigmentosum (XP) and Cockayne syndrome (CS) [13]. Thus, the names of many human NER components often reflect genetic complementation groups from these phenotypes. The process of NER is highly conserved in eukaryotes and consists of the following four steps: (a) recognition of the damaged DNA; (b) excision of an oligonucleotide of 24–32 residues containing the damage from DNA by dual incision of the damaged strand on each side of the lesion; (c) filling in of the resulting gap by DNA polymerase; and (d) ligation of the nick [13–15]. In human cells, NER reaction requires at least six core protein complexes for damage recognition and dual incision (XPA, XPC-hHR23B, RPA, TFIIH, XPG and XPF–ERCC1) and other factors for repair DNA synthe- sis and ligation (PCNA, RFC, DNA polymerase a or d and DNA ligase I) [16–18]. The molecular mechanisms of NER have been thoroughly analyzed using highly purified human proteins or recombinant polypeptides on damaged naked DNA or UV-irradiated SV40 mini- chromosomes [18–21]. NER consists of two subpathways termed global genome repair (GG-NER) that is transcription-independ- ent and removes lesions from the entire genome, and transcription-coupled repair (TC-NER) [11,13]. 6-4PPs, which distort the DNA more than CPDs, are removed rapidly, predominantly by GG-NER. In contrast, CPDs arerepairedveryslowlybyGG-NERandareremoved more efficiently from the transcribed strand of expressed genes by TC-NER [22]. The elongating transcriptional machinery is thought to facilitate the recognition of DNA lesions on the transcribed strand in TC-NER. However, detailed mechanisms of TC-NER remain undefined, due to the lack of an in vitro system for analysis. Recent biochemical and immunocytological studies demonstrate that the XPC–hHR23B complex appears to be the initiator of GG-NER [23,24], although several other models have been proposed [25,26]. It has been a curious problem how the huge multi- subunit protein complexes of NER recognize and remove DNA lesions that are formed in chromatin [27,28] (see below). In addition, organization of DNA in chromatin affects how UV light and chemical agents impart damage to DNA. In order to understand the relationship between chromatin dynamics and NER, it is crucial to elucidate effects of chromatin structure on DNA damage forma- tion and to investigate NER processes at the chromatin level. EFFECTS OF CHROMATIN STRUCTURE ON UV-INDUCED DNA DAMAGE FORMATION As mentioned above, UV light induces the formation of both cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6–4) pyrimidone photoproducts (6–4PPs) [11,28]. Their yield and distribution depend on DNA sequence, the local DNA structure and the association of DNA with chromo- somal proteins [11,27,28]. Specifically, the chromatin envi- ronment has been shown to affect UV-induced damage formation distributions in nucleosomes isolated from UV-irradiated cells [29,30]. In these mixed sequence nucleo- somes, the CPD distribution shows a striking 10.3-bp periodicity with a strong preference for sites where the minor groove is oriented away from the histone surface [29,30]. Interestingly, this distinctive periodicity coupled with the ability of UV radiation to penetrate whole nuclei provided the first evidence that the DNA structure of isolated nucleosome cores is identical to that found in nucleosomes within native chromatin [31]. On the other hand, 6-4PPs are distributed relatively uniformly within nucleosome cores and preferentially formed in linker DNA of bulk chromatin from UV-irradiated cells [32]. In order to investigate the effects of nucleosome structure on the formation of UV-induced DNA lesions, several groups have used reconstituted model nucleosomes con- taining a defined DNA sequence [33–36]. A major advant- age of such systems is that the relationship between rotational and translational position of the DNA with respect to the histone octamer is well defined for a majority of the sample, making correlation of damage incidence to structure feasible. The distribution of CPDs in reconstituted nucleosomes containing defined sequences does not show the obvious 10.3-bp periodicity observed with mixed- sequence chromatin, although CPD formation is reduced at sites where the minor groove faces the histone octamer and around the pseudo-dyad (center) of the nucleosome compared to naked DNA [33–35]. This is likely due to sequence-dependent DNA structural effects on the probab- ility of lesion formation and indicates that the chemical reactivity of DNA varies significantly about the mean behavior observed in bulk chromatin. Also, as observed with bulk chromatin structures, no effect of nucleosome assembly was observed on 6-4PP distribution in physiolog- ically spaced reconstituted dinucleosomes composed of two tandem repeats of 5S RNA genes [36,37]. Interestingly, regardless of the large effects of histone H1 on chromatin structure, the formation of either CPDs or 6-4PPs in the reconstituted dinucleosomes was not significantly affected by the binding of linker histones [36,38]. Therefore, despite local variations in lesion formation these defined chromatin systems demonstrate that chromatin structure of DNA does not greatly restrict acquisition of UV-induced lesions, even in the presence of linker histone H1 [26,33–38]. Interestingly, after the acquisition of DNA damage by UV irradiation, neither DNase I footprinting, hydroxyl radical footprinting nor micrococcal nuclease mapping shows any significant changes in the rotational or transla- tional setting between UV-irradiated and nonirradiated reconstituted chromatin templates. These results indicate that local DNA-distortions induced by UV-induced lesions do not propagate throughout the nucleosome or lead to its Ó FEBS 2002 Nucleotide excision repair and chromatin remodeling (Eur. J. Biochem. 269) 2289 dissolution [28,33–36]. Thus, the UV-induced DNA lesions formed throughout chromatin probably do not cause drastic alternations of nucleosomal structure in vivo.How- ever, it is interesting to note that assembly of nucleosomes on DNA containing UV-induced lesions can lead to changes in the association of histones. The introduction of UV damage within a 5S rDNA fragment reduced the relative affinity for binding histones and thus the efficiency of nucleosome reconstitution in vitro [34,39]. Moreover, UV irradiation of both mixed-sequence and unique sequence DNA fragments was found to affect the rotational positioning of the DNA upon reconstitution into nucleo- somes [33,35,40]. NER IN CHROMATIN Several lines of evidence clearly indicate that the presence of nucleosomes on damaged DNA severely inhibits the activity of NER machinery. Damage within UV irradiated plasmids reconstituted into nucleosomes or within SV40 minichro- mosomes is repaired much less efficiently compared to naked DNA [20,21]. NER repair studies that used UV- irradiated reconstituted nucleosomes as templates with bacterial repair enzymes or Xenopus oocytes repair extracts demonstrated that nucleosome assembly reduces efficiencies of DNA repair at many but not all CPD sites in nucleosome cores [40,41]. Removal of histone tails has little effect on the repair efficiency of UV-irradiated nucleosomes [40,41]. Interestingly, the variation of efficiency of NER for nucleosomal DNA does not reveal any periodicity related to the helical twist of the DNA [41]. Therefore, it is likely that NER components require full access to DNA com- pletely released from histone proteins, as is provided by the spontaneous uncoiling of DNA from the histone surface discussed above [5,6]. Recently, in order to unravel the molecular mechanisms of NER in chromatin, defined nucleosomal templates containing synthetic 6-4PPs at unique sites were used for NER reactions reconstituted with purified human NER core factors RPA, XPA, XPC–hHR23B, XPG, ERCC1– XPF and TFIIH [26,36]. These studies demonstrated that excision activity at the center of nucleosome cores was reduced drastically to  15% of that of naked DNA. The use of synthetic oligonucleotides containing DNA lesions makes it possible to introduce a specific type of DNA damage at a specific position within reconstituted chroma- tin. Surprisingly, strong repression of NER in physiologi- cally spaced dinucleosome templates was observed even when the 6-4PP lesion was located in the linker DNA [36]. In yeast cells, NER rates for CPDs and 6-4PPs on the nontranscribed strand are influenced by the chromatin environment and are removed more efficiently in linker DNA than in nucleosomal DNA [42,43]. These results demonstrate that extra factors other than the six human NER factors are required to overcome the structural barriers that chromatin poses to the removal of DNA damage in vivo. Although histone acetylation is generally related to chromatin accessibility, the primary effect of this modifica- tion may be to destabilize higher order structures [44]. Indeed, increasing the global levels of acetylation by general inhibition of histone deacetylases causes an approximately twofold increase in the extent of repair in hyperacetylated nucleosomes [45]. However, removal of the histone tails does not enhance repair rates on nucleosomes in a purified system [41], and histone acetylation has only modest effects on nucleosome structure and accessibility [1,6,44]. Thus ATP-dependent chromatin remodeling complexes are likely candidates for assisting NER in nucleosomes in vivo.Over 10 large protein complexes that locally disrupt or alter the association of histones with DNA depending on ATP have been purified to date. All of these chromatin remodeling complexes contain the ATPase subunit of the SNF2 superfamily that is classified into one of three distinct groups: SWI/SNF2-like (e.g. SWI/SNF, RSC and BRM), ISWI-like(e.g.NURF,CHRAC,ACF,yISWIcomplexes and RSF), and CHD-like (e.g. Mi-2/NURD) [7]. The recent purification of a complex containing an SNF2-related ATPase that may be related to DNA repair underscores a connection between repair and remodeling activities [46; see below]. It has been recently demonstrated that recombinant ACF facilitates the excision of 6-4PP lesions by the NER core factors, in particular those situated in the linker DNA [36]. ACF, ATP-utilizing chromatin assembly and remode- ling factor, consists of ISWI and Acf1 in addition to a few other polypeptides and is well conserved from Drosophila to human [47–49]. Although the exact function of ACF in cells remains unknown, this is the first biological evidence to indicate a direct connection between ATP-dependent chro- matin remodeling and NER. Interestingly, NER in Xenopus oocyte nuclear extracts can effectively repair a single CPD located near the dyad center of a positioned nucleosome [50]. NER in these extracts presumably relies on the activity of one or more ATP-dependent chromatin remodeling complexes [50]. Such activities may facilitate repair at different sites of chromatin by nucleosome movement, octamer transfer, or local twist of nucleosomal DNA in eukaryotic cells [51,52]. XPC–hHR23B can preferentially bind to UV-damaged DNA, even when DNA is wrapped around the histone octamer (K. Ura, unpublished data). Once the XPC– hHR23B complex binds to a DNA lesion of chromatin, some ATP-dependent chromatin remodeling factors may induce targeted chromatin reconfiguration around the lesion to assemble the initiation complex of NER [28] (see Fig. 1). It is likely that the extensive interactions with DNA required by the pre- and post-incision complexes and the synthesis of nascent DNA require disruption of nucleosome structure, even if damage is located within the linker region between nucleosomes [28,36] (Fig. 1, steps 4–6). Thus nucleosomes must be reformed after repair-dependent DNA synthesis. Importantly, nucleosomes are assembled selectively on damaged DNA by cell or nuclear extracts containing both chromatin assembly and NER activities [50,53]. Thus, rapid chromatin assembly coupled to DNA synthesis may suggest that the later steps of NER actually occur in nucleosomes or subnucleosomal intermediates. In this regard, it should be noted that human DNA ligase I can efficiently seal DNA nicks in nucleosomes, even in the presence of linker histone H1 [54] (Fig. 1, step 7). Recent studies further highlight the possibility of a direct functional link between chromatin remodeling activities and DNA repair. Interestingly, the TC-NER component CSB (Cockayne Syndrome B, see above) has homology to the SWI2/SNF2 family and indeed has been shown to be a DNA-dependent ATPase [55]. Moreover, this protein in 2290 K. Ura and J. J. Hayes (Eur. J. Biochem. 269) Ó FEBS 2002 isolation can affect ATP-dependent nucleosome remodeling in vitro by several criteria [55]. Although the role of CSB remodeling activity in NER remains to be established, these results provide a potentially important link between the two activities. A study in Saccharomyces cerevisiae indicates that there exists a genetic connection between ATP-dependent chromatin remodeling and DNA repair [46]. Two ÔRuvB- likeÕ proteins, Rvb1p and Rvb2p copurified as part of a complex containing the SNF2/ISWI-related protein Ino80p [46]. Consistent with the activity of bacterial RuvB, the INO80 complex contains DNA helicase activity and, moreover, ino80 null mutants display sensitivity to hydroxyurea, the alkylating agent methylmethane sulfonate, and ionizing and UV radiation [46]. In addition, recent results have shown that a large complex containing the human TIP60 histone acetylase plays a role in DNA repair and apoptosis [56]. TIP60 possesses several activities that influence the activity of repair enzymes in chromatin including ATPase, DNA helicase, and structural DNA binding activities. We expect that in the future, purified reconstituted systems using purified factors and defined nucleosomal templates will allow further systematic analyses of NER in chromatin. Results to date indicate that at least some, if not all, steps involved in NER requires active disruption of nucleosome structure by ATP-dependent chromatin remodeling complexes. Elucidation of the targeting of these complexes to sites of DNA damage, perhaps via interactions with damage-recognition complexes will greatly clarify the relationship between NER and chromatin remodeling. We also note that some damage within nucleosomes and at least the final step in the repair process, DNA ligation, appear to be at least partially compatible with nucleosome structure. Thus, some individual steps of DNA repair processes may not require expenditure of ATP by the cell to disrupt nucleosomes. ACKNOWLEDGEMENTS This work was supported by NIH grant RO1G M 52426 (J. J. H.) and by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (K. U.). We would like to dedicate this article to the memory of our mentor, colleague, and friend Alan P. Wolffe, who constantly effused a contagious passion for science and life. REFERENCES 1. Wolffe, A.P. (2000) Chromatin Structure and Function.Academic Press, San Diego, CA. 2. van Holde, K.E. (1989) Chromatin. Springer Verlag, New York. 3. Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F. & Richmond, T.J. (1997) Crystal structure of the nucleosome core particle at 2.8 A ˚ resolution. Nature 389, 251–260. 4. Hayes, J.J. & Wolffe, A.P. (1992) Transcription factor interaction with nucleosomal DNA. Bioessays 14, 597–603. 5. Polach, K.J. & Widom, J. 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EMBO J. 19, 5492–5501. 55.Citterio,E.,VanDenBoom,V.,Schnitzler,G.,Kanaar,R., Bonte, E., Kingston, R.E., Hoeijmakers, J.H. & Vermeulen, W. (2000) ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcription-coupling factor. Mol. Cell. Biol. 20, 7643–7653. 56. Ikura,T.,Ogryzko,V.V.,Grigoriev,M.,Groisman,R.,Wang,J., Horikoshi, M., Scully, R., Qin, J. & Nakatani, Y. (2000) Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis. Cell 102, 463–473. Ó FEBS 2002 Nucleotide excision repair and chromatin remodeling (Eur. J. Biochem. 269) 2293 . MINIREVIEW Nucleotide excision repair and chromatin remodeling Kiyoe Ura 1 and Jeffrey J. Hayes 2 1 Division of Gene Therapy Science, Osaka University School of Medicine, Suita,. CPDs, are removed rapidly, predominantly by GG-NER. In contrast, CPDs arerepairedveryslowlybyGG-NERandareremoved more efficiently from the transcribed strand of expressed genes by TC-NER [22]. The. RFC and RPA fill in the gap by repair synthesis. (7) Redistribution of nucleosomes or reassembly of nucleosomes, which might be mediated by chromatin assembly factor 1 (CAF-1) and/ or chromatin remodeling