the structure of the xpf-ssdna complex underscores the distinct roles of the xpf and ercc1 helix-...
TRANSCRIPT
Structure
Article
TheStructureof theXPF-ssDNAComplexUnderscoresthe Distinct Roles of the XPF and ERCC1 Helix-Hairpin-Helix Domains in ss/ds DNA RecognitionDevashish Das,1,3 Gert E. Folkers,1 Marc van Dijk,1 Nicolaas G.J. Jaspers,2 Jan H.J. Hoeijmakers,2 Robert Kaptein,1
and Rolf Boelens1,*1Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands2Department of Cell Biology and Genetics, Erasmus MC, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands3Department of Radiology, Radboud University Nijmegen Medical Center, Geert Grooteplein 10, 6525 GA, Nijmegen, The Netherlands
*Correspondence: [email protected]
DOI 10.1016/j.str.2012.02.009
SUMMARY
Human XPF/ERCC1 is a structure-specific DNAendonuclease that nicks the damaged DNA strandat the 50 end during nucleotide excision repair.We determined the structure of the complex of theC-terminal domain of XPF with 10 nt ssDNA. A posi-tively charged region within the second helix of thefirst HhH motif contacts the ssDNA phosphate back-bone. One guanine base is flipped out of register andpositioned in a pocket contacting residues from bothHhH motifs of XPF. Comparison to other HhH-con-taining proteins indicates a one-residue deletionin the second HhH motif of XPF that has alteredthe hairpin conformation, thereby permitting ssDNAinteractions. Previous nuclear magnetic resonancestudies showed that ERCC1 in the XPF-ERCC1 het-erodimer can bind dsDNA. Combining the two obser-vations gives amodel that underscores the asymme-try of the human XPF/ERCC1 heterodimer in bindingat an ss/ds DNA junction.
INTRODUCTION
Ultraviolet (UV) radiation, chemical carcinogens, and cellular
metabolites are important contributors of DNA damage in cells
(Hoeijmakers, 2001). Of particular importance is the mammalian
nucleotide excision repair (NER) pathway and associated gene
products, which coordinate the elimination of UV-radiation-
induced DNA lesions, like CPDs and 6-4 photoproducts (de
Laat et al., 1999; Scharer, 2007). The NER pathway entails a
collective action of at least 25 proteins that are assembled at
the DNA lesion (de Laat et al., 1999; Riedl et al., 2003; Volker
et al., 2001). Two proteins, ERCC1 and XPF, form a very stable
complex. The role of the ERCC1/XPF complex is well character-
ized (Bessho et al., 1997; de Laat et al., 1998a, 1998c; Enzlin and
Scharer, 2002; Gaillard and Wood, 2001; Houtsmuller et al.,
1999; Park et al., 1995; Sijbers et al., 1996; Tsodikov et al.,
2005), demonstrating that the complex functions as a struc-
ture-specific DNA endonuclease that nicks at the 50 end of the
Structure 20
damaged DNA strand during nucleotide excision repair (de
Laat et al., 1999; Riedl et al., 2003; Scharer, 2007; Sijbers
et al., 1996), leading to the excision of 24 to 32 nucleotides after
concomitant incision 30 to the damage by XPG (de Laat et al.,
1999; Scharer, 2007). In NER, XPF/ERCC1 is the last factor to
be assembled at the 50 end of the DNA lesion (Houtsmuller
et al., 1999; Riedl et al., 2003; Volker et al., 2001), where it inter-
acts with other NER proteins (de Laat et al., 1998b; Orelli et al.,
2009; Tripsianes et al., 2007; Tsodikov et al., 2007; Winkler
et al., 2001). The crucial role of XPF/ERCC1 in NER follows
from observations that both ERCC1 (McWhir et al., 1993; Weeda
et al., 1997) and XPF (Tian et al., 2004) knockoutmice showmore
severe phenotypes compared to mice lacking other NER genes
(Melis et al., 2008). Along with the well-established role of XPF/
ERCC1 in the NER pathway, additional roles in DNA processing
have been described, including interstrand cross-link repair
(Kuraoka et al., 2000; Niedernhofer et al., 2004), telomere length
control (Munoz et al., 2005;Wu et al., 2008), homologous recom-
bination repair (Ahmad et al., 2008; Clingen et al., 2005), and
gene-targeting events (Al-Minawi et al., 2008; Niedernhofer
et al., 2001). The precise DNA recognition in these mechanisms
is structurally not understood.
The C-terminal HhH domains of the human XPF and ERCC1
proteins are required and sufficient for stable heterodimer forma-
tion (de Laat et al., 1998c). Structure determination of the
minimal dimerization domains of the two proteins showed the
presence of two canonical helix-hairpin-helix motifs (HhH)
related by a pseudo-2-fold symmetry axis (Tripsianes et al.,
2005; Tsodikov et al., 2005). We and others independently re-
ported that ERCC1 could only fold in the presence of XPF, the
latter acting as a scaffold (Choi et al., 2005; Tripsianes et al.,
2005). Whereas the C-terminal domain of ERCC1 was insoluble
in isolation, the C-terminal domain of XPFwas highly soluble. We
subsequently showed that this XPF domain forms a homodimer
that is significantly more stable than is the heterodimeric XPF/
ERCC1 complex. Nuclear magnetic resonance (NMR) structural
analysis provided a molecular explanation for the higher stability
in vitro (Das et al., 2008). NMR chemical shift perturbations found
by the addition of a hairpin substrate with 22 unpaired nucleo-
tides (H22) showed that the two hairpins of the HhH domain of
ERCC1 were primarily affected by the addition of DNA (Trip-
sianes et al., 2005). Using the structural homology with other
HhH domain proteins (Shao and Grishin, 2000), including RuvA
, 667–675, April 4, 2012 ª2012 Elsevier Ltd All rights reserved 667
Structure
Structure of the XPF-ssDNA Complex
bound to a Holliday junction (Ariyoshi et al., 2000) and the
archaeal homodimeric XPF bound to DNA (Newman et al.,
2005), a model was proposed for the ERCC1-dsDNA complex
with a clear role for the two ERCC1 hairpins (Tripsianes et al.,
2005). This mode of DNA binding by the C-terminal ERCC1
HhH domain fits well with the presence of two canonical HhH
motifs. However, the role of the XPF HhH motifs in DNA binding
was less clear. The loss of the canonical hairpin structure of XPF
led us to hypothesize that XPF would recognize a different DNA
structure.
Using mass spectrometry, NMR spectroscopy, and in vitro
DNA binding assays, we show here that the XPF homodimer
forms a stable complex with single-strand DNA. We determined
the solution structure of the human XPF homodimer in complex
with ssDNA which shows that the DNA forms a right-handed
structure that wraps around the protein mainly via interactions
along the ssDNA phosphate backbone. Interestingly one
guanine base is flipped out of register and is positioned in a
hydrophobic cavity formed between canonical HhH1 and the
distorted HhH2. Combined with the previously established
ERCC1-dsDNA model (Tripsianes et al., 2005), this allowed us
to build a model for the complex of ERCC1/XPF, which explains
the positioning of this endonuclease at the ss/dsDNA junction.
RESULTS
ssDNA Binding by the C-terminal XPF HhH HomodimerPreviously, we reported the DNA binding activity of the HhH
domains of the XPF/ERCC1 heterodimer using electrophoretic
mobility shift assay (EMSA) experiments and NMR spectroscopy
(Tripsianes et al., 2005). The NMR spectra of the XPF/ERCC1
heterodimer in complexes with a DNA hairpin revealed signifi-
cant chemical shift perturbations for residues of the HhH motifs
of the ERCC1 protein indicative for dsDNA binding, whereas
smaller but consistent shifts were found for the XPF residues.
To investigate the significance of these XPF chemical shifts
perturbations, we decided to further analyze the ability of the
C-terminal XPF homodimer, (XPF)2 to bind to various DNA
substrates.
First, we performed electrophoretic mobility shift assays using
the same probes for the (XPF)2 homodimer as used previously for
the XPF/ERCC1 heterodimer (Tripsianes et al., 2005). Similar to
the XPF/ERCC1 heterodimer, the (XPF)2 homodimer has a sub-
micromolar affinity for dsDNA, containing a bubble. However,
in contrast to the ERCC1/XPF heterodimer, which has only
weak affinity for ssDNA in electrophoretic mobility shift assays
(Tripsianes et al., 2005), we noted that the (XPF)2 homodimer
binds to ssDNA substrates. For a long 39 nt ssDNA fragment it
even binds with a micromolar range affinity (Figure S5 available
online). The ability of (XPF)2 to form a stable complex in the pres-
ence of an excess of 39 nt ssDNA was also established using
mass spectrometry. Next to the presence of some free (XPF)2,
matrix-assisted laser desorption/ionization time of flight mass
spectrometry experiments (MALDI-TOF-MS) revealed the pres-
ence of a major ion signal at m/z 15433.33 attributable to a 2:1
XPF/39 nt ssDNA complex (Figure 1A). These results show that
the (XPF)2 homodimers can form a stable complex with a single
long single-strand DNA fragment. The 2:1 stoichiometry can be
explained by the fact that the 39 nt ssDNA can loop around
668 Structure 20, 667–675, April 4, 2012 ª2012 Elsevier Ltd All rights
(XPF)2 and different parts of the 39 nt ssDNA fragment bind to
the two DNA binding sites of the dimer.
For the NMR structure determination of the XPF-ssDNA
complex, a 10 nt substrate, CAGTGGCTGA, was used. Though
this fragment has much weaker affinity (estimated Kd > 50 mM),
it leads as shown below in the NMR experiments to a 2:2
complex, where the symmetry of the (XPF)2 homodimer is main-
tained, which has considerable advances for the NMR analysis.
The formation of the complex could be easily followed by 31P
NMR. Relatively large 31P chemical shift changes are noted for
the DNAphosphate backbone upon addition of the (XPF)2 homo-
dimer. For further analysis, the 31P and 1H spectra of the free and
bound ssDNA fragment resonance assignments were required.
For this, we recorded two-dimensional (2D) 1H-1H NOESY and
the 2D 31P-1H HMQC spectra (Figures S1A, S1B, and S2A).
The obtained 1H chemical shifts for free and (XPF)2 bound
CAGTGGCTGA are shown in the Tables S1 and S2. The largest
chemical shift changes upon binding can be noted for nucleo-
tides 3–7, suggesting that this region of the ssDNA fragment is
involved in XPF binding (Table S3 and Figure 1C).
For XPF, DNA binding is also evident by comparing the 1H-15N
HSQC NMR spectra of free and DNA bound (XPF)2 homodimers
(Figure 2A). The 15N-1H HSQC spectra show chemical shift
changes for a small subset of peaks, demonstrating the speci-
ficity of the XPF homodimer for ssDNA. Since the spectra
show a single set of peaks during the titrations, the two 10 nt
ssDNA fragments should bind symmetrically to the homodimers
in a 2:2 stoichiometry. The chemical shift perturbations (CSP) are
most prominent for residues of the second helix of the canonical
HhHmotif, namely, Lys850, Asn851, Ser854, Leu855, Met856, His857,
His858, and Val859 (Figure 2B).
NMR titrations of the XPF/ERCC1 heterodimer with the same
10 nt ssDNA show chemical shift perturbations for similar resi-
dues as observed for XPF homodimers (Figures 2B and S3).
Based on this similarity, the complex of the homodimer (XPF)2with ssDNA is anticipated to be a good model for a structural
analysis of ssDNA binding by XPF in the XPF/ERCC1 hetero-
dimer. Compared to the heterodimer, the homodimer-ssDNA
complex has considerable advantages for the NMR spectral
analysis and structure because of the symmetry, resulting in
a lower number of signals and reduced spectral overlap.
Structure of the XPF-10 nt ssDNA ComplexThe structure of the (XPF)2 homodimer bound to 10 nucleotide
single-strand DNA (CAGTGGCTGA) was determined on the
basis of 3184 distance restraints and 178 dihedral angle
restraints. A summary of all structural and restraints statistics
is given in Table 1. The ensemble backbone rmsd for the 20
conformers is 0.57A for the well-folded part of the protein and
comparable to that of 0.39 A for the free structure (Das et al.,
2008). Figure 3A shows an overlay of the final 20 lowest-energy
conformers after water refinement.
The structure of the (XPF)2 bound to two single DNA strands
show a similar fold as free (XPF)2 (Das et al., 2008), with a back-
bone rmsd of �0.69 A between the two. For residues 845–861
and 8450–8610, which are close to ssDNA, we note minor
changes in the conformation of the side chains (rmsd �1.14 A).
In the XPF protein, the residues, which are part of the conserved
canonical and noncanonical HhH motif, form a cavity (Das et al.,
reserved
A
B
C
10.0 20.0 30.0 40.0
m/z
Inte
nsity
500.0
1000.0
1500.0
(ssDNA)(ssDNA)
2+
(Free XPF)2
+
+
11998.29
23995.09
15433.33
18881.18
(XPF-ssDNA Complex) +
30887.69
2 +(XPF-ssDNA Complex)
37792.43
Free DNA
31P
XPF-DNA complex
−0.7 −0.8 −0.9 −1.0 −1.1 −1.2 −1.3 −1.4 −1.5 ppm1.82.02.22.42.6
Nucleotides
1 2 3 4 5 6 7 8 9 10
5’C
A
G
T
G
G
C
T
G A
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
H C
SP
Sum
/ N
ato
ms
1
kDa
Figure 1. The C-terminal XPF HhH Homodimer
Binds ssDNA
(A) MALDI-TOF (MS) spectrum of the XPF-ssDNA
complex. The signals are assigned based on the expected
mass/charge ratio of the free and bound species.
(B) Overlay of 1D phosphorus spectra, free (red) and
bound (blue) states of the (XPF)2-(CAGTGGCTGA)2complex, indicating changes in the phosphate backbone
in the free and bound forms of the ssDNA.
(C) Plot of the 1H chemical shift differences of the10 nt
ssDNA fragment in free and bound forms. The chemical
shift differences were summed for all atoms of each
nucleotide of the10 nt ssDNA and normalized by dividing
by the number of atoms.
See Figure S1 and Tables S1, S2, and S3 for additional
NMR data and Figure S5 for an EMSA assay of ssDNA
binding by (XPF)2.
Structure
Structure of the XPF-ssDNA Complex
2008; Tripsianes et al., 2005). In the DNA-bound structure, the
base of Gua5 is positioned in this cavity and has a large interac-
tion surface area of �140A2 with the protein (Figure 3B). Altered
conformations are seen for the Lys850 and Asn879 side chains in
the free and DNA-bound XPF. This altered side-chain conforma-
tion of Lys850 allows for a hydrogen bond contact to the phos-
phate group of Cyt7. The slight reorientation of the Asn879 side
chain results in a hydrogen bond with Gua5, as is observed in
�30% of the conformers of the final ensemble.
Figure 3B shows a closer view of the ssDNA in the complex.
The DNA binding surface shows a buried surface area of
640A2 for each subunit. For nucleotides 2–7, which contact the
protein, the phosphate backbone is well defined with a rmsd of
1.18 A, whereas the nucleotides 8–10 have no well-defined
structure. The nucleotide 3–7 region adopts a right-handed
helical structure (Figure 3B) as supported by strong sequential
Structure 20, 667–675, April 4
protein-DNA NOEs between the H10 ribose
protons of Gua5 and Gua6 and side-chain
protons of Lys850 and Arg853, respectively (Fig-
ure S2B). Analysis of the DNA geometry shows
that the right-handed helical structure has
a rather extended conformation, which is quite
distinct from B-DNA (Table S4 and Figure S4).
This geometry cannot be easily adapted in
dsDNA, which can certainly contribute to the
preference of XPF for ssDNA.
Interactions at Interface of theXPF-ssDNA ComplexFigures 3B and 4A show how the ssDNA is posi-
tioned on the surface of XPF. The arrangement
of the ssDNA on the protein is a result of several
electrostatic contacts between protein and
DNA, mainly at the phosphate backbone. For
example, the side chain of Lys850 interacts
with the phosphate group of Cyt7, and the
side chain of Arg853 contacts the phosphate
groups of both Gua5 and Gua6 in �80% of
the conformers. A summary of the protein-
DNA contacts is shown in Figure 4B. Interest-
ingly, the Ser854 hydroxyl group interacts with
the oxygen O50 atom of Thy4. The hydrogen bonding of the OH
of Ser854 to DNA is confirmed by a slow exchange of the Ser
OH proton with water leading to an observable NOE between
the OH and the Ser854 CH2 protons. Without hydrogen bonding
stabilization, such OH protons are normally not observed in solu-
tion NMR spectra. Additionally, the side chain of His857 makes
a hydrogen bond interaction with Gua3, whereas the His858
side chain contacts the phosphate group of Gua3. Consistent
with the observed interaction between His857 side chain and
ssDNA, we find an NOE between the His857 HD1 proton and
the side-chain beta-protons only in the protein-DNA complex
(Figure S2C).
Most of the DNA bases are sticking outwards. A remarkable
aspect of this protein-DNA complex is, however, the position
of the guanine base of Gua5. This base is flipped out of register
with respect to the other bases and positioned in a pocket of
, 2012 ª2012 Elsevier Ltd All rights reserved 669
Table 1. Structural Statistics of the (XPF)2-(CAGTGGCTGA)2Conformers
rmsd (A) with respect to mean (backbone)a
(XPF)2 0.57
(XPF-ssDNA)2 1.17
No. of experimental restraintsb
DNA
Intramolecular DNA-DNA NOE 192
Protein-DNA
Total intermolecular protein-DNA 42
Homodimeric XPF2
Intraresidue NOE 664
Sequential NOEs (ji � jj = 1) 778
Medium-range NOEs (1 < ji � jj < 4) 739
Long-range NOEs (ji � jj > 4) 514
Interprotein 255
Total NOEs 3,184
Dihedral angle restraints 178
Restraint violations (A)
NOE distances with violations >0.5 A 4.3 ± 0.6
Dihedrals with violations >5� 0
rmsd for experimental restraints (A)
All distance restraints (A) 0.07 ± 0.005
Torsion angles (�) 0.46 ± 0.03
rmsd from idealized covalent geometry
Bonds (A) 0.004 ± 0.000
Angels (�) 0.7 ± 0.0
Improper (�) 0.4 ± 0.0
CNS energies after water refinement
Evdw (kcal/mol) �755.7 ± 19.5
Eelec (kcal/mol) �5708.5 ± 105.4
Ramachandran analysis
Residues in the favored regions (%) 93.3 ± 1.5
Residues in additional allowed regions (%) 4.8 ± 1.6
Residues in generously allowed regions (%) 1.50 ± 0.5
Residues in disallowed regions (%) 0.04 ± 0.2armsd values (in A) were calculated for XPF (835-894) and ssDNA(2-7).bNumber of restraints for the dimer, as used in the calculations.
A
B
8.2 8.0 7.8 7.6 7.4 7.2
122
120
118
116 I890 S875
Q885
K884 D839 F840
A867
A863
T892M844
L886
L855
L865
R853
L868
V859A883
E872
I876
1H
15N
8.4 8.2 8.0 7.8
115.5
115.0
114.5
114.0
113.5
H858
S854
M856
H857
1 H
Free XPF
XPF-DNA complexR853 side chain
CSP
Residue
1.4
1.2
1.0
0.8
0
0.2
0.4
0.6 > average + 2* Std.devi
> average
> average + Std.devi
ppm
missing
1.4-0.5
0.5-0.3
0.3-0.2
0.2-0.02
DSETLPESEKYNPGPQDFLLKMPGVNAKNCRSLMHHVKNIAELAALSQDELTSILGNAANKQLYDFIHTSFAEVVSKGKG
α β γ δ ε
βα
γ
ε
δ
C
N
Figure 2. NMR Chemical Shift Perturbations of the C-terminal XPF
HhH Homodimer upon Addition of ssDNA
(A) Part of a 1H-15N heteronuclear single quantum correlation (HSQC)
spectrum of the (XPF)2-ssDNA complex (blue) and of the free XPF protein (red).
The arrows indicate the observed changes in the titrations of XPF with ssDNA.
(B) Chemical shift perturbation (CSP) upon 10 nt ssDNA binding. CSP values
plotted on the lowest-energy structure of the XPF subunit. CSP values
for the amide proton (HN) and nitrogen (N) are calculated as CSP=ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffið53DppmHNÞ2 + ðDppmNÞ2
q, where the chemical shift difference (D ppmHN)
and (D ppmN) are the proton and nitrogen nuclei in part per million, respec-
tively, for the free and bound states of the protein. The color scheme is from
gray (CSP < 0.2) to red (CSP > 0.48). Black indicates residues for which the
CSP values are missing.
See Figure S3 and Table S4 for 1H-15N HSQC spectrum of the XPF-ERCC1
heterodimer binding to 10 nt ssDNA.
Structure
Structure of the XPF-ssDNA Complex
XPF, an observation that is clearly supported by the unambig-
uous protein-DNA NOE between the Asn879 side-chain atom
(HB) and �NH2 protons of the Gua5 in 2D 1H-1H NOESY and
three-dimensional (3D) 13C-edited-1H-1H NOESY spectra (Fig-
ure S2B). In the NMR structure, the position of the guanine
base within this pocket is very well-defined and is stabilized by
van der Waals contacts and a H-bond of the NH2 group of
Asn879 with the O6 atom of Gua5.
DISCUSSION
Structural Comparison to dsDNA Binding by HhH MotifsIn contrast to XPF, most proteins containing HhH motifs bind
dsDNA (Shao and Grishin, 2000). Several residues within the
two hairpins, as well as in the adjacent helices, contribute to
this dsDNA binding. For example, in the complex of the recom-
670 Structure 20, 667–675, April 4, 2012 ª2012 Elsevier Ltd All rights
bination protein RuvA with Holliday junction DNA, two basic resi-
dues, Lys119 and Arg123, located in the helices of the HhH motif,
bind to a negatively charged DNA phosphate oxygen atoms
(Ariyoshi et al., 2000). Similar protein-DNA contacts have been
reported for the archaeal XPF homodimers (Newman et al.,
2005), for UvrC (Singh et al., 2002) and XPF/ERCC1 (Tripsianes
et al., 2005). Interestingly, in the present XPF-ssDNA complex,
Lys850 and Arg853, which are both residues of XPF located in
equivalent positions as Lys119 and Arg123 of RuvA, contact
ssDNA. In addition, we observe a number of contacts from resi-
dues in the second helix of the first canonical HhH motif of XPF
(Figure 2B). No significant chemical shift perturbations were
observed for the hairpins of (XPF)2, indicating a distinct DNA
binding mode as compared to other HhHmotifs. Most molecular
reserved
Figure 3. Three-dimensional Structure of
the (XPF)2-(CAGTGGCTGA)2 Complex
(A) Stereoview of the final 20 conformers of the
ensemble. The protein subunits are colored blue,
and the DNA is colored green; the hairpins in both
protein subunits are in red.
(B) Surface representation of the XPF-CAGT
GGCTGA complex. The sugar phosphate back-
bone of the DNA is colored gray, the XPF subunit is
colored blue, and the surface is colored light gray.
See Figure S4 and Table S5 for the distribution of
the a (O30-P-O50-C50) and g (O50-C5-C40-C30)torsion angles for nucleotides 3–7 in the (XPF)2-
ssDNA complex.
Structure
Structure of the XPF-ssDNA Complex
contacts were mainly with the phosphate backbone of ssDNA in
an extended geometry. With dsDNA, these interactions would
lead to a disruption of the B-DNA geometry, and this may be
the explanation for the noted preference of XPF for ssDNA.
Structural Comparison to Protein-ssDNA ComplexesThere is a large set of proteins that form complexes with ssDNA
(Bochkarev et al., 1997; Braddock et al., 2002a, 2002b; Brand-
sen et al., 1997; Kerr et al., 2003; Mitton-Fry et al., 2002). In
most of these complexes, DNA bases that mediate protein-
DNA interactions point toward the protein. However, in an alter-
nate situation, DNA bases can also point away from the protein
surface such that the DNA phosphate groups mediate electro-
static contacts with the protein side chains (Werten and Moras,
2006). The structure of RecA-ssDNA complex is a good
example, where it has been noted that the three stacked DNA
bases are sandwiched between the RecA b hairpins with their
Watson-Crick edges solvent exposed and that one of the bases
contacts aliphatic groups of long-chain amino acids like Lys198,
Ile199, and Thr 208 (Chen et al., 2008). Notably, in the present
XPF-ssDNA complex, the phosphate backbone for nucleotide
2 to 7 of the 10 nt ssDNA is largely buried, whereas most bases
of the ssDNA are pointing away from the protein surface. The
ssDNA in the complex exists in a right-handed helical conforma-
tion. Such a right-handed helical ssDNA conformation has been
seen also for the hnRNPK-ssDNA complex (Braddock et al.,
2002a). Thus, the XPF surface presents a scaffold for single
strand DNA binding and the helical conformation of the bound
DNA is determined by the protein side chains, as seen before
in the structure of the CdC13-telomere DNA complex (Mitton-
Structure 20, 667–675, April 4, 2012
Fry et al., 2002). The ssDNA preference
of XPF is clearly distinct from the dsDNA
preference of ERCC1.
A Model for the Splayed Arm DNASubstrate Recognition by theFunctional, Full-Length XPF/ERCC1 HeterodimerIn this study, we determined solution
structure of XPF protein bound to 10 nt
ssDNA. NMR titration experiments also
showed that XPF when present in the
XPF/ERCC1 complex binds the 10 nt
ssDNA in a very similar way. Therefore,
the present structural data of the complex of the (XPF)2 homo-
dimer with ssDNA can serve as a model explaining how the
HhH domain of XPF binds ssDNA at a ss/ds DNA junction in
the ERCC1/XPF complex. Tsodikov et al. (2005) suggested
a model of XPF/ERCC1 binding to a splayed arm DNA substrate
to account for their biochemical and structural findings. In this
model, the HhH domain of XPF is in contact with a single
strand fragment of the splayed arm DNA, which is consistent
with our present structural findings. It has also been reported
that the ERCC1 HhH domain can interact with both dsDNA
and ssDNA (Tripsianes et al., 2005; Tsodikov et al., 2005).
Based on NMR titration data, we suggested that the conserved
HhH domain of the ERCC1 protein contact the phosphate back-
bone of the dsDNA in the minor groove as noted for the RuvA
(Ariyoshi et al., 2000) and archaeal XPF proteins (Newman
et al., 2005).
When combining the current structural data of the (XPF)2-
ssDNA complex with the previous findings of Newman et al.
(2005), Tsodikov et al. (2005, 2007), and Tripsianes et al. (2005,
2007), we can build a model for the complex of XPF/ERCC1,
including the nuclease domains at a splayed arm DNA substrate
(Figure 5). A major highlight of this model is that the XPF HhH
domain interacts with a defined 50/ 30 polarity with one of the
single strand arms of the splayed arm DNA. The ERCC1 HhH
domain interacts with the minor grove at the double stranded
region of a splayed arm duplex DNA. The XPF catalytic residues
on the nuclease domain are in proximity to the cleavage site in
the damaged DNA strand, in a position in accordance with
previous biochemical analysis of this DNA cleavage by XPF/
ERCC1 (de Laat et al., 1998a).
ª2012 Elsevier Ltd All rights reserved 671
A
B5‘
3‘
K850
R853
H858
H857
S854
1
2
3
4
5
6
7
8
9
10
N879
S875
I876
G878
L877
K850N848
N851
N879
H858
A2
G3
T4
G6R853 S854
H857
G5C7
C
5‘
3‘
Figure 4. The Interaction Interface of the XPF-CAGTGGCTGA
Complex
(A) Detailed structural view showing the protein DNA surface. The protein side
chains are colored yellow.
(B) Summary of the interactions between XPF and DNA. Residues mediating
phosphate interactions are shown by black arrows; a blue arrow indicates
nonbonding interactions involving protein residues and DNA. The purple
arrows indicate base hydrogen bond contact. The flipped nucleotide is en-
closed in the red box.
See Figure S2 for supporting NMR data.
ERCC1 Central
XPF Catalytic
5’
XPF HhH
ERCC1 HhH
ssDNA
h1
h2
Cleavage site
5’ 3’
5’
3’
Figure 5. A Model for XPF/ERCC1 Binding to a Splayed Arm DNA
Substrate
The ERCC1 HhH domain (red), containing two hairpins H1 and H2 (colored
magenta), contacts dsDNA. The XPF HhH domain (blue) is positioned on the
ssDNA (green), thereby stabilizing the ssDNA. The DNA strand colored brown
is the damaged strand to be cleaved by XPF nuclease domain (light blue). The
cleavage site marked by a thick black arrow is close to catalytic residues
(orange). Themodeled 6nts ssDNA (white) connects 30 end of the nondamaged
ssDNA strand (green) to the 50 end of the dsDNA (green). The ERCC1 central
domain (light yellow) can interact with XPA and ssDNA strand on the NER
complex.
Structure
Structure of the XPF-ssDNA Complex
Role of XPF in Positioning XPF/ERCC1 on the NERComplexXPF/ERCC1 cleaves DNA near the ss/ds DNA junction with
a defined polarity. Despite the available structures of archaeal
XPF bound to DNA, and various structures of parts of human
XPF and ERCC1, the mechanism underlying polarity remains
elusive (Tripsianes et al., 2005, 2007; Tsodikov et al., 2005,
2007). We propose that the substrate preference of the DNA
binding domains of ERCC1 and XPF could contribute to the
672 Structure 20, 667–675, April 4, 2012 ª2012 Elsevier Ltd All rights
correct positioning of the heterodimeric XPF/ERCC1 complex
with respect to the single-double strand junction (de Laat
et al., 1998a). This substrate preference well-clarifies the role
of XPF in positioning XPF/ERCC1 at the ss/ds DNA junction in
the NER complex.
EXPERIMENTAL PROCEDURES
Sample Preparation
Expression and purification of the C-terminal XPF823-905 as (XPF)2 homodimers
has been described before (Das et al., 2008). The protein fraction was concen-
trated to nearly 450 ml, with a final protein monomer concentration of �1 mM
using Amicon filters with a 3 kD cutoff. Using this protocol both 15N-labeled
and 13C/15N-labeled (XPF)2 samples were prepared.
The 10 nt sequence 50CAGTGGCTGA and the 39 nt sequence 50TGCG
AATTCATATGCAATATTCAGTGGCTGAGCTACTGG were purchased from
Operon Biotechnologies GmBH (Cologne, Germany).
Mass Spectrometry
The MALDI-TOF mass spectra were recorded on a Voyager-DE PRO mass
spectrometer (Applied Biosystems) with implemented delayed extraction
technique, equipped with a N2 laser (337 nm, 3 ns pulse width). The mass
spectra (positive-ion mode) were recorded in a linear mode at an accelerating
voltage of 25 kV using an extraction delay of 300 ns. The (XPF)2 homodimer
and 39 nt ssDNA samples (�0.6 ml) were mixed in a 1:1 ratio on the target plate
reserved
Structure
Structure of the XPF-ssDNA Complex
with the matrix solution consisting of a-cyano-4-4 hydroxycinnaminic acid
(10mg/ml) dissolved in acetonitrile/water/trifluoroacetic acid (50:49.7:0.3, v/v).
NMR Spectroscopy
NMR data were collected at 20.6�C on a Bruker DRX 600 MHz spectrometer
equipped with a z-gradient triple-resonance cryoprobe and a Bruker
Avance 900 MHz spectrometer equipped with a z-gradient triple-resonance
probe. The final NMR sample contained �1 mM (XPF)2-ssDNA. A set of triple
resonance experiments—HNCACB, CBCACONH, HNCO, HN(CA)CO, HNCA,
HN(CO)CA, HBHA(CBCACONH), HBHANH, HN(CA)HA, (H)CC(CO)NH-
TOCSY, H(CCCO)NH-TOCSY, (H)CCH-TOCSY, and H(C)CH-TOCSY—as
described previously (Das et al., 2008), were acquired at 600 MHz to reassign
the backbone and side chain 1H, 13C and 15N resonances for the complex.
A set of NOE spectra—3D-NOESY-(13C,1H)-HSQC, 3D-(13C)-HMQC-NO-
ESY-(15N,1H)-HSQC, 2D 15N-filtered/13C edited-(1H,1H)-NOESY (tmix =
80 ms, 100 ms), and 13C/15N double half filter (1H-1H)- NOESY (tmix = 80 ms,
100 ms)—were acquired at a Bruker DRX 600 MHz spectrometer using a
triple-resonance cryoprobe, and a 3D NOESY-(15N,1H)-HSQC (tmix = 80 ms,
100ms) was acquired at a Bruker Avance 600MHz spectrometer as described
previously (Das et al., 2008). 1H-decoupled one-dimensional (1D) 31P spectra
and 2D 1H-31P HMQC spectra of free and bound DNA were acquired at
a Bruker DRX 500 MHz spectrometer equipped with a QXI probe. 2D NOESY
(tmix = 80 ms, 100 ms, 150 ms, 200 ms) and 2D TOCSY (tmix = 20 ms, 40 ms,
70 ms) spectra on free DNA were acquired on a Bruker Avance 900 MHz
spectrometer. 2D-NOESY (13C/15N decoupled, 13C decoupled, coupled)
and 2D-TOCSY (15N/13C-filtered) were acquired on a �1mM (XPF)2-(CAGT
GGCTGA)2 sample in a buffer containing 2 mM NaPi (pH 5.2), �80 mM
NaCl, 95%/5% H2O/D2O, and a small amount of complex protease inhibitor
(Roche). All the spectra were processed using XWINNMR 3.5 (Bruker) and
analyzed as described previously (Das et al., 2008).
For the NMR titrations with DNA, we used 15N-labeled (XPF)2. The15N-1H
HSQC spectra at the different titration points were acquired and analyzed as
described previously (Tripsianes et al., 2005).
Structure Calculation
The structure of the (XPF)2-ssDNA complex was determined as follows and
essentially using a strategy as described before (Kopke Salinas et al., 2005;
Romanuka et al., 2009). First, the structure of the (XPF)2 homodimer part
without ssDNA was determined using distance restraints (based on 1H-1H
NOEs) and dihedral restraints (based on TALOS analysis of the backbone
chemical shifts) using the program CYANA/CANDID (Herrmann et al., 2002),
similarly as described for the free (XPF)2 homodimer (Das et al., 2008). Next,
the two 10 nt ssDNA fragments were docked onto the XPF homodimer using
a large set of unambiguous and ambiguous intermolecular protein-DNA NOE
restraints with the program CNS using an Aria protocol (Brunger et al., 1998)
as implemented in the Haddock 2.0 software suite (van Dijk et al., 2006; de
Vries et al., 2007). Finally, the conformers in the ensemble were refined with
CNS in explicit solvent and the conformers clustered. In this final calculation,
the unstructured C- and N-terminal residues of the protein, as evidenced
from our previous structural data and the absence of any nonsequential inter-
residue 1H-1H NOEs, were not included. A summary of the experimental
restraints used for the final structure calculations is given in Table 1. Additional
restraints for maintaining nucleic acid base planarity, the correct sugar pucker
conformation (C20-endo), and the phosphate backbone conformation were
used as described in van Dijk et al. (2006). The dihedral angles of the sugar-
phosphate backbone were obtained from a 2D 31P-1H HMQC spectrum (Fig-
ure S2A) and converted to restraints as described in Braddock et al. (2002a).
The error ranges attributed to these restraints were defined independently
for the four distinct docking stages described below.
(1) Preparation of the DNA starting structures: an extended ssDNA
structure generated using CNS was subjected to a single molecule
HADDOCK run using the intramolecular DNA NOEs. This procedure
generated 40 structures during the simulated annealing stage. All 40
structures were subjected to water refinement. The error range for
the sugar-phosphate backbone dihedral restraints was set to (ainp ±
50, binp ± 50, ginp ± 35, dinp ± 10, εinp ± 50, xinp ± 50). The 20 lowest-
Structure 20, 66
energy water-refinedDNA structures were selected for the calculations
of the (XPF)2-ssDNA complex.
(2) Sampling conformational space of the complex: using intramolecular
DNA/DNA NOE distance restraints and the ambiguous and unambig-
uous intermolecular protein/DNA NOE restraints 1000 docking solu-
tions of the complex were generated using rigid body docking.
From these the best 200 structures were selected according to the
HADDOCK score and subjected to simulated annealing using CNS,
as implemented in Haddock, followed by a gentle water refinement.
All residues of the ssDNA were set as fully flexible during the simulated
annealing stage of CNS. Because of the imposed DNA restraints, only
the C10- (N9/N1) bond angle between the nucleic acid base, the internal
sugar, and the sugar-phosphate backbone dihedral angles can adapt
at this stage. The error range of all sugar-phosphate backbone dihedral
restraints was set to a broad range, Xinp ± 100�. All parameters were as
described in van Dijk et al. (2006). Finally, the 200 solutions were clus-
tered using a 7.5 A rmsd cutoff on the pairwise backbone rmsd matrix
(Ca and P atoms). This resulted in a total of three clusters. The dielectric
constant (ε) as used in the rigid-body docking and simulated annealing
stage was set to 78. To reduce computational time the sampling in this
docking run was only directed toward one of the two symmetric XPF
binding sites (2-component docking).
(3) Restricting the conformational space. Sixteen DNA conformations ex-
tracted from the best cluster of stage 2 were used as input for the next
round of docking calculations. This ensemble of 16 DNA conformations
was extended to 20 by including the best solution from each of the two
other clusters (clusters 2 and 3) to minimize the risk of excluding
possible favorable conformations that ended up in the other clusters.
The docking proceeded in the samemanner as in stage 2, but the error
ranges for the phosphate backbone dihedral restraints were now
reduced to (ainp ± 80, binp ± 80, ginp ± 50, dinp ± 40, εinp ± 80, zinp ± 80).
(4) Final refinement. Using a duplicated set of protein/ssDNA NOEs,
ssDNA was docked on both sides of the homodimeric (XPF)2 using
a 3-component docking. For this round, five ssDNA conformations
extracted from the best solutions of stage 3 were used as starting
structures. Noncrystallographic symmetry restraints were used to
enforce a symmetrical orientation of all atoms of the two ssDNA mole-
cules in the complex as described in Kopke Salinas et al. (2005). The
structure calculation proceeded in the same manner as the previous
two stages, but the error range of the phosphate backbone dihedral
restraints was reduced to Xinp ± 50� for all dihedral angles, similarly
to Braddock et al. (2002a). The structures were clustered, and a final
ensemble containing the 20 lowest-energy structures was obtained.
The structures were analyzed using WHATCHECK (Hooft et al., 1996)
and PROCHECK (Laskowski et al., 1996).
Modeling of the XPF/ERCC1 DNA Complex
The model of the complex of XPF/ERCC1 with splayed arm DNA was made
using the program Pymol (http://www.pymol.org). The model is constructed
from the known structures of the human XPF-ERCC1 HhH domains (1z00),
the ERCC1 central domain (2jpd), the archaeal XPF homodimer bound to
dsDNA (2bgw), and the present structure of (XPF)2 bound to 10 nt ssDNA.
The position of dsDNA to ERCC1 is based upon the X-ray structure of A, pernix
XPF in complex with dsDNA duplex (PDB code 2bgw; Newman et al., 2005),
and the structure of free ERCC1/XPF (PDB code 1z00; Tripsianes et al.,
2005). The position of XPF HhH motifs on the ssDNA is established from the
present study. The gap from the 30 end of ssDNA to the 50 end of dsDNA
was filled with a 6 nt ssDNA sequence, whereas the alternate choice would
lead to a significant large nucleotide linker. Our choice for the model was the
shortest linker.
ACCESSION NUMBERS
Coordinates have been deposited in the Protein Data Bank (PDB) with the
accession code 2KN7, and the chemical shifts and structural restraints have
been deposited in the Biological Magnetic Resonance Bank with the entry
numbers 16449 and 18343.
7–675, April 4, 2012 ª2012 Elsevier Ltd All rights reserved 673
Structure
Structure of the XPF-ssDNA Complex
SUPPLEMENTAL INFORMATION
Supplemental Information includes five figures and five tables and can be
found with this article online at doi:10.1016/j.str.2012.02.009.
ACKNOWLEDGMENTS
The authors are grateful to Profs. A.M.J.J Bonvin, J. Romanuka, E. AB, and
K. Tripsianes for the assistance and discussions during the project. We thank
V. Blanchard and Prof. J.H. Kamerling for use of the MALDI-TOF mass spec-
trometer. This work was financially supported by the European Commission
(project 031220, Spine2-complexes), the Center for Biomedical Genetics,
and the Netherlands Organisation for Scientific Research, Chemical Sciences
(NWO-CW).
Received: October 24, 2011
Revised: January 22, 2012
Accepted: February 17, 2012
Published: April 3, 2012
REFERENCES
Ahmad, A., Robinson, A.R., Duensing, A., van Drunen, E., Beverloo, H.B.,
Weisberg, D.B., Hasty, P., Hoeijmakers, J.H., and Niedernhofer, L.J. (2008).
ERCC1-XPF endonuclease facilitates DNA double-strand break repair. Mol.
Cell. Biol. 28, 5082–5092.
Al-Minawi, A.Z., Saleh-Gohari, N., and Helleday, T. (2008). The ERCC1/XPF
endonuclease is required for efficient single-strand annealing and gene
conversion in mammalian cells. Nucleic Acids Res. 36, 1–9.
Ariyoshi, M., Nishino, T., Iwasaki, H., Shinagawa, H., and Morikawa, K. (2000).
Crystal structure of the holliday junction DNA in complex with a single RuvA
tetramer. Proc. Natl. Acad. Sci. USA 97, 8257–8262.
Bessho, T., Sancar, A., Thompson, L.H., and Thelen, M.P. (1997).
Reconstitution of human excision nuclease with recombinant XPF-ERCC1
complex. J. Biol. Chem. 272, 3833–3837.
Bochkarev, A., Pfuetzner, R.A., Edwards, A.M., and Frappier, L. (1997).
Structure of the single-stranded-DNA-binding domain of replication protein
A bound to DNA. Nature 385, 176–181.
Braddock, D.T., Baber, J.L., Levens, D., and Clore, G.M. (2002a). Molecular
basis of sequence-specific single-stranded DNA recognition by KH domains:
solution structure of a complex between hnRNP K KH3 and single-stranded
DNA. EMBO J. 21, 3476–3485.
Braddock, D.T., Louis, J.M., Baber, J.L., Levens, D., and Clore, G.M. (2002b).
Structure and dynamics of KH domains from FBP bound to single-stranded
DNA. Nature 415, 1051–1056.
Brandsen, J., Werten, S., van der Vliet, P.C., Meisterernst, M., Kroon, J., and
Gros, P. (1997). C-terminal domain of transcription cofactor PC4 reveals
dimeric ssDNA binding site. Nat. Struct. Biol. 4, 900–903.
Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-
Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al.
(1998). Crystallography & NMR system: A new software suite for macromolec-
ular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921.
Chen, Z., Yang, H., and Pavletich, N.P. (2008). Mechanism of homologous
recombination from the RecA-ssDNA/dsDNA structures. Nature 453, 489–4.
Choi, Y.J., Ryu, K.S., Ko, Y.M., Chae, Y.K., Pelton, J.G., Wemmer, D.E., and
Choi, B.S. (2005). Biophysical characterization of the interaction domains
and mapping of the contact residues in the XPF-ERCC1 complex. J. Biol.
Chem. 280, 28644–28652.
Clingen, P.H., De Silva, I.U., McHugh, P.J., Ghadessy, F.J., Tilby, M.J.,
Thurston, D.E., and Hartley, J.A. (2005). The XPF-ERCC1 endonuclease and
homologous recombination contribute to the repair of minor groove DNA inter-
strand crosslinks in mammalian cells produced by the pyrrolo[2,1-c][1,4]
benzodiazepine dimer SJG-136. Nucleic Acids Res. 33, 3283–3291.
674 Structure 20, 667–675, April 4, 2012 ª2012 Elsevier Ltd All rights
Das, D., Tripsianes, K., Jaspers, N.G., Hoeijmakers, J.H., Kaptein, R., Boelens,
R., and Folkers, G.E. (2008). The HhH domain of the human DNA repair protein
XPF forms stable homodimers. Proteins 70, 1551–1563.
de Laat, W.L., Appeldoorn, E., Jaspers, N.G., and Hoeijmakers, J.H. (1998a).
DNA structural elements required for ERCC1-XPF endonuclease activity.
J. Biol. Chem. 273, 7835–7842.
de Laat, W.L., Appeldoorn, E., Sugasawa, K., Weterings, E., Jaspers, N.G.,
and Hoeijmakers, J.H. (1998b). DNA-binding polarity of human replication
protein A positions nucleases in nucleotide excision repair. Genes Dev. 12,
2598–2609.
de Laat, W.L., Sijbers, A.M., Odijk, H., Jaspers, N.G., and Hoeijmakers, J.H.
(1998c). Mapping of interaction domains between human repair proteins
ERCC1 and XPF. Nucleic Acids Res. 26, 4146–4152.
de Laat, W.L., Jaspers, N.G.J., and Hoeijmakers, J.H.J. (1999). Molecular
mechanism of nucleotide excision repair. Genes Dev. 13, 768–785.
de Vries, S.J., van Dijk, A.D., Krzeminski, M., van Dijk, M., Thureau, A., Hsu, V.,
Wassenaar, T., and Bonvin, A.M. (2007). HADDOCK versus HADDOCK: new
features and performance of HADDOCK2.0 on the CAPRI targets. Proteins
69, 726–733.
Enzlin, J.H., and Scharer, O.D. (2002). The active site of the DNA repair endo-
nuclease XPF-ERCC1 forms a highly conserved nuclease motif. EMBO J. 21,
2045–2053.
Gaillard, P.H., and Wood, R.D. (2001). Activity of individual ERCC1 and XPF
subunits in DNA nucleotide excision repair. Nucleic Acids Res. 29, 872–879.
Herrmann, T., Guntert, P., and Wuthrich, K. (2002). Protein NMR structure
determination with automated NOE assignment using the new software
CANDID and the torsion angle dynamics algorithm DYANA. J. Mol. Biol. 319,
209–227.
Hoeijmakers, J.H.J. (2001). Genome maintenance mechanisms for preventing
cancer. Nature 411, 366–374.
Hooft, R.W., Vriend, G., Sander, C., and Abola, E.E. (1996). Errors in protein
structures. Nature 381, 272.
Houtsmuller, A.B., Rademakers, S., Nigg, A.L., Hoogstraten, D., Hoeijmakers,
J.H.J., and Vermeulen, W. (1999). Action of DNA repair endonuclease ERCC1/
XPF in living cells. Science 284, 958–961.
Kerr, I.D., Wadsworth, R.I., Cubeddu, L., Blankenfeldt, W., Naismith, J.H., and
White, M.F. (2003). Insights into ssDNA recognition by the OB fold from a
structural and thermodynamic study of Sulfolobus SSB protein. EMBO J. 22,
2561–2570.
Kuraoka, I., Kobertz, W.R., Ariza, R.R., Biggerstaff, M., Essigmann, J.M., and
Wood, R.D. (2000). Repair of an interstrand DNA cross-link initiated by
ERCC1-XPF repair/recombination nuclease. J. Biol. Chem. 275, 26632–
26636.
Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R., and
Thornton, J.M. (1996). AQUA and PROCHECK-NMR: programs for checking
the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486.
McWhir, J., Selfridge, J., Harrison, D.J., Squires, S., and Melton, D.W. (1993).
Mice with DNA repair gene (ERCC-1) deficiency have elevated levels of p53,
liver nuclear abnormalities and die before weaning. Nat. Genet. 5, 217–224.
Melis, J.P., Wijnhoven, S.W., Beems, R.B., Roodbergen, M., van den Berg, J.,
Moon, H., Friedberg, E., van der Horst, G.T., Hoeijmakers, J.H., Vijg, J., and
van Steeg, H. (2008). Mouse models for xeroderma pigmentosum group A
and group C show divergent cancer phenotypes. Cancer Res. 68, 1347–1353.
Mitton-Fry, R.M., Anderson, E.M., Hughes, T.R., Lundblad, V., and Wuttke,
D.S. (2002). Conserved structure for single-stranded telomeric DNA recogni-
tion. Science 296, 145–147.
Munoz, P., Blanco, R., Flores, J.M., and Blasco, M.A. (2005). XPF nuclease-
dependent telomere loss and increased DNA damage in mice overexpressing
TRF2 result in premature aging and cancer. Nat. Genet. 37, 1063–1071.
Newman, M., Murray-Rust, J., Lally, J., Rudolf, J., Fadden, A., Knowles, P.P.,
White, M.F., and McDonald, N.Q. (2005). Structure of an XPF endonuclease
with and without DNA suggests a model for substrate recognition. EMBO J.
24, 895–905.
reserved
Structure
Structure of the XPF-ssDNA Complex
Niedernhofer, L.J., Essers, J., Weeda, G., Beverloo, B., de Wit, J., Muijtjens,
M., Odijk, H., Hoeijmakers, J.H.J., and Kanaar, R. (2001). The structure-
specific endonuclease Ercc1-Xpf is required for targeted gene replacement
in embryonic stem cells. EMBO J. 20, 6540–6549.
Niedernhofer, L.J., Odijk, H., Budzowska, M., van Drunen, E., Maas, A., Theil,
A.F., de Wit, J., Jaspers, N.G.J., Beverloo, H.B., Hoeijmakers, J.H.J., and
Kanaar, R. (2004). The structure-specific endonuclease Ercc1-Xpf is required
to resolve DNA interstrand cross-link-induced double-strand breaks. Mol.
Cell. Biol. 24, 5776–5787.
Orelli, B., McClendon, T.B., Tsodikov, O.V., Ellenberger, T., Niedernhofer, L.J.,
and Scharer, O.D. (2009). The XPA-binding domain of ERCC1 is required for
nucleotide excision repair but not other DNA repair pathways. J. Biol. Chem.
285, 3705–3712.
Park, C.H., Bessho, T., Matsunaga, T., and Sancar, A. (1995). Purification and
characterization of the XPF-ERCC1 complex of human DNA repair excision
nuclease. J. Biol. Chem. 270, 22657–22660.
Riedl, T., Hanaoka, F., and Egly, J.M. (2003). The comings and goings of nucle-
otide excision repair factors on damaged DNA. EMBO J. 22, 5293–5303.
Romanuka, J., Folkers, G.E., Biris, N., Tishchenko, E., Wienk, H., Bonvin, A.M.,
Kaptein, R., and Boelens, R. (2009). Specificity and affinity of Lac repressor for
the auxiliary operators O2 and O3 are explained by the structures of their
protein-DNA complexes. J. Mol. Biol. 390, 478–489.
Kopke Salinas, R., Folkers, G.E., Bonvin, A.M.J.J., Das, D., Boelens, R., and
Kaptein, R. (2005). Altered specificity in DNA binding by the lac repressor:
a mutant lac headpiece that mimics the gal repressor. Chem. Bio. Chem. 6,
1628–1637.
Scharer, O.D. (2007). Achieving broad substrate specificity in damage recog-
nition by binding accessible nondamaged DNA. Mol. Cell 28, 184–186.
Shao, X., and Grishin, N.V. (2000). Common fold in helix-hairpin-helix proteins.
Nucleic Acids Res. 28, 2643–2650.
Sijbers, A.M., de Laat, W.L., Ariza, R.R., Biggerstaff, M., Wei, Y.F., Moggs,
J.G., Carter, K.C., Shell, B.K., Evans, E., de Jong, M.C., et al. (1996).
Xeroderma pigmentosum group F caused by a defect in a structure-specific
DNA repair endonuclease. Cell 86, 811–822.
Singh, S., Folkers, G.E., Bonvin, A.M.J.J., Boelens, R., Wechselberger, R.,
Niztayev, A., and Kaptein, R. (2002). Solution structure and DNA-binding prop-
erties of the C-terminal domain of UvrC from E.coli. EMBO J. 21, 6257–6266.
Structure 20
Tian, M., Shinkura, R., Shinkura, N., and Alt, F.W. (2004). Growth retardation,
early death, and DNA repair defects in mice deficient for the nucleotide exci-
sion repair enzyme XPF. Mol. Cell. Biol. 24, 1200–1205.
Tripsianes, K., Folkers, G., Ab, E., Das, D., Odijk, H., Jaspers, N.G.,
Hoeijmakers, J.H., Kaptein, R., and Boelens, R. (2005). The structure of the
human ERCC1/XPF interaction domains reveals a complementary role for
the two proteins in nucleotide excision repair. Structure 13, 1849–1858.
Tripsianes, K., Folkers, G.E., Zheng, C., Das, D., Grinstead, J.S., Kaptein, R.,
and Boelens, R. (2007). Analysis of the XPA and ssDNA-binding surfaces on
the central domain of human ERCC1 reveals evidence for subfunctionalization.
Nucleic Acids Res. 35, 5789–5798.
Tsodikov, O.V., Enzlin, J.H., Scharer, O.D., and Ellenberger, T. (2005). Crystal
structure and DNA binding functions of ERCC1, a subunit of the DNA struc-
ture-specific endonuclease XPF-ERCC1. Proc. Natl. Acad. Sci. USA 102,
11236–11241.
Tsodikov, O.V., Ivanov, D., Orelli, B., Staresincic, L., Shoshani, I., Oberman, R.,
Scharer, O.D., Wagner, G., and Ellenberger, T. (2007). Structural basis for the
recruitment of ERCC1-XPF to nucleotide excision repair complexes by XPA.
EMBO J. 26, 4768–4776.
van Dijk, M., van Dijk, A.D.J., Hsu, V., Boelens, R., and Bonvin, A.M.J.J. (2006).
Information-driven protein-DNA docking using HADDOCK: it is a matter of
flexibility. Nucleic Acids Res. 34, 3317–3325.
Volker, M., Mone, M.J., Karmakar, P., van Hoffen, A., Schul, W., Vermeulen,
W., Hoeijmakers, J.H.J., van Driel, R., van Zeeland, A.A., and Mullenders,
L.H. (2001). Sequential assembly of the nucleotide excision repair factors
in vivo. Mol. Cell 8, 213–224.
Weeda, G., Donker, I., de Wit, J., Morreau, H., Janssens, R., Vissers, C.J.,
Nigg, A., van Steeg, H., Bootsma, D., and Hoeijmakers, J.H.J. (1997).
Disruption of mouse ERCC1 results in a novel repair syndrome with growth
failure, nuclear abnormalities and senescence. Curr. Biol. 7, 427–439.
Werten, S., and Moras, D. (2006). A global transcription cofactor bound to
juxtaposed strands of unwound DNA. Nat. Struct. Mol. Biol. 13, 181–182.
Winkler, G.S., Sugasawa, K., Eker, A.P., de Laat, W.L., and Hoeijmakers,
J.H.J. (2001). Novel functional interactions between nucleotide excision DNA
repair proteins influencing the enzymatic activities of TFIIH, XPG, and
ERCC1-XPF. Biochemistry 40, 160–165.
Wu, Y., Mitchell, T.R., and Zhu, X.D. (2008). Human XPF controls TRF2 and
telomere length maintenance through distinctive mechanisms. Mech. Ageing
Dev. 129, 602–610.
, 667–675, April 4, 2012 ª2012 Elsevier Ltd All rights reserved 675