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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;

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M I N I R E V I E W

Nucleotide excision repair and chromatin remodeling

Kiyoe Ura1and Jeffrey J Hayes2

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

I N T R O D U C T I O N

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  13

4loops 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 portransi-tions 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 103)106, 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

N U C L E O T I D E E X C I S I O N R E P A I R

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)

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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 transcription-independ-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

are repaired very slowly b y GG-NER and are removed

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

E F F E C T S O F C H R O M A T I N S T R U C T U R E

O N U V - I N D U C E D D N A D A M A G E

F O R M A T I O N

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

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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]

N E R I N C H R O M A T I N

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, yISWI complexes 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

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isolation can affect ATP-dependent nucleosome remodeling

in vitroby 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

A C K N O W L E D G E M E N T S

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.

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Fig 1 NER factors are indicated for human but each stepappears to be

conserved in yeast (1) Two major types of UV-induced DNA lesions

CPDs and 6–4PPs (red asterisks), are formed throughout chromatin

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