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

MINIREVIEW
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:
(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.
1600 FEBS Journal 273 (2006) 1600–1608 ª 2006 The Authors Journal compilation ª 2006 FEBS
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).
C J. Park & B S. Choi The protein shuffle in NER pathway
FEBS Journal 273 (2006) 1600–1608 ª 2006 The Authors Journal compilation ª 2006 FEBS 1601
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 connecting 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 protein shuffle in NER pathway C J. Park & B S. Choi
1602 FEBS Journal 273 (2006) 1600–1608 ª 2006 The Authors Journal compilation ª 2006 FEBS
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.
C J. Park & B S. Choi The protein shuffle in NER pathway
FEBS Journal 273 (2006) 1600–1608 ª 2006 The Authors Journal compilation ª 2006 FEBS 1603
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).
The protein shuffle in NER pathway C J. Park & B S. Choi
1604 FEBS Journal 273 (2006) 1600–1608 ª 2006 The Authors Journal compilation ª 2006 FEBS
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).
C J. Park & B S. Choi The protein shuffle in NER pathway
FEBS Journal 273 (2006) 1600–1608 ª 2006 The Authors Journal compilation ª 2006 FEBS 1605
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)
2
domains [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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