Biophysical Characterization of Human XRCC1 and Its Binding to Damaged and Undamaged DNA

ABSTRACT The human DNA repair protein, hXRCC1, which is required for DNA single-strand break repair and genetic stability was produced as a histidine-tagged polypeptide in Escherichia coli, purified by affinity chromatography, and subjected to sedimentation and spectroscopic analyses. This study represents the first biophysical examination of full-length XRCC1. Sedimentation equilibrium measurements indicated that hXRCC1 exists as a monomer at lower protein concentrations but forms a dimer at higher protein concentrations with a Kd of 5.7  10-7 M. The size and shape of hXRCC1 in solution were determined by analytical ultracentrifugation studies. The protein exhibited an intrinsic sedimentation coefficient, s0 20,w, of 3.56 S and a Stokes radius, Rs, of 44.5 Å, which together with the Mr of 68000 suggested that hXRCC1 is a moderately asymmetric protein with an axial ratio of 7.2. Binding of model ligands, representing single-strand breaks with either a nick or a single nucleotide gap, quenched protein fluorescence, and binding affinities and stoichiometries were determined by carrying out fluorescence titrations as a function of ligand concentration. XRCC1 bound both nicked and 1 nucleotide-gapped DNA substrates tightly in a stoichiometric manner (1:1) with Kd values of 65 and 34 nM, respectively. However, hXRCC1 exhibited lower affinities for a duplex with a 5 nucleotide gap, the intact duplex with no break, and a singlestranded oligonucleotide with Kd values of 215, 23

The X-ray repair cross-complementing group 1 (XRCC1)
gene was the first mammalian gene isolated that affects
cellular sensitivity to ionizing radiation (1). The 633 amino
acid protein encoded by the human XRCC1 gene is required
for maintenance of genome stability (2) and efficient repair
of oxidative DNA base damage and DNA single-strand
breaks by the base excision repair (BER)1 and single-strand
break repair (SSBR) pathways, respectively (3, 4). Both
pathways are essential for the repair of DNA damage inflicted
by ionizing radiation and alkylating agents, including many
chemotherapeutic drugs currently in use (5). Other results
support a role for XRCC1 protein in genetic stability in
noncycling and postmitotic stages of the cell cycle (6).
Current evidence strongly indicates that XRCC1 functions
as a chaperone or scaffolding protein capable of interacting
with several proteins participating in different repair pathways.
It forms complexes with poly(ADP-ribose) polymerase
(PARP) (7, 8), DNA polymerase â (9, 10), DNA ligase III
(11), polynucleotide kinase (12), human AP endonuclease
(13), and proliferating cell nuclear antigen (PCNA) at DNA
replication foci to facilitate SSBR at S phase (14).
Structural analysis of XRCC1 focused initially on one of
its BRCA1 carboxyl-terminal (BRCT) conserved domains.
Several other DNA repair and cell cycle regulator proteins
contain BRCT domains (15, 16). The first three-dimensional
structure of a BRCT domain was obtained for the C-terminal
region (residues 538-633) of XRCC1 (17) containing the
BRCT II domain. This structure provided a framework for
modeling other BRCT domains and interactions between
BRCT domains on different proteins, including the physical
association between XRCC1 and DNA ligase IIIR (11, 18,
19). The DNA binding and polymerase â binding sites of
XRCC1 have been mapped to its N-terminal domain (9, 20).
The NMR solution structure of this domain, located within
residues 84-183, indicated preferential binding to a DNA
ligand bearing a single-strand break (20).
The contact regions of hXRCC1 with a number of other
proteins have also been determined. These include PARP 1
and 2, OGG1 glycosylase and AP endonuclease I, which can
bind to the BRCT I domain (21, 22), and the forkheadassociated
domain of polynucleotide kinase, which binds
? This work was supported by the Canadian Institutes of Health
Research, the Alberta Cancer Board, the Alberta Cancer Foundation,
the Alberta Heritage Foundation for Medical Research, and the National
Cancer Institute of Canada. C.E.C. is Canada Research Chair in
Oncology.
* To whom correspondence should be addressed at the Department
of Experimental Oncology, Cross Cancer Institute, 11560 University
Ave., Edmonton, Alberta T6G 1Z2, Canada. Phone: [phone].
Fax: [phone]. E-mail: [e-mail] and rajam.mani@
cancerboard.ab.ca.
? Department of Experimental Oncology, Cross Cancer Institute, and
Department of Oncology, University of Alberta.
§ Genome Damage and Stability Centre, University of Sussex.
1 Abbreviations: hXRCC1, human X-ray repair cross-complementing
group 1; BER, base excision repair; SSBR, single-strand break repair;
PARP, poly(ADP-ribose) polymerase; Tris, tris(hydroxymethyl)aminomethane;
EDTA, ethylenediaminetetraacetic acid; SDS, sodium
dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; Hepes, 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid; nt, nucleotide.
Biochemistry 2004, 43, 16505-16514 16505
10.1021/bi048615m CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/02/2004
within a C-terminal domain (residues 402-529) of hXRCC1
(23). This latter binding is greatly stimulated by casein kinase
2-mediated phosphorylation of XRCC1 (23).
Despite the fact that several peptide domains have been
identified and two of these have been characterized at the
molecular level, there are still many physical properties of
hXRCC1 that remain to be resolved. This work represents
the first biophysical examination of full-length hXRCC1 in
solution. We carried out detailed hydrodynamic studies to
establish its oligomeric state in nondenaturing medium,
mimicking its cellular milieu. Our data indicate that hXRCC1,
at low protein concentration, exists as a monomer and is
moderately asymmetric but at higher concentrations exists
predominantly as a dimer. The hXRCC1 protein exhibited
strong affinity for DNA with single-strand breaks (nick or 1
nucleotide gap) with 1:1 protein:ligand binding stoichiometry.
MATERIALS AND METHODS
Expression of His-Tagged XRCC1 in Escherichia coli. The
pET16BXH construct (24) carrying a histidine (His10) tag
at the C-terminus of human XRCC1 was transfected into
host E. coli strain BL21(DE3) (Novagen). The bacteria were
grown at 37 °C to an OD600 of 0.6 in 60 mL of LB medium
containing ampicillin at a concentration of 100 íg/mL and
then kept at 4 °C overnight. The pelleted cells were used to
inoculate 4 L of LB media and then grown at 37 °C to an
OD600 of 0.6. XRCC1 expression was induced at 37 °C for
90 min by addition of isopropyl 1-thio-â-D-galactopyranoside
(Sigma, St. Louis, MO) to a final concentration of 1 mM.
After the cells were harvested by centrifugation at 5000g at
4 °C for 10 min, the cells were resuspended in 40 mL of
ice-cold sonication buffer (50 mM Hepes-NaOH, pH 8.0,
0.5 M NaCl, 0.1 mM EDTA, 10% glycerol), quick frozen
in liquid nitrogen, and thawed on ice followed by addition
of imidazole, dithiothreitol, and phenylmethanesulfonyl
fluoride to a final concentration of 1 mM for each reagent.
The bacteria were disrupted by sonication on ice, and the
soluble fraction was obtained by centrifugation at 10000g
for 20 min.
Purification of His-Tagged XRCC1 Protein. Recombinant
XRCC1 was isolated from the supernatant using ProBond
nickel-chelating resin (Invitrogen Life Technologies, Carlsbad,
CA) according to the manufacturer''s instructions and
as described previously (24). Briefly, supernatant from 2 L
of culture was mixed with 3 mL of nickel-charged affinity
resin and stirred on ice for approximately 1 h. The slurry
was loaded into a 5 mL column at a flow rate of 3 mL/min,
and the flow-through (40 mL) was collected. The column
was washed with 20 mL of sonication buffer and 30 mL of
wash buffer (50 mM Hepes-NaOH, pH 7.0, 0.1 M NaCl,
0.1 mM EDTA, 1 mM dithiothreitol, 10% glycerol) containing
40 mM imidazole, pH 8.0, at a flow rate of 0.5 mL/min.
The column was washed further with 15 mL of wash buffer
containing 80 mM imidazole, and the bound protein was
subsequently eluted with 15 mL of wash buffer containing
250 mM imidazole in 1.5 mL fractions. The protein purity
was assessed by electrophoresis of 10 íL of each fraction
on a 10% SDS-polyacrylamide gel and Coomassie Blue
staining. The desired protein concentration and buffer
exchange (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM
MgCl2, and 1 mM dithiothreitol) were achieved by using a
30 kDa cutoff Millipore ultrafree concentrator.
Hydrodynamic Studies. Fringe counts were performed
using a Beckman XLI analytical ultracentrifuge and doublesector
capillary synthetic boundary sample cells as described
by Babul and Stellwagen (25). Prior to ultracentrifugation,
protein samples were dialyzed for 48 h in 50 mM Tris-HCl
buffer (pH 7.5), 100 mM NaCl, 5 mM MgCl2, and 1 mM
dithiothreitol. The absorbance of each sample was measured
using 1.0 cm path length cuvettes. Samples (150 íL) were
loaded into one sector of the sample cell, and 400 íL of the
dialysate was loaded into the other sector. Runs were
performed at 8000 rpm, and scans were taken when fringes
were resolved across the boundary region between protein
solution and solvent. The number of fringes produced across
the boundary was measured and converted to concentration
using an average increment of 3.31 fringes mg-1 mL-1. From
a plot of the number of fringes versus optical density, a value
of 7.90 was established as the extinction coefficient,
1%
1cm,280nm, for hXRCC1.
Sedimentation Velocity Measurements. Sedimentation velocity
experiments were carried out at 20 °C and 50000 rpm
using the XLI analytical ultracentrifuge and absorption optics
following the procedures described by Laue and Stafford (26)
and as also outlined in the instruction manual (Spinco
Business Center of Beckman Instruments, Inc., Palo Alto,
CA). Four hundred microliters of sample solution and 400
íL of dialysate were loaded into two-sector CFE centerpiece
sample cells containing sapphire windows. Runs were
performed for 4 h during which time a minimum of 30 scans
were taken. The sedimentation velocity data were analyzed
according to Williams et al. (27) to determine the sedimentation
coefficient, s. The intrinsic sedimentation coefficient,
s0
20,w, which represents the sedimentation coefficient corrected
to water at 20 °C, was then calculated from the
observed S value as described by Laue et al. (28).
Sedimentation Equilibrium Studies. Sedimentation equilibrium
experiments were carried out at 5 °C using absorption
optics. Samples (110 íL) were loaded into six-sector CFE
cells, allowing three concentrations of sample to be run
simultaneously. Runs were performed at 9000 and 11000
rpm, and each speed was maintained until there was no
significant difference in scans taken 2 h apart to ensure
equilibrium was achieved. The sedimentation equilibrium
data were evaluated with the Nonlin analysis program using
a nonlinear least-squares curve-fitting algorithm (29). The
program Sednterp (Sedimentation Interpretation Program,
version 1.01) was employed to calculate the partial specific
volume of the protein from the amino acid composition using
the method of Cohn and Edsall (30).
Hydrodynamic Calculations and Ellipsoid Modeling. The
observed sedimentation coefficient, s, determined from
sedimentation velocity data will correspond to the maximum
S value that can be obtained for the given molecular mass
of the protein and correspondingly the protein would have
the minimum frictional coefficient, f0. Translational frictional
ratios (f/f0) were calculated from the experimental Stokes
radius obtained from sedimentation velocity experiments
(Rs,sed) according to Mani and Kay (31). The frictional ratio
f/f0, which is equivalent to Smax/s20,w, indicates the maximum
shape asymmetry of the protein. The total shape asymmetry
depends on two factors, a geometrical shape asymmetry and
expansion due to hydration. A globular protein with different
ellipsoid shapes can be modeled from f/f0 or f/fshape values
16506 Biochemistry, Vol. 43, No. 51, 2004 Mani et al.
(28), using the software program Sednterp 1.01, in which
the semimajor to semiminor (a/b) axial ratio of a prolate or
oblate ellipsoid of revolution is determined using the
respective power series approximation of the tabulated data
for a/b as a function of (f/f0 - 1) or (f/fshape - 1) for each
ellipsoid. To calculate f/fshape using Sednterp, the ä value,
which corresponds to hydration in grams of water per gram
of protein, was based on the amino acid composition of
XRCC1 (32). The Sednterp program provides a graphical
presentation of the hydrodynamic model from the volume
of an ellipsoid (4/3ðab2), which is equivalent to the volume
of the hydrated protein.
Fluorescence Studies. Steady-state fluorescence spectra
were measured at room temperature on a Perkin-Elmer LS-
55 spectrofluorometer (Freemont, CA) with 5 nm spectral
resolution for excitation and emission using 0.1-0.2 íM
solutions of purified recombinant hXRCC1. Protein fluorescence
was excited at 295 nm, and fluorescence emission
spectra were recorded in the 300-400 nm range; changes
of fluorescence were usually monitored at the emission
maximum (326 nm). In studying the effects of DNA ligands
(Table 1) on protein fluorescence intensities, additions to
hXRCC1 samples were made from ligand stock solutions,
keeping the protein dilution below 3%, and fluorescence
intensities were corrected for dilution factors. Background
quenching, if present (
nicked DNA . 5 nt-gapped DNA > intact duplex > singlestranded
oligonucleotide. The values indicate that there is a
substantial difference in binding affinity for damaged versus
undamaged or single-stranded substrates. While the presence
of a 5¢-phosphate at a 1 nt gap had very little influence,
widening the gap to five nucleotides reduced the affinity of
the protein to the same level as the undamaged substrate.
The binding stoichiometry determined for all of these
substrates was also 1:1, suggesting XRCC1 binds its
substrates in a stoichiometric manner.
In addition, we also determined the binding affinity of
XRCC1 for 1 nt-gapped DNA in the presence of singlestranded
oligonucleotide by fluorometric titration. For this
experiment, we first added 0.5 íM single-stranded oligonucleotide
(2  Kd) to XRCC1, and the tryptophan
emission intensity obtained at 326 nm in the presence of
single-stranded oligonucleotide was taken as the control
value. Quenching of the fluorescence intensity was then
monitored as a function of the concentration of added 1 ntgapped
DNA to determine the binding affinity of the 1 ntgapped
DNA to XRCC1 in the presence of single-stranded
oligonucleotide. The Kd value obtained in this instance was
55 ( 5 nM, suggesting that XRCC1 was capable of binding
1 nt-gapped DNA with high affinity in the presence of
competing single-stranded oligonucleotide. On the other
hand, the presence of 1 nt-gapped DNA (75 nM; 2  Kd)
interfered with the XRCC1-single-stranded oligonucleotide
interaction. The Kd value obtained with the single-stranded
oligonucleotide in this instance was 1 íM, indicating that
the binding affinity was reduced approximately 4-fold
compared with the value of 260 nM in the absence of
competing 1 nt-gapped substrate.
Circular Dichroism Studies. Information concerning the
secondary structure of hXRCC1 was obtained from far-UVCD
data, and a typical far-UV-CD spectrum of hXRCC1
is shown in Figure 6. hXRCC1 exhibited two large, negative
CD bands centered around 208 and 218 nm, indicating the
presence of R-helical organization. The observed molar
ellipticities, [õ]M, at these two wavelengths were - 8700 (
300 and -6100 ( 300 deg cm2 dmol-1, respectively. The
CD spectra were analyzed according to the method of
Compton and Johnson (33). The protein possessed 40%
R-helix and 30% â-structure, and the remaining 30%
represented random structure.
Since, hXRCC1 exhibited strong affinity for 1 nt-gapped
and nicked DNA and significantly lower affinities for the
intact duplex with no break and a single-stranded oligonucleotide,
we studied the effect of 1 nt-gapped and nicked
DNA on hXRCC1 protein conformation. It is evident from
Figure 6 that the addition of 1 nt-gapped DNA induced a
conformational change in hXRCC1; the molar ellipticity
values [õ]M at 208 and 218 nm were reduced to -7000 (
300 and -5200 ( 300 deg cm2 dmol-1, respectively.
Analysis of the CD data indicated an increase in R-helical
content accompanied by a decrease in â-structure, and the
calculated values were R-helix (45%) and â-structure (25%)
and the random structure corresponded to 30%. The binding
FIGURE 4: Fluorescence titration of hXRCC1 vs duplex with a
single nucleotide gap. (A) hXRCC1 (78 nM) against gapped DNA
in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2, and 1
mM dithiothreitol. The protein was excited at 295 nm, and the
fluorescence intensity was monitored at 326 nm (see inset). The
fraction bound (i.e., relative fluorescence quenching) vs ligand
concentration is plotted. (B) Sample plot of fluorescence data from
titration with gapped DNA. F0, F, and F¥ are the relative
fluorescence intensities at 326 nm of hXRCC1 alone, hXRCC1 in
the presence of a given concentration of gapped DNA, and hXRCC1
saturated with gapped DNA, respectively. The plot is according to
Chipman et al. (52). (C) The observed change in fluorescence
intensity, ¢F, at a given ligand concentration divided by the
maximum change at saturating ligand concentration, ¢Fmax, is
plotted against the molar ratio of ligand to protein.
16510 Biochemistry, Vol. 43, No. 51, 2004 Mani et al.
of nicked DNA produced changes similar to those observed
with gapped DNA in the CD spectrum of hXRCC1 (data
not shown).
DISCUSSION
This study provides the first biophysical examination of
full-length hXRCC1, a protein that plays an important role
in base excision repair (BER) and single-strand break repair
(SSBR) of damaged DNA. The hXRCC1 cDNA, which
encodes a protein of 633 amino acids with a molecular mass
of 69.5 kDa (11), was expressed in E. coli. However, the
size of the purified recombinant protein as estimated by
SDS-PAGE was 85 kDa, in agreement with the earlier
reported values (11, 34). This anomalous behavior in SDSPAGE
is not unique to XRCC1. Anomalous electrophoretic
behavior has also been reported for several other proteins
(43-45). For example, pig heart calpastatin with 713 amino
acid residues (Mr 77122) exhibits an anomalous behavior in
SDS gels, and the estimated molecular mass is 107 kDa (43).
The observed slow migration in SDS gels could be a
reflection of their unique amino acid compositions, being
poor in aromatic acids and rich in proline and acidic residues.
The proline content of XRCC1 is 9.5 mol %, and the acid
residue content (Asp and Glu) is 14.3 mol %. This high
content of negatively charged amino acids may restrict
binding of SDS to hXRCC1, resulting in deviation from
normal mobility expected for a protein of this size.
In the ultracentrifuge, the hXRCC1 sedimented with an
intrinsic sedimentation coefficient, s0
20,w, of 3.56, suggesting
that it must be moderately asymmetric, since a globular
protein like bovine serum albumin with a molecular mass
of 66000 Da sediments much faster with an intrinsic
sedimentation coefficient of 4.6. The sedimentation value
decreased upon dilution, implying that the subunits are in a
rapidly reversible equilibrium between monomeric and
oligomeric forms. For a nonassociating system the s values
should increase as the protein concentration decreases, since
the frictional coefficient will decrease as the protein concentration
is lowered. To understand the nature of the
aggregation, sedimentation equilibrium experiments were
carried out over a range of protein concentrations.
The self-association of hXRCC1 in 50 mM Tris-HCl, 0.10
M NaCl, 5 mM MgCl2, and 2 mM dithiothreitol at pH 7.5
was characterized in detail. Sedimentation equilibrium studies
demonstrated that hXRCC1 exists in a monomer-dimer
equilibrium. The multiple sedimentation equilibrium data
FIGURE 5: Fluorescence titration of hXRCC1 with DNA ligands: (A, B) hXRCC1 (78 nM) vs nicked DNA; (C, D) hXRCC1 (95 nM) vs
duplex; (E, F) hXRCC1 (95 nM) vs single-stranded oligonucleotide.
Table 2: Binding of Ligands to hXRCC1 at 25 °C in 50 mM
Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2, and 1 mM DTTa
ligand Kd (nM)
1 nt-gapped DNA 34 ( 3
1 nt-gapped DNA (5¢-P)b 52 ( 4
5 nt-gapped DNA 215 ( 10
nicked DNA 65 ( 5
duplex 230 ( 10
single-stranded oligonucleotide 260 ( 10
a Kd values (mean ( SE, n ) 3) were determined by fluorescence
titration. b 5¢-P indicates the presence of a 5¢-phosphate at the DNA
terminus.
FIGURE 6: Far-UV CD spectrum of hXRCC1 (2) and hXRCC1 +
10 íM 1 nt-gapped DNA (9). The concentration of hXRCC1 was
0.45 mg/mL in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM
MgCl2, and 1 mM dithiothreitol.
Biophysical Characterization of Human XRCC1 Biochemistry, Vol. 43, No. 51, 2004 16511
sets, obtained at three initial loading concentrations as well
as two rotor speeds, were fitted to a monomer-dimer model,
and the Ka value of 1.75  106 M-1 indicates a strong
association between monomers to form dimers. The association
free energy (-8.5 kcal/mol) strongly favors dimer
formation, such that at concentrations exceeding 0.6 mg/
mL the reaction was nearly complete with 86% of the protein
being dimerized. Zhang et al. (17) have determined the threedimensional
structure and fold of the C-terminal BRCT
domain of hXRCC1. In the crystal structure there are two
BRCT domains in the asymmetric unit forming a dimer. In
the present study, we have demonstrated that intact hXRCC1
at higher concentrations can also exist as a dimer. It remains
to be determined if hXRCC1 dimerization is mediated by
its C-terminal BRCT domain. It is probable that the hXRCC1
C-terminal BRCT domain is also the site of interaction
between full-length hXRCC1 and the complementary BRCT
domain in DNA ligase III, because these two domains in
truncated proteins have been shown to form a stable
heterodimeric complex (18). Our finding that full-length
XRCC1 protein in solution can also exist as a dimer suggests
that the DNA ligase III binding site at the C-terminal BRCT
domain in XRCC1 is conserved in the intact protein.
Although XRCC1 tends to dimerize at higher concentrations,
we believe that, under physiological conditions, XRCC1 will
exist as a monomer in association with DNA ligase III.
However, we still need to determine the relative amounts of
both proteins in the cell and also determine their binding
affinity and stoichiometry. The conserved BRCT domains
in XRCC1 and other proteins enable them to dimerize, both
within a single polypeptide and between different polypeptides,
and there is evidence that these BRCT dimers facilitate
phosphorylation-specific interactions and may have a regulatory
role during repair (46, 47).
Our finding that hXRCC1 is moderately asymmetric with
the calculated dimensions of roughly 235  30 Å and an
axial ratio of about 8:1, regardless of the model chosen, could
have a bearing on its function. For a scaffolding protein
capable of interacting with several proteins, it is advantageous
to have an extended rodlike structure, thereby providing more
surface area for other proteins to bind without much steric
hindrance. Williams et al. (48) have predicted that proteins
with more than two BRCT domains are likely to assume
rodlike structures. Our data suggest that this prediction may
be extended to proteins possessing two BRCT domains. At
least four different proteins, PARP, DNA polymerase â,
polynucleotide kinase, and DNA ligase III, in addition to
XRCC1, are involved in recognizing and binding to radiationinduced
single-strand break DNA. The scaffolding protein
hXRCC1 may not only bring all the players together but
may also regulate them (12, 49). Defined regions of hXRCC1
are involved in binding these proteins. As mentioned, the
N-terminal region is involved in binding DNA polymerase
â, the C-terminal BRCT-II domain is responsible for binding
to DNA ligase III and XRCC1 interacts with PARP through
a central BRCT-I domain (8), and the linker region between
the two BRCT domains which contains the CK2 phosphorylation
sites interacts with the forkhead-associated domain
of PNK (23).
The NMR solution structure of the XRCC1 N-terminal
domain was studied in detail by Marintchev et al. (20). This
domain specifically binds DNA with single-strand breaks
(gapped and nicked), as evidenced by gel-shift assays (20).
In the present study, we have determined the binding affinity
and the stoichiometry of binding of these ligands to the fulllength
protein by fluorescence measurements, and these
parameters are essential for understanding the role of
hXRCC1. The binding was specific for 1 nt-gapped and
nicked DNA, and the protein showed significantly lower
affinities for the intact duplex with no break, a singlestranded
oligonucleotide, and a 5 nt-gapped duplex. Our
finding that hXRCC1 exhibits relatively low affinity toward
a single-stranded oligonucleotide implies that recognition of
single-strand breaks by hXRCC1 is intrinsic to the nature
of the single-strand break itself, rather than the single
strandedness that might occur through DNA breathing at the
break site. Furthermore, the fact that a gap of five nucleotides
causes a marked reduction in binding affinity suggests that
XRCC1 contacts both the 3¢ and 5¢ termini of the strand break
and this gap has to be <5 nucleotides for optimum binding.
The fact that hXRCC1 itself has high affinity (Kd values
in the nanomolar range) for single-strand break DNA
suggests that it could have a sensor function by itself. Current
models of SSBR propose that PARP-1 functions as the
primary single-strand break sensor (50). Arrival of XRCC1
at these PARP-bound sites may lead to an exchange of
XRCC1 for PARP, with XRCC1 then functioning as a
scaffolding protein directing the enzymes that carry out repair
(8, 12). However, the absence of PARP-1 does not prevent
SSBR but only reduces the rate of repair 2-3-fold (51). In
these circumstances XRCC1 may be acting as the strand
break sensor in addition to its structural function.
Now that we have obtained some basic physical parameters
for hXRCC1, it will be important to study in detail the mode
of interaction of hXRCC1 with other proteins involved in
single-strand break binding and repair. A similar approach
using fluorescence spectroscopy and circular dichroism
measurements should now allow us to examine binary,
ternary, and quaternary complexes of these proteins.
ACKNOWLEDGMENT
We thank Emmanuel Guigard of the Biochemistry Department
(University of Alberta) for sedimentation studies,
Wayne Moffat, Spectral Services Supervisor, Department of
Chemistry (University of Alberta), for CD analysis, and Dr.
J. N. Mark Glover, Department of Biochemistry (University
of Alberta), for valuable discussions.
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16514 Biochemistry, Vol. 43, No. 51, 2004 Mani et al.

Rajam S. Mani,*,? Feridoun Karimi-Busheri,? Mesfin Fanta,? Keith W. Caldecott,§ Carol E. Cass,? and
Michael Weinfeld*,?
Department of Experimental Oncology, Cross Cancer Institute, and Department of Oncology, UniVersity of Alberta,
Edmonton, Alberta T6G 1Z2, Canada, and Genome Damage and Stability Centre, UniVersity of Sussex, Science Park Road,
Falmer, Brighton BN1 9RQ, U.K.