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;

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

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

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,

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

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

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].

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 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-

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