Crystal Structure of Human XLF:
A Twist in Nonhomologous DNA End-Joining
Sara N. Andres,1Mauro Modesti,2Chun J. Tsai,3,4Gilbert Chu,3,4and Murray S. Junop1,*
1Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada
2Genome Instability and Carcinogenesis, CNRS FRE 2931, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
3Department of Medicine
4Department of Biochemistry
Stanford University School of Medicine, Stanford, CA 94305, USA
DNA double-strand breaks represent one of the
most severe forms of DNA damage in mamma-
lian cells. One pathway for repairing these
breaks occurs via nonhomologous end-joining
(NHEJ) and depends on XRCC4, LigaseIV, and
Cernunnos, also called XLF. Although XLF stim-
ulates XRCC4/LigaseIV to ligate mismatched
and noncohesive DNA ends, the mechanistic
basis for this function remains unclear. Here
we report the structure of a partially functional
224 residue N-terminal fragment of human XLF.
Despite only weak sequence similarity, XLF1–170
shares structural homology with XRCC41–159.
However, unlike the highly extended 130 A˚heli-
cal domain observed in XRCC4, XLF adopts
a more compact, folded helical C-terminal re-
gion involving two turns and a twist, wrapping
back to the structurally conserved N terminus.
Mutational analysis of XLF and XRCC4 reveals
a potential interaction interface, suggesting
of mismatched ends.
Generation of DNA double-strand breaks (DSBs) poses
a serious threat to chromosomal integrity. If left unre-
paired, such breaks can generate destabilizing chromo-
somal rearrangements that may lead to tumorigenesis
(Gao et al., 2000; Lieber, 1998). DSBs occur in response
to exogenous genotoxic agents and as intermediates in
the genomic rearrangements associated with V(D)J
recombination. Eukaryotic cells maintain two systems
for repairing DSBs: homologous recombination, which
facilitates accurate restoration of DNA (Jeggo, 1998),
and nonhomologous end-joining (NHEJ), which facilitates
error-prone repair without a homologous DNA template.
NHEJ requires a core set of five proteins: Ku, DNA-
PKcs, XRCC4, LigaseIV, and Cernunnos, an XRCC4-like
factor hereafter called XLF. Ku initially binds broken DNA
ends, preventing nucleolytic degradation (Liang and
Jasin, 1996), and then recruits DNA-PKcs, which stabi-
lizes synapsis of the DNA ends (DeFazio et al., 2002).
DNA end processing enzymes, including Artemis, en-
hance NHEJ by making DNA ends compatible for ligation
2005). However, in the presence of Ku, XLF stimulates
XRCC4/LigaseIV to ligate mismatched and noncohesive
DNA ends (Tsai et al., 2007). XLF can bind DNA and inter-
act with the XRCC4/LigaseIV complex; however, the
nature of these interactions and the mechanism by which
XLF functions remain poorly understood (Hentges et al.,
2006; Callebaut et al., 2006; Ahnesorg et al., 2006; Lu
et al., 2007).
XLF was identified as the gene mutated in patients
exhibiting immune deficiencies and microcephaly, symp-
toms consistent with impaired NHEJ (Ahnesorg et al.,
2006; Buck et al., 2006). Secondary structure predictions
suggested that XLF and XRCC4 are structurally related,
despite limited sequence similarity (Ahnesorg et al.,
2006). In addition, XLF and XRCC4 exhibit other similari-
ties. Both proteins exist as dimers and have DNA binding
activity dependent on the presence of long (>80 bp) DNA
fragments (Hentges et al., 2006; Lu et al., 2007). Although
XLF and XRCC4 are able to interact, the nature and func-
tional importance of this quaternary structure remain
unknown. Current data suggest that XLF interacts with
XRCC4/LigaseIV primarily through contact with XRCC4,
but the role of DNA and LigaseIV in this interaction has
not been rigorously evaluated (Deshpande and Wilson,
2007; Lu et al., 2007).
Here we report the crystal structure of a partially func-
tional N-terminal fragment of human XLF1–224at 2.5 A˚
resolution. This fragment fails to efficiently bind DNA or
stimulate ligation of noncohesive DNA ends but retains
the ability to directly interact with XRCC4. The N-terminal
region of XLF (amino acids 1–170) adopts a structure
almost identical to XRCC4, as predicted from the amino
acid sequences (Ahnesorg et al., 2006; Callebaut et al.,
2006). However, amino acids 170–224 diverge greatly
from the expected elongated helix seen in XRCC4. Two
helices within this region of XLF fold back onto and twist
Molecular Cell 28, 1093–1101, December 28, 2007 ª2007 Elsevier Inc. 1093
around the main helical stalk of XLF, occluding the analo-
gous LigaseIV binding interface observed in XRCC4. The
structure of XLF therefore explains why LigaseIV is unable
to bind XLF in the same manner observed for XRCC4.
Based on the structures of XLF and XRCC4, we then
conducted mutational analysis aimed at further defining
surfaces involved in the protein-protein interactions.
of XLF interact with XRCC4 and DNA, respectively. Taken
of a functional DNA repair complex.
RESULTS AND DISCUSSION
Crystal Structure of XLF1–224Shows Formation
of a Stable Dimer
Human XLF contains 299 residues (Ahnesorg et al., 2006;
Buck et al., 2006). Comparison with other eukaryotic ho-
mologs reveals a high degree of sequence similarity within
the N-terminal ?220 residues. Interestingly, this does
not extend to the C-terminal portion of XLF. As expected,
similarity within the N-terminal region is most pronounced
among hydrophobic amino acids (Figure 1A). Several
clusters of more highly conserved residues, including
both hydrophobic and charged/polar amino acids, occur
in the N-terminal region (57–65, 108–123, and 160–186)
as well as at a single basic region in the extreme C termi-
nus (residues 288–295) (Figures 1A and 1C). Based on
these observations, we created a truncation to remove
the less conserved C-terminal 75 residues of human
XLF, with the hope of reducing structural flexibility and
thereby facilitating crystallization.
Although native XLF1–224formed crystals, the quality
and resolution of diffraction data were poor; thus, all
data used for both structure solution and refinement
came from selenomethionine-substituted protein. Experi-
mental phases were determined by using single-wave-
length anomalous diffraction (SAD). Crystals formed in
space group P212121, with 2 monomers of XLF in each
asymmetric unit. Regions of disorder within the crystal
prevented modeling of residues 85–92 in chain A and res-
idues 80–92 in chain B. These residues are illustrated
as dashed loops in the final structure (Figure 1C). The
overall structure was refined to R and Rfree values of
25.3% and 28.7%, respectively (PDB 2R9A). Table 1 con-
tains a complete list of data collection and model refine-
XLF1–224is comprised of two principal domains. The
globular N-terminal head domain consists of a seven-
stranded antiparallel b sandwich with a single helix at the
N terminus and a helix-turn-helix motif inserted between
strands 4 and 5 (Figure 1B). The loop region connecting
a3 and b5 is not well ordered and therefore not present
in the final model. Three a helices combine to form an
elongated C-terminal domain, the first of which (a4)
extends ?60 A˚away from the N-terminal head. This rather
long helix terminates with a conserved glycine at position
171. The following extended coiled region (residues 171–
185) reverses the overall trajectory of the polypeptide
chain, bringing the second (a5) and third (a6) helices of
the C-terminal domain back into contact with a4. The
last helix (a6) of the C-terminal domain wedges itself
between a4 and b1, forcing the N-terminal head domain
to adopt an elevated position (Figure 1C). In addition,
of XLF reside near the N-terminal head domain.
XLF formed a dimer within the crystallographic asym-
metric unit. This quaternary structure has also been ob-
served for XLF homologs in S. pombe and S. cerevisiae
(Cavero et al., 2007; Sulek et al., 2007), suggesting that
human XLF forms a homodimer in the absence of DNA
and other NHEJ proteins. XLF1–224monomers are related
by a 2-fold noncrystallographic axis and interact primarily
through conserved hydrophobic regions within a4, listed
in detail in Figure S1 available online. The dimer is also
held in place by compensatory interactions between a4
of one monomer and a5 of the opposing monomer.
Further stabilization occurs from the clamp formed by a5
and a6 as they twist upward and wrap around a4 from
the opposite subunit. The dimer interface buries a surface
area of ?6500 A˚2, the greatest contribution coming from
interactions thatresult fromthefolding back andclamping
of a5 and a6 onto a4. The combined effects of burying
extensive hydrophobic surface and structural support
gained by wrapping a5 and a6 onto a4 suggest that the
XLF dimer is highly stable. However, the possibility re-
mains that opposing tails (a5 and a6) from two XLF dimers
could interchange to form a tetramer similar to XRCC4
(Junop et al., 2000). Preliminary data from gel filtration
analysis suggest that XLF can form concentration-depen-
dent higher-order complexes (Figure S2).
Interestingly, three patches of highly conserved resi-
dues in XLF1–224do not contribute significantly to the
dimer interface (Figure 1). Many of the conserved residues
in these regions are exposed and likely involved in other
interactions. Patch III (residues 160–186) is N-terminal to
the region in XLF (residues 186–209) corresponding to
the region in XRCC4 that binds to LigaseIV. However,
Patch III forms a hinge that folds back to occlude the
potential LigaseIV binding surface. Although Patch I and
II are noncontiguous in primary structure, these regions
form a continuous surface in the tertiary structure of XLF
(Figure 1C). Many of the conserved residues in these
two patches consist of exposed polar/charged residues
that are not expected to contribute to protein folding. A
stretch of three exposed hydrophobic residues (amino
acids 115, 116 and 118; see Figure S1) is also found in
this region, specifically within the short loop joining b6
and b7. These features raise the possibility that the Patch
I and II surfaces mediate interactions with XRCC4 or other
The XLF C-Terminal Domain Is Required for DNA
Binding and Mismatched End Ligation
To determine the role of the conserved N-terminal region
of XLF in NHEJ, we tested XLF1–224for its ability to
1094 Molecular Cell 28, 1093–1101, December 28, 2007 ª2007 Elsevier Inc.
Crystal Structure of Human XLF
stimulate ligation of mismatched DNA ends, bind DNA,
and interact with XRCC4/LigaseIV. Tsai et al. (2007)
recently used an in vitro end-joining assay to demonstrate
that XLF stimulates ligation of noncohesive and mis-
matched ends. Using this assay, we found that full-length
XLF and XLF1–224stimulated ligation of mismatched ends
by ?300- and 3-fold, respectively (Figure 2A). The de-
creased activity of XLF1–224was surprising given the
Figure 1. Structure and Sequence Conservation of the N-Terminal Region of Human XLF
(A) Sequence alignment of XLF homologs. Conserved residues are colored as follows: hydrophobic, yellow; negative charge, red; positive charge,
blue; proline and glycine,brown;threonine and serine, green; cysteine, light blue; and glutamine and asparagine, purple. Regionsof highly conserved
residues are underlined in green, orange, red, and black. For clarity, XLF residues 249–282 from S. cerevisiae are not shown in the alignment.
(B) Stereo image of a single XLF1–224. b strand and a helix are in red and blue, respectively.
(C) XLF dimer observed in crystal asymmetric unit. Chains A and B are shown in teal and yellow. Conserved patches are labeled and colored to cor-
relate with (A).
Molecular Cell 28, 1093–1101, December 28, 2007 ª2007 Elsevier Inc. 1095
Crystal Structure of Human XLF
general absence of conservation in the C-terminal 75
amino acids deleted from XLF1–224. However, the extreme
C terminus of XLF contains a small conserved basic clus-
ter, which was proposed to represent an NLS (Ahnesorg
et al., 2006).
We compared the DNA binding activities of XLF1–224,
full-length XLF, and XRCC4 and found that the ability of
to bind DNA was dramatically decreased
(Figure 2B), suggesting that the C-terminal 75 residues
of XLF are necessary for DNA binding. In agreement with
these findings, biochemical studies of the XLF homolog
in S. cerevisiae indicate that the analogous C-terminal re-
gion is necessary and sufficient for DNA binding (Sulek
et al., 2007).
XLF interacts with XRCC4 and the XRCC4/LigaseIV
complex (Ahnesorg et al., 2006). Using a two-step
approach, we examined the protein-protein interactions
retained by XLF when its C-terminal 75 residues are ab-
sent. In the first step, individually purified proteins were
mixed and then resolved by native PAGE (Figure 2C). In
the second step, protein bands were excised from the
gel and resolved by electrophoresis under denaturing
conditions (Figure 2D). XRCC4 and LigaseIV654–911were
included as a positive control, because they form a stable
complex (Modesti et al., 2003). When XRCC4 and Liga-
seIV654–911were combined and resolved under native
conditions, a new band was observed with altered mobil-
ity compared to either individual protein on its own
(Figure 2C, compare lane 4 to lanes 1 and 3). When this
band was resolved under denaturing conditions, both
XRCC4 and LigaseIV654–911were present as expected
(Figure 2D, lane 4). We then tested various combinations
of XRCC4, LigaseIV654–911, and XLF1–224for their ability
to form stable complexes (Figures 2C and 2D). The con-
served N-terminal fragment of XLF1–224retains the ability
to interact stably with XRCC4 and to the complex of
XRCC4 bound to LigaseIV654–911. On the other hand,
XLF1–224and LigaseIV654–911failed to form a stable com-
plex, consistent with studies examining the interaction
between full-length XLF and LigaseIV (Lu et al., 2007;
Deshpande and Wilson, 2007).
In summary, the C-terminal 75 residues of human XLF
are required for stimulation of mismatched DNA ligation
and DNA binding, but not for interaction with XRCC4.
Interestingly, this region of XLF exhibits little sequence
conservation with other homologs, except for the extreme
XLF and XRCC4 Structures Exhibit Similarities
of XLF1–224and XRCC41–211. Although the first 120 resi-
dues of XLF and XRCC4 exhibit only 13.4% sequence
identity, the predicted secondary structures display al-
most perfect homology with an overall root-mean-square
deviation of 1.2 A˚for the Ca atoms (SuperPose; Maiti
et al., 2004). However, examination of the entire tertiary
structure reveals unexpected and dramatic differences
between XRCC4 and XLF. Two key differences become
evident upon alignment of XLF and XRCC4 with either
the tail domains (Figure 3B) or the head domains
(Figure 3C). The first difference is a large 45?outward
rotation of the XLF helical tail domain (Figure 3C). This
structural difference is due to insertion of a1 and a6 be-
tween the head and stalk domains, elevating both heads
within the XLF dimer (Figure 3B, black arrow). This creates
a flattened, elongated surface (?25 3 70 A˚) extending
from either end of the dimer (Figure 3B green arrows).
We speculate that the newly formed surface may serve
as a binding interface.
The second major difference is the folding of the C-ter-
minal helical domain of XLF. In XRCC4 the C-terminal do-
main exists as a single helix that extends away from the
head domain with a small but important conformational
deviation at the point that interacts with LigaseIV
(Figure 3B). The analogous region of XLF is broken into
three helices, with a5 and a6 folding back onto a4 of the
Table 1. Crystallographic Data and Refinement
Cell parameters (A˚)a = 63.41, b = 86.93,
c = 91.87 a = b = g = 90
Molecules in A.U.2
Resolution range (A˚)a
Wilson scaling B factor (A˚2) 37.0
Model and Refinement
Resolution range (A˚)25.0–2.50
Reflection test set1031
Number of protein atoms3464
Number of waters 288
Rmsd bond lengths (A˚) 0.016
Rmsd bond angles (A˚) 1.613
Average B factor (A˚2)40.3
aStatistics for the highest data resolution shell are shown
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Crystal Structure of Human XLF
opposing dimer subunit. XLF helix a5 aligns with the Liga-
seIV binding region of XRCC4 (Figure 3A). Interestingly,
XLF helix a5 is less conserved than Patch III of the XLF
hinge (Figure 1A), suggesting that a5 might not interact
with LigaseIV. In addition, the helical stalk of XLF folds
back onto itself to produce a more compact structure
with a greatly increased dimer interface. Importantly, the
folding and twisting of a5 and a6 back onto a4 prevents
LigaseIV from binding to XLF in the same way it binds to
XRCC4 (Modesti et al., 2003; Sibanda et al., 2001). This
result supports our biochemical data showing that XLF
and LigaseIV do not interact strongly (Figure 2C) and is
consistent with data from other groups (Lu et al., 2007;
Deshpande and Wilson, 2007). Nevertheless, the folded
teins or with itself to form tetramers, as discussed above.
Model for the XLF/XRCC4/LigaseIV Complex
Recent studies by Deshpande and Wilson (2007) suggest
that the head region of human XRCC4 is necessary and
sufficient for stable interaction with the N-terminal region
of human XLF. We conducted a mutational analysis of
both XRCC4 and XLF to define their interacting surfaces
in greater detail and thereby provide data for a model of
the XRCC4/XLF complex.
We targeted mutations in XRCC4 and XLF to exposed,
conserved residues not predicted to be involved in dimer-
ization or LigaseIV binding (Figure 3A and Figure S3). In
total, 17 XRCC4 mutants and ten XLF mutants were ana-
lyzed for their ability to bind XLF, XRCC4, LigaseIV, and
DNA. XLF mutants were also tested for stimulation of mis-
matched end ligation. Figure 4 summarizes the results.
Whereas most mutations did not alter function, amino
acid substitutions at K63, K65, and K99 of XRCC4 disrup-
ted interaction with XLF. These mutations nevertheless
preserved full LigaseIV and DNA binding activity. Within
the tertiary structure of XRCC4, these residues cluster at
the base of the head domain (Figure 3D). Of the ten XLF
mutants, three (L174A, R178A, and L179A located in
Patch III) were shown to be essential for XLF activity, but
not for binding to DNA or XRCC4. This phenotype is
consistent with these residues mediating interaction with
Ku70/80 and/or LigaseIV. Only a single XLF mutant,
L115A, disrupted XRCC4 binding. The L115A mutant re-
tained DNA binding activity and showed no alteration in
tant for protein-protein interaction, but not for the folding
of XLF (data not shown). L115 is located within the small
cluster of three exposed hydrophobic residues found in
the b6-b7 loop of Patch II (Figures 1C and 3D). Mutations
that disrupted the interaction between XRCC4 and XLF
were all located in the head domains of XRCC4 and XLF.
This result suggests that the head domains are essential
pande and Wilson (2007). As shown in Figure 3D, the b6-
b7 loop region of XLF is predicted to interact with the a3
helices of XRCC4 (residues 138–150). Conversely, resi-
dues K63, K65, and K99 of XRCC4 appear to make
contacts with the conserved Patch I and II regions of
XLF. Further investigation is required to verify these inter-
Our structural and biochemical data further define the
XRCC4-XLF interacting regions and suggest a working
model for how these proteins assemble during NHEJ.
The most probable way of bringing XLF into contact with
XRCC4, without introducing steric clashes with the
BRCT domains of LigaseIV, is to assemble the proteins
in a stacked head-to-head fashion (Figure 3D). This model
Figure 2. Functional Analysis of XLF1–224
(A) Stimulation of noncohesive end-joining
requires the C terminus of XLF.
(B) DNA binding of XLF requires the C-terminal
75 amino acids. XLF1–224and/or XRCC4 was
tested by mobility gel shift using increasing
amounts of protein (2-fold increase starting at
4 pmol in lane 2) and 100 ng of linearized
dsDNA (2.6 kbp).
(C and D) Protein-protein interactions with
XLF1–224, XRCC4, and LigaseIV654–911. Native
PAGE (C). Bands from lanes 4, 5, and 7 in (C)
were cut out and resolved by SDS-PAGE (D).
Molecular Cell 28, 1093–1101, December 28, 2007 ª2007 Elsevier Inc. 1097
Crystal Structure of Human XLF
Figure 3. Structure-Based Comparison of Human XLF1-224
(A) Structure-based sequence alignment of XLF1–224and XRCC41–211. Conserved residues are colored according to the designation in Figure 1.
Based on the S. cerevisiae structure of Lif1-Dnl4 (tandem BRCT), triangles indicate putative residues of XRCC4 that mediate LigaseIV interaction.
Reflecting asymmetric interactions in Lif1-Dnl4, triangles are colored either black or gray for interaction with subunits A or B of XRCC4. XRCC4
and XLF mutations are indicated by triangles in blue and orange, respectively.
(B) Overlay of XLF1–224(orange) and XRCC41–201(blue) bound to LigaseIV755–782(yellow). The helical tail region of XLF (residues 128–171) was
used to structurally align the corresponding region of XRCC4. The structure of XRCC4 bound to LigaseIV (PDB 1Z56) is reported in Dore et al.
(C) Overlay of N-terminal head domains of XLF1–152and XRCC41–142, in orange and blue, respectively. Arrow indicates a 45?difference in the
trajectory of XLF and XRCC4 C-terminal tail domains.
1098 Molecular Cell 28, 1093–1101, December 28, 2007 ª2007 Elsevier Inc.
Crystal Structure of Human XLF
satisfies the current biochemical data indicating that head
regions of XRCC4 and XLF interact with each other, and
conforms to the structural restrictions imposed by shape
complementarity. The model also predicts an interaction
ures 3D and 3E). Such an interaction would be weaker
than the highly stable binding mode between XRCC4
and LigaseIV, in agreement with a recent report (Desh-
pande and Wilson, 2007).
In this model, an XRCC4 dimer potentially binds two
dimers of XLF (Figure 3D). Furthermore, a single dimer of
XLF potentially binds two dimers of XRCC4, thus generat-
ing a continuous structure or filament of alternating
ament would be expected to cooperatively stabilize bind-
ing of XRCC4 and XLF to DNA.In support of this proposed
arrangement of XLF and XRCC4, these proteins have
been shown to exhibit highly cooperative DNA binding
and are only able to interact stably with unusually large
DNA substrates (?100 bp) (Modesti et al., 1999; Lu
et al., 2007; Sulek et al., 2007).
To explain how XLF stimulates ligation of blunt and mis-
matched ends, we propose the following model. Ku stabi-
lizes binding of the first XRCC4/LigaseIV complex to the
DNA ends (Nick McElhinny et al., 2000). For cohesive
ends, alignment by base pairing would facilitate immedi-
ate ligation by LigaseIV even in the absence of XLF. For
blunt or mismatched ends, an XLF/XRCC4 filament would
assemble on the DNA. Both XLF and XRCC4 can bind to
internal sites on DNA (Figure 2B), and assembly of the
XLF/XRCC4 filament would stabilize DNA binding. The
DNA ends could slide with respect to each other within
the filament. Because each XLF and XRCC4 subunit of
the filament includes a DNA binding domain, juxtaposition
(D) Stereo image of XRCC4-XLF interface suggested by mutational analysis. XLF-A and XLF-B are contributed from separate dimers. Mutations
inhibiting XRCC4-XLF interaction are circled in gray.
(E) Model for assembly of an XRCC4-XLF-LigaseIV filament.
Figure 4. Mutational Analysis of XRCC4
and XLF Binding Surfaces
XRCC4 and XLF mutants either bound (+) or
did not bind (?) DNA, LigaseIV654–911, XLF, or
XRCC4. Stimulation of end-joining, illustrated
as a bar graph, was measured from 0- to
150-fold for the joining of an EcoRV-KpnI-
digested DNA substrate.
Molecular Cell 28, 1093–1101, December 28, 2007 ª2007 Elsevier Inc. 1099
Crystal Structure of Human XLF
of the DNA ends would optimize the energy of DNA bind-
ing. Mismatched or blunt ends could slide into a position
that would permit LigaseIV to join one or both strands,
depending on the structure of the DNA ends (Tsai et al.,
2007). We are currently conducting additional experi-
ments to confirm this model.
Protein Expression and Purification
See the Supplemental Data for details of vector construction. Purifica-
tion was achieved by Ni2+affinity and gel filtration chromatography
(see Supplemental Data for further details). A description of XRCC4
and XLF mutagenesis and mutant purification is given in the Supple-
Electrophoretic Mobility Shift Assays
DNA binding reactions were assembled with EMSA buffer (20 mM Tris
[pH 8.0], 50 mM KCl, 0.1 mM dithiothreitol, 10 mg/mL BSA, and 5%
glycerol), 100 ng of HindIII (NEB) linearized pUC-18, and varying
amounts of protein (Figure 2B). Reactions were resolved by electro-
phoresis on a 0.8% TBE agarose gel for 1 hr at 80 V.
2D Gel Analysis of Protein-Protein Interactions
Purified proteins were combined as indicated in Figure 2B in buffer
(20 mM Tris [pH 8.0], 50 mM KCl, 0.1 mM DTT, 10 mg/mL BSA, and
5% glycerol). Reactions were resolved by 6% native PAGE in TBE.
Shifted gel bands were excised and boiled in SDS-PAGE gel loading
dye for 5 min prior to 12% SDS-PAGE gel electrophoresis.
Crystallization and Data Collection of XLF1–224
Crystals were grown by using the hanging drop vapor diffusion
method. Equal volumes of protein (3.4 mg/mL XLF, 150 mM KCl,
10 mM Tris [pH 8.0], 5 mM DTT, and 1 mM EDTA) and crystallization
solution (0.1 M HEPES [pH 7.5], 20% PEG 10000, and 200 mM
NDSB-201) were dehydrated over 800 ml of 1.8 M (NH4)2SO4. Crystals
(700 3 50 3 20 mm) grew after 1–2 days at 4?C. Diffraction data were
collected at NSLS, X8C (Brookhaven, NY).
Structure Determination and Model Refinement
SeMet sites were located by using HYSS (Adams et al., 2002; Grosse-
Kunstleve and Adams, 2003). Phasing and density modification were
carried out with CNS (Brunger et al., 1998). Iterative rounds of manual
model building and refinement were carried out with WinCoot (Emsley
and Cowtan, 2004) and REFMAC (Murshudov et al., 1997), until R and
Rfreevalues converged (Jones et al., 1991). Structural figures were
generated with PyMOL (DeLano, 2002).
In Vitro End-Joining Reactions
End-joining reactions were performed as previously described with
EcoRV-EcoRV or EcoRV-KpnI substrate (Tsai et al., 2007). See the
Supplemental Data for further details.
Supplemental Data include Supplemental Experimental Procedures
and three figures and can be found with this article online at http://
We wish to thank Leonid Flaks for his assistance in data collection at
X8C, NSLS, Brookhaven, NY. This work was funded by a grant from
the Canadian Institutes of Health Research to M.S.J. (MOP-53209)
and an NSERC fellowship to S.N.A.
Received: June 14, 2007
Revised: September 7, 2007
Accepted: October 10, 2007
Published: December 27, 2007
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Crystal Structure of Human XLF