Báo cáo khoa học: The protein shuffle Sequential interactions among components of the human nucleotide excision repair pathway pdf

XPA, XPB, XPC, XPD, XPE, XPF and XPG are known to participate in various Keywords damage recognition; dual incision; nucleotide excision repair; protein–protein interaction; replication

The protein shuffle

Sequential interactions among components of the human

nucleotide excision repair pathway

Chin-Ju Park and Byong-Seok Choi

Department of Chemistry, National Creative Initiative Center, Korea Advanced Institute of Science and Technology, Guseong-dong,

Yuseong-gu, Daejon, Korea

In mammalian cells, nucleotide excision repair (NER)

is the major DNA repair pathway for the removal of

bulky adducts induced by UV light or other

environ-mental carcinogens [1–3] NER proteins display both

versatility and specificity in that they (a) recognize

various types of DNA damage and (b) discriminate

between these lesions and the abundant undamaged

DNA present in the genome (including the intact

DNA strand opposite the lesion) Depending on the

precise location of the damaged DNA, the NER

pro-cess is referred to as either transcription-coupled repair (TCR) or global genomic repair (GGR) The TCR process specifically repairs blemishes on the transcribed DNA strands of active genes, while GGR eliminates lesions from the entire genome As defects in NER are known to cause inherited diseases, such as xeroderma pigmentosum (XP), it is crucial that researchers deci-pher the mechanisms of NER at the molecular level

XP proteins A–G (i.e XPA, XPB, XPC, XPD, XPE, XPF and XPG) are known to participate in various

Keywords

damage recognition; dual incision;

nucleotide excision repair; protein–protein

interaction; replication protein A; structure;

xeroderma pigmentosa

Correspondence

B.-S Choi, Department of Chemistry,

National Creative Initiative Center, Korea

Advanced Institute of Science and

Technology (KAIST), 373–1 Guseong-dong,

Yuseong-gu, Daejon 305–701, Korea

Fax: +82 42 8692810

Tel: +82 42 8692868

E-mail: byongseok.choi@kaist.ac.kr

(Received 12 December 2005, accepted

16 February 2006)

doi:10.1111/j.1742-4658.2006.05189.x

Xeroderma pigmentosum (XP) is an inherited disease in which cells from patients exhibit defects in nucleotide excision repair (NER) XP proteins A–G are crucial in the processes of DNA damage recognition and incision, and patients with XP can carry mutations in any of the genes that specify these proteins In mammalian cells, NER is a dynamic process in which a variety of proteins interact with one another, via modular domains, to carry out their functions XP proteins are key players in several steps of the NER process, including DNA strand discrimination (XPA, in complex with replication protein A), repair complex formation (XPC, in complex with hHR23B; XPF, in complex with ERCC1) and repair factor recruit-ment (transcription factor IIH, in complex with XPG) Through these pro-tein–protein interactions, various types of bulky DNA adducts can be recognized and repaired Communication between the NER system and other cellular pathways is also achieved by selected binding of the various structural domains Here, we summarize recent studies on the domain structures of human NER components and the regulatory networks that utilize these proteins Data provided by these studies have helped to illu-minate the complex molecular interactions among NER factors in the con-text of DNA repair

Abbreviations

CPD, cyclopyrimidine dimer; GGR, global genomic repair; (HhH) 2 , helix–hairpin–helix domain; MBD, minimal DNA-binding domain; NER, nucleotide excision repair; PH, pleckstrin homology; PTB, phosphotyrosine binding; RPA, replication protein A; TCR, transcription-coupled repair; TFIIH, transcription factor IIH; Ub, ubiquitin; UBA, ubiquitin association; UV-DDB, UV-damaged DNA-binding protein; XP, xeroderma pigmentosum.

aspects of DNA damage recognition and incision, and

patients with XP can carry mutations in any of the

genes that specify these proteins Cell lines established

from patients with mutations in one of these genes are

referred to as XP-A, XP-B, XP-C, XP-D, XP-E, XP-F,

or XP-G cells, depending on which gene houses the

mutations These cell lines have served as essential

tools in studies of NER

Results from a wide variety of biochemical and

bio-physical studies have illuminated mechanistic aspects

of DNA damage recognition and incision in eukaryotic

cells, and are reviewed herein We will mainly discuss

human NER in this review These studies reveal that

NER is a dynamic process in which pivotal proteins

are assembled and disassembled as needed [4,5]

NER reactions: an overview

The NER mechanism in mammalian cells involves (a)

DNA damage recognition and assembly of the protein

complex that carries out DNA incision around the

lesion, (b) incision of the damaged DNA strand on

both sides of the injury, which results in damage

exci-sion, and (c) synthesis and ligation of a stretch of

DNA to repair the gap created by the excision In

TCR, stalled RNA polymerase II acts as a marker for

recognition of the lesion by the DNA repair

machin-ery With respect to the GGR pathway, although the

order of arrival and departure of each factor at a

lesion remains controversial, it is widely accepted that

GGR in human cells occurs as follows DNA

damage-induced helical distortion is recognized by the XPC–

hHR23B complex, and transcription factor IIH

(TFIIH; which consists of nine subunits), XPA (a

possible homodimer) and replication protein A (RPA,

which consists of three subunits) arrive sequentially at

the site of the damage and constitute the pre-incision

complex Endonuclease XPG and the XPF–ERCC1

complex are responsible for the 3¢ and 5¢ DNA

inci-sions, respectively Binding of XPG induces the release

of XPC–hHR23B, whereas XPF–ERCC1 triggers

exci-sion of the damaged DNA and the release of XPA

and TFIIH Subsequently, the newly formed gap in

the DNA is filled by DNA polymerase d⁄ e, replication

factor C, proliferating cell nuclear antigen, RPA and

DNA ligase I (Fig 1)

For the NER process to be executed successfully,

multiple protein–protein and protein–DNA

interac-tions must occur in the appropriate order For

exam-ple, XPC interacts with the p62 subunit of TFIIH and,

in turn, p62 interacts with XPG The results of

intri-cate studies designed to characterize these interactions

are reviewed below

The XPC–hHR23B complex: a sensor

of helical distortion

XPC and its partners XPC is a 125 kDa protein that interacts with a variety

of factors, including hHR23B, TFIIH and DNA XPC

is known to form a stable complex with the hHR23B and centrin2 proteins (see below) Although the XPC subunit is solely responsible for binding of the XPC– hHR23B complex to sites of DNA damage, hHR23B stimulates XPC to function in NER and is also necessary for XPA–RPA-mediated displacement of the

Fig 1 Scheme of the global genomic repair (GGR) pathway The sequential arrivals and departures of nucleotide excision repair (NER) components are marked with arrows Proteins are defined throughout the text Adapted by permission from Macmillan Pub-lishers Ltd: EMBO Journal, [4], copyright (2003).

XPC–hHR23B complex from damaged DNA during

the early stages of the NER process [5] (Fig 2A)

hHR23B is a 58 kDa human homolog of the yeast

NER protein, RAD23 In addition to an XPC-binding

domain, hHR23B has an N-terminal ubiquitin

(Ub)-like domain and two Ub-association domains (UBA1

and UBA2) Therefore, hHR23B is a modular protein,

and solution structures of its domains and possible

intramolecular binding surfaces have been described [6]

(Fig 2B,C) Recently, the centrin 2 protein, which exists in a complex with XPC and hHR23B, was shown to stimulate the NER activity of XPC by enhancing damage recognition [7]

A recent report revealed that, upon UV irradiation, XPC undergoes reversible ubiquitylation, and this reaction depends on the presence of a UV-damaged DNA-binding protein (UV-DDB) The UV-DDB com-plex consists of the DDB1 (p127) and DDB2 (p48) proteins When UV irradiates cells, it is associated with Cullin 4A, Roc1 and Cop9 signalosome, which are components of ubiquitin ligase (E3) [8] The UV-DDB binds specifically to lesions caused by UV irradiation, such as (6–4) photoproducts and cyclopyrimidine dimers (CPDs) Studies of XP-E cells, which have mutations in the DDB2 gene, have revealed that the UV-DDB represents an initial damage sensor, especi-ally for CPD lesions However, the mechanism by which UV-DDB and XPC are functionally linked, in terms of damage recognition, in the GGR process remains to be elucidated

Sugasawa et al showed that the ubiquitylation of XPC is involved in the transfer of the UV-induced DNA lesion from UV-DDB to XPC Even though UV-induced multi-ubiquitylation of XPC occurs through the UV-DDB-associated E3 complex, it does not serve as a signal for protein degradation The UBA domains of hHR23B are thought to protect XPC from the ubiquitin⁄ proteasome system XPC is also modified by SUMO-1 – a member of the small ubiqu-itin-like modifier family of proteins – following UV irradiation, and this modification event is dependent

on XPA activity [9], which is known to be necessary for preventing UV-induced XPC degradation There-fore, sumoylation is believed to play a role in stabiliza-tion of the XPC protein These various UV-induced post-transcriptional modifications of XPC appear to

be crucial for the serial binding and release of proteins

to and from the DNA-damage site, before and after XPC binding However, the precise molecular interac-tions that orchestrate this intricate game of musical chairs are not yet fully understood (Fig 2C)

The 3D structures of the core XPC-binding domains

of hHR23B and hHR23A have been solved [10,11] (Fig 2C) These two XPC-binding domains each con-sist of five similar alpha helices, as well as differentially distributed hydrophobic surfaces that make direct con-tact with the XPC The DNA-binding domain of XPC overlaps with its hHR23B interaction domain [12] However, a dearth of structural information for the hHR23B-binding site of XPC makes it difficult to determine precisely how these proteins interact with each other

A

B

C

Fig 2 Structures of the nucleotide excision repair (NER) players.

(A) Domain structure of the human xeroderma pigmentosum C

(XPC) protein Binding sites for interaction partners are shown with

arrows (B) Domain structure of the hHR23B protein (C) Solution

structures for each domain of hHR23B UbL, ubiquitin-like domain.

The Protein Data Bank (PDB) entry code for Ubl is 1P1A UBA,

ubiquitin association domain UBA1 and UBA2 structures were

derived for hHR23; the PDB entry codes are 1IFY and 1 DV0,

respectively The PDB entry code for the XPC-binding domain of

hHR23B is 1PVE All figures were generated from PDB files using

SWISS-PDB VIEWER and POV-RAY The linker regions, which were not

structurally determined, are shown by dotted lines Important

bind-ing partners are also indicated as connectbind-ing arrows Ubiquitin

interacts with UBA1 and UBA2, as well as PubS2, in the

protea-some complex UbL can bind with the same partners, UBA1, UBA2

and PubS2 The XPC-binding domain binds to XPC.

The XPC–hHR23B complex is known to interact

preferentially with damaged DNA substrates, such as

(6–4) photoproducts or acetaminoflorene adducts

[1,13,14] However, XPC-hHR23B recognizes CPDs

poorly, which implies that recognition of such lesions

requires additional factors [15] By using a series of

artificial DNA substrates that contained mismatched

bases opposite a CPD, Sugasawa et al performed a

series of experiments, the results of which suggest that

the increased structural distortion caused by having

a mismatched base opposite a CPD enhances XPC–

hHR23B binding to these lesions [15] This hypothesis

was supported by the results of an NMR structural

study of DNA decamers that was designed to elucidate

the influence of mismatched bases on the DNA

struc-tures containing CPD [16] Although the hydrogen

bonds between CPDs and the mismatched bases are

maintained, helical bending, backbone conformation

and the major and⁄ or minor grooves differ between

CPDs that have correct bases and CPDs that have

mismatched bases on the opposite DNA strand

There-fore, these structural properties might play a role in

determining the binding affinity of XPC–hHR23B for

DNA Furthermore, it is known that DNA bending is

induced by UV-DDB binding to damaged DNA sites

Taken together, these findings suggest that the

struc-tural properties of DNA-damaged substrates, whether

intrinsic or the result of protein binding, function in

the recruitment of the XPC–hHR23B complex to sites

of DNA damage

The protein shuffle

In the GGR pathway, one study has shown that

XPC–hHR23B interacts with the p62 subunit of

TFIIH and recruits TFIIH to sites of helical

distort-ion [17] Another study has suggested that XPC–

hHR23B is able to interact with XPA during the

transition from an initial damage-recognition

inter-mediate (involving XPC and TFIIH) to the

forma-tion of an ultimate incision complex [5] In NER

assays reconstituted in vitro, XPC does not remain

in contact with the DNA substrate during the dual

incision reaction, as this initial damage sensor is

released from the excision machine when XPG and

XPA associate with the damaged DNA [4,5,18] It is

still not known how hHR23B triggers XPC

displace-ment from damaged DNA upon arrival of the XPA–

RPA complex or which domains of XPC and XPA

are responsible for interacting each other More

research is required to elucidate the various steps of

this handing-off process that occurs in the initial

steps of NER

TFIIH: shuttling between repair and transcription

TFIIH consists of nine protein subunits: XPB, XPD, p62, p52, p44, p34, cdk7, cyclin H and MAT1 XPB and XPD are DNA helicases, and their ATP-depend-ent DNA unwinding activities have been reviewed pre-viously [19] In addition to its helicase activities, TFIIH is directly responsible for the recruitment of XPG and XPA to the nascent DNA damage excision complex [20,21] A recent NMR study revealed that the N-terminal region of the p62 subunit of TFIIH contains a pleckstrin homology⁄ phosphotyrosine bind-ing (PH⁄ PTB) domain that associates with XPG [22] The PH⁄ PTB domain adopts a b-sandwich fold that (a) contains two nearly orthogonal b-sheets made up

of seven antiparallel b-strands and (b) is closed off at one end by a long C-terminal a-helix (Fig 3) Because this domain also interacts with acidic transcriptional activator proteins, such as p53 and VP16, the involve-ment of the PH domain in NER raises interesting questions regarding the dual role of TFIIH in tran-scription and DNA repair It is known that TFIIH complexes which have been released from the NER dual-incision complex can support mRNA synthesis by RNA polymerase II in a reconstituted transcription assay Moreover, it was shown recently that yeast TFIIH houses a Ub ligase (E3) activity that plays a regulatory role in the transcription of DNA damage response genes Specifically, the RING finger motifs in

Fig 3 Transcription factor IIH (TFIIH) (A) Molecular composition of TFIIH and its interacting partners (B) Solution structure of the N-terminal region of the p62 subunit The PDB entry code is 1PFJ.

the Ssl1 subunit of yeast TFIIH are responsible for the

observed Ub ligase activity [23] (Ssl is a homolog of

the p44 subunit of human TFIIH) This finding

sug-gests that TFIIH participates in DNA repair, not only

through its commonly required helicase activities, but

also through the transcriptional regulation of DNA

repair genes

XPA-RPA: a linchpin of the NER

network of interactions

XPA is a 36 kDa zinc metalloprotein that interacts

with many other NER subunits, such as RPA (see

below), ERCC1 (a binding partner of XPF, a 5¢

endo-nuclease) and TFIIH (see above) [21,24,25] The

N-ter-minal region of XPA (residues 1–97) is responsible for

the interaction with RPA32 and ERCC1 The central

part of the protein (residues 98–219) consists of zinc

finger and loop-rich subdomains, which are able to

bind to RPA70 and DNA [24,26] (Fig 4) The NMR

structure of this domain showed the existence of a

pos-itively charged cleft and confirmed that DNA binding

occurs in the loop-rich subdomain and that RPA70

interactions occur in the zinc-binding core [27] NMR

studies also showed that the ERCC1-binding region of

XPA is unstructured and forms a transient

intramole-cular association with the DNA-binding domain of

XPA [28] These results suggest that ERCC1 binding

to XPA would be possible only after damaged DNA

displaces the XPA ERCC1-binding region from its

DNA-binding domain

RPA is an abundant, heterotrimeric ssDNA-binding

complex that is composed of 70-, 32- and 14 kDa

polypeptide subunits (RPA70, RPA32 and RPA14) The ssDNA-binding activity resides mainly in the cen-tral region of the 70 kDa subunit, which contains two tandem oligonucleotide binding folds [29–31] The oligonucleotide binding folds consist of five b strands coiled to form a closed b barrel that is capped by an

a helix located between the third and fourth b strands ssDNA binds to the protein via extensive electrostatic interactions and stacking contacts The rest of RPA70, as well as its DNA-binding domain, interact with protein partners that are involved in DNA repair, recombination and replication pathways (Fig 4) [32–36]

In the NER system, RPA participates in both early and late steps of the process For example, early in the NER process, RPA assists TFIIH in the opening of the DNA helix around the damage site [36] Further-more, in the presence of the XPA minimal DNA-binding domain (XPA-MBD), RPA70AB (residues 181–422) shows a tendency to interact with the undam-aged strand opposite the DNA damage site [32] This result implies that RPA protects the intact DNA strand from inadvertent nuclease attack With respect

to XPA–RPA interactions, NMR analysis of RPA70 (residues 1–326) and XPA–MBD (residues 98–219) fragments revealed that the XPA-MBD site of RPA overlaps with its ssDNA-binding region Therefore, XPA–RPA interactions appear to be modulated by ssDNA–RPA binding [34] RPA32 (residues 172–270) also interacts with the N-terminal region of XPA in a manner similar to the mode of RPA32 binding to human uracil-DNA glycosylase and Rad52 This result reveals that RPA participates in multiple DNA repair

Fig 4 Domain structure of the human xeroderma pigmentosum A (XPA) protein and solution structure of the XPA minimal DNA-binding domain (XPA–MBD) (PDB entry: 1XPA) 3D structures of each domain

in the human replication protein A (RPA) protein are shown These include the C-terminal part of RPA32 (PDB entry: 1DPU), the N-terminal part of RPA70 (PDB entry: 1EWI), the RPA70AB–dC8 complex (PDB entry: 1JMC), and the trimerization core, which consists of the C-terminal part

of RPA70, the N-terminal part of RPA32, and the N-terminal part of RPA14 (PDB entry: 1LIO).

pathways by selective binding to functionally distinct

partner proteins [37]

In later steps of the NER pathway, the XPA–RPA

complex interacts with XPG and the XPF–ERCC1

com-plex Through a stable interaction with TFIIH, XPG is

already present in the NER complex prior to the arrival

of the XPA subunit [20] The interaction between XPA–

RPA and XPG contributes to their association with the

DNA substrate mutually [4] RPA remains in NER

complexes after the dual incision reactions and

partici-pates in the DNA resynthesis step (Fig 1)

XPG and the XPF–ERCC1 complex:

structure-specific nucleases

XPG is a 133 kDa protein and a member of the FEN-1

family of structure-specific nucleases As is the case

with other members of the FEN-1 family, XPG has

two highly conserved nuclease motifs known as the

N- and I regions [38] Although these regions are

sep-arated by only a short helical loop in other FEN-1

family members, the N- and I regions in XPG are

separated by a large insertion that was shown to be

responsible for the binding of XPG to the other

TFIIH subunits [39] In addition to mediating

pro-tein–protein interactions, the spacer region may also

contribute to the substrate specificity of XPG These

findings demonstrate that XPG acts as a modular

pro-tein, helping to orchestrate progression through the

NER process via its functionally independent domains

which interact specifically with other NER proteins

and DNA substrates [39]

XPF–ERCC1 is the last protein complex to join the NER incision complex, and it does so by interacting specifically with both XPA and RPA [40] XPG is also required for the recruitment of XPF–ERCC1 to the site of DNA damage and accomplishes this task by inducing a structural change in the pre-incision com-plex XPF–ERCC1 cleaves DNA at sites 5¢ to the lesion The XPF subunit consists of three domains, namely (a) an N-terminal helicase-like domain, (b) a central nuclease domain, and (c) a C-terminal helix– hairpin–helix [(HhH)2] domain; the ERCC1 subunit consists of only two domains, namely (a) a central region that is similar to the XPF nuclease domain, but

is devoid of residues characteristic of proteins with nuclease activity, and (b) a C-terminal (HhH)2 domain (Fig 5) The C-terminal (HhH)2 domains of both XPF and ERCC1 mediate binding between the two proteins, mainly by hydrophobic interactions [41]

Recently, a crystal structure of the crenarchaeal XPF homodimer, alone and bound to double-stran-ded DNA (dsDNA) [42], the central domain of human ERCC1 as well as the (HhH)2 domain het-erodimer of human XPF–ERCC1 [43], and a solu-tion structure of human XPF–ERCC1 (HhH)2 domain complex [44], were published (Fig 5) The central domain of human ERCC1 closely resembles the nuclease domains of XPF from humans and other organisms, despite low percentages of sequence identity

Investigations into DNA interaction of the protein complex have provided an insight into the roles of ERCC1 in the NER process Tsodikov and his

Fig 5 Domain structures of the human

xeroderma pigmentosum F (XPF) and

ERCC1 proteins Crystal structure of a

complex containing the C-terminal domains

of human XPF and ERCC1 (PDB entry:

2A1J) (left); crystal structure of the central

domain of human ERCC1 (PDB entry: 2A1I)

(right).

collaborators reported that positively charged and

aro-matic residues in the central domain of ERCC1 are

spe-cially responsible for its interaction with ssDNA [43] It

was also observed that the each component of the

XPF–ERCC1 (HhH)2 complex displayed the ability to

bind to ssDNA in their crystal structure Chemical shift

perturbation data of Tripsianes and his collaborators is

not fully consistent with the previous model They

indi-cated that the (HhH)2 domain of ERCC1 has the

DNA-binding activity that is not possessed by (HhH)2

domain of XPF [44] Even though there is an

inconsis-tency which remains to be identified, these results show

that ERCC1 serves to localize the XPF nuclease

domain properly by binding the ssDNA strand through

the central and (HhH)2domains [45]

Conclusion and perspectives

Recent studies have emphasized that components of

the NER process interact with one another in a

dynamic manner and participate in other DNA

meta-bolizing pathways using their diverse structural

domains The structural studies described above were

instrumental in deciphering the details of the various

molecular interactions among NER players, such as

those that occur in the XPC–hHR23B, XPA–RPA and

XPF–ERCC1 complexes

The observations, that hHR23B contains

Ub-relat-ed modules and that XPC undergoes ubiquitylation,

raise the possibility that the protein degradation

pro-teasome pathway can communicate with the NER

pathway Another intriguing finding is that a number

of the NER proteins are multifunctional For

exam-ple, TFIIH plays a critical role in both RNA

poly-merase II transcription and the DNA repair process

by interacting with suitable protein partners

Mul-tiple roles for RPA have also been documented For

example, RPA functions in NER as well as other

DNA processing reactions by selective binding to a

variety of proteins

Results from the collection of studies described in

this article highlight several important questions that

need to be answered if researchers are to fully define

the NER process To achieve this, scientists will need

to produce a detailed map of the sequential protein

assembly processes that occur during damage

recogni-tion and repair

Acknowledgements

This work was supported by the National Creative

Research Initiative Program to B.-S.C from the

Minis-try of Science and Technology, Korea

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