From the Harold C. Simmons Arthritis Research Center, the
Department of Internal Medicine and Program in
Immunology, University of Texas Southwestern Medical Center, Dallas,
Texas 75235 and ¶ The Center for Blood Research and Department of
Genetics, Harvard University Medical School,
Boston, Massachusetts 02115
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ABSTRACT |
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The gene product of XRCC4 has been implicated in both V(D)J recombination and the more general process of double strand break repair (DSBR). To date its role in these processes is unknown. Here, we describe biochemical characteristics of the murine XRCC4 protein. XRCC4 expressed in insect cells exists primarily as a disulfide-linked homodimer, although it can also form large multimers. Recombinant XRCC4 is phosphorylated during expression in insect cells. XRCC4 phosphorylation in Sf9 cells occurs on serine, threonine, and tyrosine residues.
We also investigated whether XRCC4 interacts with the other factor known to be requisite for both V(D)J recombination and DSBR, the DNA-dependent protein kinase. We report that XRCC4 is an efficient in vitro substrate of DNA-PK and another unidentified serine/threonine protein kinase(s). Both DNA-PK dependent and independent phosphorylation of XRCC4 in vitro occurs only on serine and threonine residues within the COOH-terminal 130 amino acids, a region of the molecule that is not absolutely required for XRCC4's DSBR function. Finally, recombinant XRCC4 facilitates Ku binding to DNA, promoting assembly of DNA-PK and complexing with DNA-PK bound to DNA. These data are consistent with the hypothesis that XRCC4 functions as an alignment factor in the DNA-PK complex.
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INTRODUCTION |
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V(D)J recombination is the process of assembling the variable (V), diversity (D), and joining (J) gene segments of the immunoglobulin and T cell receptor variable region genes during development of B and T lymphocytes (1, 2). The proteins responsible for V(D)J joining are collectively referred to as the V(D)J recombinase, and are shared by both the B and T cell lineages. It has recently become clear that the lymphocyte-specific proteins, recombination activating genes 1 and 2 (RAG1 1 and RAG 2) (3, 4) are directly responsible for initiation of the V(D)J recombination reaction (5-8) together directing cleavage at the recombination signal sequence-coding juncture; whereas resolution of recombination intermediates also requires several ubiquitously expressed DNA repair factors. Evidence linking DNA repair activities with V(D)J recombination arose from the characterization of cells from homozygous severe combined immune deficient (SCID) mice (9-11). C.B-17 SCID mice which are immunodeficient because of defective V(D)J recombination, were shown to also be defective in repairing DSBs in lymphoid and non-lymphoid cells (12, 13). Further evidence for an overlap in the activities of V(D)J recombination and DSB repair came from the observation that radiosensitive rodent cell lines which were defective in DSB repair could not support V(D)J recombination induced by co-transfecting RAG 1 and RAG 2; whereas, radiosensitive mutants proficient in DSB rejoining were normal in this capacity (14-16). Four factors have been delineated which are required for both V(D)J recombination and DSBR; three of these encode components of the DNA-dependent protein kinase (17-25). DNA-PK is a nuclear serine/threonine protein kinase whose catalytic activity is dependent upon its association with DNA discontinuities such as DSBs (26, 27). The catalytic subunit of DNA-PK is an ~460-kDa protein termed DNA-PKCS and the DNA-binding component is a heterodimer, Ku which is comprised of two polypeptides (Ku70 and Ku86). Because of its unique dependence on DNA ends and other DNA discontinuities for activation, it has been proposed that the role of DNA-PK in DNA repair is to modulate, via phosphorylation, the function and/or expression of other factors in response to DNA damage (18). This hypothesis has not been tested. The fourth factor required for both V(D)J recombination and DSBR is the product of the XRCC4 gene. This factor has no homology to known proteins and its function is unknown (28) although it has recently been reported that XRCC4 interacts with DNA ligase IV (29, 30). Although the functions of DNA-PK and XRCC4 in V(D)J recombination have not been elucidated, it is intuitive that their role is in the resolution of coding and signal ends generated by RAG-mediated cleavage.
In both DNA-PK- and XRCC4-defective cells, low levels of rearrangements (both coding and signal joints) can be isolated (i.e. "leaky" SCID phenotype) (31, 32). Fine structure of these joints has provided insight into mechanistic details of V(D)J recombination. In SCID mice, rare coding joints often have large deletions and/or long P elements (palindromic sequences at the point of ligation which apparently result from internal nicking of the hairpinned coding intermediate) (31, 32). Thus, it has been proposed that these long P elements reflect a diminution of hairpin resolution consistent with the observation that coding ends with hairpinned termini accumulate in SCID lymphocytes (33, 34). Rare coding joints isolated from XRCC4 defective cells are distinct from those found in SCID mice in that there is no evidence of extensive P nucleotides. Instead, it appears that short sequence homologies at the DNA termini contribute to the resolution of both coding and signal ends in XRCC4-deficient cells (28). Although homology mediated joining is often observed in coding joints, it is not observed in signal ligations (35-37). Thus, Li et al. (28) have postulated that one function of XRCC4 may be to align free DNA ends, and in the absence of XRCC4 short sequence homologies might serve to facilitate DNA end joining.
Here we report that recombinant XRCC4 and DNA-PK interact in vitro. The multimeric nature of XRCC4 suggests a model where XRCC4 functions as an alignment factor of DNA-PK bound to DNA serving to align DNA ends during DNA repair and V(D)J recombination. These data, in conjunction with the recent reports of XRCC4's interaction with DNA ligase IV (29, 30), suggest a model whereby XRCC4 targets cellular ligase activities to damaged DNA bound by the DNA-dependent protein kinase.
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MATERIALS AND METHODS |
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Expression of XRCC4 in Baculovirus
Polymerase chain reaction mutagenesis was utilized to engineer
EcoRI cloning sites and a 9X histidine tag into the murine XRCC4 cDNA (28) for cloning into the baculovirus
transfer vector pAcGP67B (Pharmingen, San Diego, CA), or
NcoI and XbaI sites for cloning into the
pH1360x transfer vector (38). Similarly, polymerase chain reaction
mutagenesis was utilized to generate the XRCC4 truncated proteins. The
oligonucleotides used were: 5
-XRCC4HIS, ggggaattcatcaccatcaccatcaccatcaccatatggaaaggaaagtaagcagaatctac; 3
-XRCC4, taggaattctatctaatcaaagagatcttctgggct; 5
-XRCC4,
ggggaattcaaaggaaagtaagcagaatctac; 3
-XRCC4HIS,
taggaattctaatggtgatggtgatggtgatggtgatgatcaaagagatcttctgggct; 5
-XRCC4NcO, tagccagccatggaaaggaaagtaagc;
3
-XRCC4HISXba,
tagtctagactaatggtgatggtgatggtgatggtgatgatcaaaagagatcttctgggct; 5
-65XRCC4HIS,gggggaattcatcaccatcaccatcaccatcaccataaatacattgatgagctgagaaaggca; 3
-171XRCC4, atcgaattccaactaggcttctttggcactcacacatttctc; and
3
-204XRCC4 actgaattcctactgctggacttcatttagc.
pAcGP67B provides a leader sequence from the viral acidic glycoprotein
gp67 and should yield secreted versions of nuclear proteins. We have
observed, however, that recombinant proteins expressed from this vector
are usually not secreted and proteins must be purified from cell
lysates using Ni-NTA resin (see "Protein Purification"). Still,
this vector was used as it results in consistently higher levels of
recombinant protein than can be obtained with other transfer
vectors.2 An additional
recombinant virus encoding full-length XRCC4 but without the gP67
leader sequence was generated using the pH1360x transfer vector
(38). The resulting plasmids were co-transfected with linearized AcMNPV
viral DNA via liposomes (Invitrogen, San Diego, CA) into Sf9
insect cells. Recombinant viruses were then isolated using standard
techniques. Baculovirus expression resulted in a fusion protein
consisting of 10 amino acids from the gp67 leader peptide, nine
histidine residues, and the murine XRCC4 polypeptide. The Ku86 and Ku70
baculoviruses were the generous gifts of Dr. J. Donald Capra
(University of Texas Southwestern Medical Center, Dallas, TX) and have
been described previously (39).
Expression of XRCC4 in XR-1 Cells
EcoRV/NotI fragments from the baculovirus transfer vectors encoding XRCC4 fusion proteins were subcloned into the NotI and SmaI sites in the cloning cassette of plasmid pcDNAI. Resulting plasmids were co-transfected with the pNeo plasmid into the XR-1 Chinese hamster ovary mutant cell line which is defective in XRCC4 with LipofectAMINE according to the manufacturer's conditions (Life Technologies, Inc., Gaithersburg, MD). Stable lines were established by G418 selection.
Assessment of Irradiation Sensitivity
2 × 103 cells suspended in 2 ml of complete media were exposed to various amounts of ionizing radiation using a 137Ce source calibrated at 558.8 R/min. Immediately after irradiation, cells were seeded in 20 ml of complete media containing 10% fetal calf serum in 150-mm2 tissue culture dishes. After 7 days, cell colonies were fixed with 2% formaldehyde followed by 100% methanol. Subsequently, the colonies were stained with trypan blue and colony numbers were assessed. Irradiation sensitivities were assessed in triplicate and are presented as percent surviving fraction based on plating efficiencies of non-irradiated controls.
Protein Purification
For protein purification, Sf9 cells which had been
infected ~60 h earlier were collected and washed with
phosphate-buffered saline. Typically, 2 × 108
infected cells were prepared at a time. Cell pellets were frozen and
thawed three times in liquid nitrogen in 10 ml of the following buffer:
20 mM Tris (pH 7.9), 0.5 M NaCl, 5 mM imidazole, and 0.5 mM phenylmethylsulfonyl
fluoride. The lysates were centrifuged for 30 min at 8,000 × g. Lysates were incubated for 1 h at 4 °C with 0.5 ml of Ni-NTA resin (Qiagen, Chatsworth, CA) and the resin washed 3 times in the same buffer with the addition of 50 mM
imidazole. Soluble recombinant XRCC4 protein was eluted from the resin
in 0.5 ml of the same buffer with 500 mM imidazole three
times. The three eluted fractions were pooled and then dialyzed against
25 mM Tris (pH 8.0), 150 mM KCl, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 10% glycerol at 4 °C. Dialyzed material was
aliquotted and stored at 70 °C. 10 µl of lysates, beads, and
eluate were analyzed by SDS-PAGE (Fig. 1). The results depicted were
typical for virus XRCC4HIS and for
C2. Purification of
the other recombinant proteins described here consistently resulted in
much lower protein yields. Because of the low yields from viruses
N,
C1, and
N
C1, contaminating Sf9 proteins comprised a
larger percentage of the total protein than with viruses
XRCC4HIS or
C2. For this reason, control Ni+
preparations of wild type-infected Sf9 cells were also prepared and used as controls in kinase assays (Fig. 5) and EMSA assays (not
shown).
In Vitro Kinase Assays
XRCC4 Assays--
Typically, 1 µg of purified XRCC4 was
incubated with 300 ng of DNA-PK (Promega Corp., Madison, WI), and with
or without 100 ng of linearized pTZ19R plasmid DNA (Pharmacia Biotech,
Piscataway, NJ) in a total volume of 20 µl of Z0.05 (24). The
mixtures were incubated with 37.5 µCi of [
-32P]ATP
for 1 h at room temperature, then mixed with an equal volume of
2 × SDS-PAGE buffer without 2-mercaptoethanol. In assays
utilizing truncated forms of XRCC4, 400 ng of each recombinant protein
was incubated with 300 ng of DNA-PK. Samples were loaded onto
SDS-polyacrylamide gels and run at 200 volts for 45 min. Following
electrophoresis, gels were dried and then exposed to Kodak X-Omat AR
film. In some assays, wortmannin was included at a final concentration
of 500 nM.
c-Jun assay--
Approximately 300 ng of purified DNA-PK
(Promega, Madison, WI) was incubated in the presence of 10 µg of
bovine serum albumin and 2 µl of DNA-PK reaction premix (13 mM spermidine and 4 mM MgCl2
dissolved in 0.05 × phosphate-buffered saline) with 1 µg of
purified rhAP1 (c-Jun, Promega, Madison, WI), 2 µl of 2.5 mM ATP, 0.5 µl (6000 Ci/mmol; DuPont)
[-32P]ATP, with or without 1 µg of purified XRCC4
and with or without 100 ng of linearized plasmid DNA. Samples were
incubated for 10 min at 30 °C, then mixed with equal volumes of
2 × SDS-PAGE buffer. Samples were loaded onto an SDS-8%
polyacrylamide gel and run at 200 volts for 45 min. Following
electrophoresis, gels were dried and exposed to Kodak X-Omat AR
film.
DNA-PK Pulldown Assay--
The "DNA-PK pulldown assay" was
performed essentially as described by Finnie et al. (24)
using the 0176 normal equine fibroblast and 1863 SCID equine fibroblast
cell lines (40). Briefly, cell pellets (1 × 107) were
resuspended in 100 µl of extraction buffer and then frozen in liquid
nitrogen and thawed at 37 °C three times. The extract was
microcentrifuged at 15,000 rpm for 7 min at 4 °C and the supernatant was frozen at 70 °C. Extracts containing ~7 mg of protein were absorbed onto DNA-cellulose beads and washed. 400 ng of XRCC4 was added
to the mixture and incubated for 1 h at room temperature. Following incubation, an equal volume of 2 × SDS-PAGE sample
buffer was added and the phosphorylated proteins were analyzed on
SDS-8% polyacrylamide gels.
Phosphoamino Acid Analysis-- Kinase assays were performed as above. Following polyacrylamide gel electrophoresis, the gel was transferred to polyvinylidene difluoride paper and exposed to film. Small strips of polyvinylidene difluoride paper which corresponded to bands of interest on the autoradiograph were excised and wet in methanol for 30 s and 1 min in water. The samples were incubated in 200 µl of 6 N HCl at 110 °C for 1-2 h and dried overnight. Pellets were counted in a scintillation counter and resuspended in acid (2.2% formic acid, 7.8% acetic acid) to a concentration of 2000 cpm/µl. 2 µl each of phosphoserine, threonine, and tyrosine standards (1 mg/ml) were mixed with 1 µl of sample and spotted onto a thin layer chromatography plate. The plate was then electrophoresed at 1200 volts constant voltage at 4 °C for 65 min. The plate was dried, sprayed with 0.3% ninhydrin in acetone, and exposed to film.
Metabolic Labeling of Baculovirus-infected Cells
Sf9 cells were infected with either wild type baculovirus or recombinant baculoviruses in SF900 serum-free media (Life Technologies, Inc., Gaithersburg, MD). After 24 h, the media was replaced with Grace's insect media containing [32P]orthophosphate (200 mCi/ml). Cells were harvested 24 h later. Metabolically labeled XRCC4 was affinity purified by Ni+ chromatography as described above.
Electrophoretic Mobility Shift Assays (EMSAs)
EMSAs were done by the method of Landolfi et al. (41)
with the following modifications. Binding reactions were done in
0.5 × buffer C (20 mM HEPES, pH 7.9, 25% glycerol,
420 mM NaCl, 1.5 mM MgCl2) instead
of buffer D, no nonspecific competitor was added, and bovine serum
albumin was included at a concentration of 3 mg/ml. The probe was a
double strand oligonucleotide that included a 12-base pair spacer V(D)J
recombination signal sequence and had the following sequence:
5-GATCTGGCCTGTCTTACACAGTGCTACAGACTGGAACAAAAACCCTGCAG-3
(8). Completely analogous results were obtained with a second unrelated oligonucleotide probe derived from a T cell receptor V
promoter. Binding reactions were assembled on ice and then incubated at
room temperature for 30 min. For EMSAs examining the ability of XRCC4
to bind DNA directly or to affect Ku binding to DNA, 5 µg of purified
XRCC4 was used without or with 1, 5, or 10 µg of
baculovirus-expressed Ku purified by absorption to DNA-cellulose (39).
Rabbit anti-Ku antisera was the generous gift of Dr. J. Donald Capra
(University of Texas Southwestern Medical Center, Dallas, TX). For
EMSAs examining DNA-PK assembly, 4, 2, 1, 0.5, or 0.25 µg of purified
full-length XRCC4 fusion protein or purified
C2 were incubated with
60 ng of DNA-PK (Promega Corp., Madison, WI). Protein-DNA complexes
were resolved on 4% native polyacrylamide gels in 0.5 × TBE.
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RESULTS |
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Characterization of Baculovirus Expressed Murine XRCC4--
To
assess the biological role of XRCC4, expression of this protein in the
baculovirus system was undertaken. To that end, several different
9X-HIS tagged versions of the XRCC4 cDNA (28) were generated and
inserted in the pAcGP67B or pH1360x transfer vectors. Recombinant
viruses were isolated using standard techniques (see "Materials and
Methods"). These are diagrammatically depicted in Fig.
1A. Constructs
XRCC4HIS and
C2 expressed relatively high levels of
recombinant protein. Constructs
C1,
N, and
N
C1 consistently expressed considerably lower levels of recombinant protein.
Ni+-agarose affinity chromatography was used to purify
recombinant XRCC4 expressed in insect cells. When expressing
full-length XRCC4, a prominent ~57-kDa band was consistently apparent
in lysates of Sf9-infected cells under reducing conditions (Fig.
1B, pre-absorption). Ni+-agarose absorption
dramatically reduced the ~57-kDa band in cell lysates (Fig. 1B,
post-absorption), but the band was apparent in the population of
proteins bound to the Ni+-agarose (Fig. 1B,
bound). XRCC4HIS was efficiently eluted from the
Ni+-agarose using imidazole (Fig. 1B, eluted).
Identification of the ~57-kDa protein as the XRCC4HIS
fusion protein was confirmed by amino acid sequence analysis. In
addition, mass spectrometric analysis indicated that the mass of the
protein was actually ~39 kDa, consistent with the predicted mass of
the XRCC4HIS fusion protein (39.5 kDa). The anomalous
mobility of recombinant XRCC4HIS in SDS-PAGE is likely due
to its low isoelectric point (5.6).
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Purified XRCC4HIS Is a Phosphoprotein That Exists
Primarily as a Homodimer but Which Can Form Large Multimers--
In
our initial analyses of the recombinant XRCC4HIS fusion
protein, it was apparent that the protein: 1) existed as dimers and
multimers, and 2) was constitutively phosphorylated during expression
in Sf9 cells. To illustrate these points, Fig.
2 presents analyses in both native
polyacrylamide gels (right panel) and SDS-PAGE (left
panel), of Ni+-agarose purified XRCC4HIS
isolated from [32P]orthophosphate metabolically labeled
Sf9 cells. As a control, Ni+-agarose fractions of
metabolically labeled wild type-infected cells are also shown. Under
nonreducing conditions in SDS-PAGE, XRCC4HIS migrates in
several heterogeneous forms (Fig. 2, top left panel, XRCC4
2ME). A majority of the XRCC4HIS protein had an
apparent mass of ~130 kDa, although a small amount of the protein was
consistently present with an apparent mass of ~57 kDa. This suggests
that XRCC4HIS exists primarily as a disulfide-linked homodimer. In agreement with this interpretation, mass spectrometric analysis of nonreduced XRCC4HIS in SDS buffer showed that
the major species had an actual mass of ~78 kDa (data not shown). There are also three additional bands with electrophoretic mobilities slower than the 207-kDa marker. It is unlikely that these larger bands
represent contaminants because under reducing conditions, the protein
migrates as a single homogeneous species (Fig. 2, top left panel,
XRCC4 +2ME). Furthermore, the Ni+ fractions of wild
type-infected cells include no proteins with similar electrophoretic
mobilities. We considered that addition of the short leader sequence
from gp67 might alter normal trafficking of XRCC4HIS
resulting in inappropriate disulfide bonding of the recombinant
protein; thus an additional virus was generated that did not include
the leader sequence from gp67. Although XRCC4 expression was
considerably lower from this virus, mobility of this protein on
nonreducing SDS-PAGE was analogous to that observed with the initial
recombinant XRCC4HIS protein (data not shown). Thus,
XRCC4HIS exists primarily as a homodimer, but it can also form covalent multimers. Furthermore, both dimeric and multimeric forms
of the XRCC4HIS fusion protein are constitutively
phosphorylated in Sf9 cells (Fig. 2, lower left panel,
XRCC4
2ME). We also analyzed the metabolically labeled protein
in native polyacrylamide gels. As can be seen (Fig. 2, right
panel, XRCC4), nondenatured XRCC4HIS migrates as two
distinct species: one which migrates between the 101 and 207 kDa
markers, and one which migrates just above the 207-kDa marker. There is
considerably less heterogeneity in native XRCC4HIS as
compared with the nonreduced, denatured protein; it is possible that
this heterogeneity may be due to incomplete disulfide bonding of
dimers and/or multimers. In any case, the mobilities of nondenatured
XRCC4HIS in native polyacrylamide gels are consistent with
homodimers and homotetramers.
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DNA-PK and Other Kinases Phosphorylate XRCC4 in Vitro--
In the
initial analysis of the XRCC4 gene, a potential DNA-PK
phosphorylation motif was noted (28). Because of the link between
DNA-PK and XRCC4, we investigated whether purified XRCC4HIS was a substrate for DNA-PK. XRCC4HIS was phosphorylated
when it was incubated in vitro with purified DNA-PK and
linearized plasmid DNA (Fig.
3A). Both dimeric and
multimeric forms of XRCC4HIS are phosphorylated.
Phosphorylation was largely, but not entirely, dependent on the
presence of linear DNA (Fig. 3A, +DNA versus DNA).
This is consistent with authentic DNA-PK mediated
phosphorylation. However, the DNA-PK pulldown assay using SCID
extracts, which lack DNA-PKCS, demonstrate that other
kinases are also able to phosphorylate purified XRCC4HIS
in vitro as well.
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Phosphoamino Acid Analysis-- We next performed phosphoamino acid analysis of XRCC4HIS which had been phosphorylated in vitro or in Sf9 cells by metabolic labeling with [32P]orthophosphate. In vitro XRCC4HIS was phosphorylated in the presence of DNA-PK, with or without linearized DNA. The phosphorylated proteins were hydrolyzed and then analyzed by thin layer chromatography (Fig. 4). As can be seen, phosphorylation of XRCC4HIS in vivo occurs on serine, threonine, and tyrosine residues. In contrast, both DNA-PK dependent and independent phosphorylation of XRCC4HIS in vitro occurs entirely on serine and threonine residues. Phosphotyrosine is consistently undetectable from XRCC4HIS phosphorylated in vitro. The ratio of serine to threonine phosphorylation is 3:1 in vitro in the presence or absence of DNA as well as in Sf9 cells. The ratio of serine to tyrosine phosphorylation in Sf9 cells is 1:1.
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Localization of XRCC4 Phosphorylation Sites--
We next assessed
phosphorylation of the truncated forms of XRCC4 (illustrated in Fig.
1A). Recombinant proteins were isolated via
Ni+-Sepharose chromatography; purified proteins (400 ng)
were then assessed as DNA-PK substrates (Fig.
5). Since expression levels of the N,
C1, and
N
C1 fusion proteins were relatively low, Ni+ affinity purified proteins were less pure than with
full-length XRCC4HIS. Thus, a Ni+ fractionation
of wild type baculovirus-infected cells was also included as a negative
control (Fig. 5, lanes 1 and 2). Of note, the
only "SQ" potential DNA-PK site in both human and murine XRCC4 is
at amino acid 53 (illustrated in Fig. 1A); there is a second SQ site in murine XRCC4 at amino acid 92. As can be seen, only full-length XRCC4HIS and the
N fusion protein were
phosphorylated in vitro. Phosphorylation of
N occurred in
the absence of linearized DNA but was augmented in the presence of
linearized DNA. In contrast, neither
C1 nor
C2 were
phosphorylated in the presence or absence of DNA when incubated
in vitro with purified DNA-PK (Fig. 5) even though each
spans the consensus SQ DNA-PK sites at amino acids 53 and 92. In
addition,
N/
C1 was also not phosphorylated in vitro.
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XRCC4 Facilitates Ku DNA End Binding Activity as Well as Association of DNA-PKCS to Ku-- The ability of purified XRCC4HIS to bind DNA or affect the affinity of Ku for DNA ends was examined using EMSAs with baculovirus expressed Ku purified by absorption to DNA-cellulose as described previously (39). Under these conditions, XRCC4HIS had no apparent affinity for the double-stranded oligonucleotide probe used in this assay (Fig. 6A). XRCC4HIS did, however, facilitate Ku binding to linear DNA. When a constant amount of XRCC4HIS was added to increasing amounts of recombinant Ku in the presence of the probe, higher levels of the migration retarded species were observed (Fig. 6A). Poly(dI-dC) and rabbit anti-Ku antisera inhibited formation of the migration retarded species, but plasmid DNA and preimmune rabbit sera did not (data not shown). Addition of increasing amounts of an irrelevant protein, had no effect. The effect of XRCC4HIS on Ku binding is not entirely specific in that a similar but less dramatic effect was also observed when XRCC4HIS was included in EMSAs examining recombinant c-jun binding to AP1 sites (data not shown).
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DISCUSSION |
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In this report, we have biochemically characterized the murine XRCC4 gene product. XRCC4 produced in insect cells is extensively post-translationally modified both by phosphorylation and cross-linking. The protein is not glycosylated via asparagine linkages and preliminary evidence suggests that XRCC4 lacks O-linked sugar modifications as well. XRCC4 exists primarily as homodimers, but it can also multimerize into multimers via disulfide linkages. Examination of XRCC4 in native polyacrylamide gels suggests that the protein exists primarily as dimers and tetramers. XRCC4 expressed in insect cells is constitutively phosphorylated on serine, threonine, and tyrosine residues.
XRCC4 is an efficient in vitro substrate for the DNA-dependent kinase. However, extracts from several cell lines which are completely deficient in the catalytic subunit of DNA-PK are also capable of phosphorylating XRCC4 in vitro. Thus, in vitro, XRCC4 can be modified via phosphorylation on serine and threonine by at least two different kinases. Phosphorylation by DNA-PK does not occur on a SQ consensus DNA-PK site, and is most likely located within the carboxyl-terminal 130 amino acids. Finally, it is unlikely that phosphorylation is requisite for the DSBR activity of XRCC4 in that the XRCC4 truncation spanning amino acids 1-204, which is not phosphorylated in vitro or in Sf9 cells, significantly reverses the XR-1 DSBR defect.
XRCC4 facilitates binding of Ku to free DNA ends without altering mobility of Ku·DNA complexes. This is reminiscent of the recently defined JAB1 factor which increases c-jun's avidity for AP1 sites without forming a stable complex with c-jun and its target sequence (45). More interestingly, the association of DNA-PKCS with DNA-bound Ku is facilitated by XRCC4. This may be because of enhanced binding of Ku to DNA but could also be a direct effect of XRCC4 on DNA-PKCS. Additionally, XRCC4 can apparently form stable complexes with DNA-PK bound to DNA in vitro. Finally, a truncated form of XRCC4 which cannot functionally complement the XRCC4 defect in XR-1 cells, essentially lacks the capacity to enhance DNA-PK assembly and to form complexes with DNA-PK and DNA in vitro. Thus, the ability of XRCC4 to directly interact with DNA-PK may be functionally germane.
It has been demonstrated that maximal DNA-PK activity requires approximately equal ratios of Ku and DNA-PKCS (46). Albeit it is speculation at this point, the dimeric nature of XRCC4 might suggest that XRCC4 could interact with two separate DNA-PK complexes, possibly stabilizing or aligning the two DNA strands by bridging two DNA-PK complexes prior to ligation. Another possibility is that tetramers of XRCC4 might stabilize 4 units of DNA-PK in the V(D)J recombination complex. The fact that rare coding and signal joints in XR-1 cells likely utilize (and possibly require) short sequence homologies to facilitate ligation, may reflect an inability in these cells to stabilize the two strands. This would also agree with a model invoking XRCC4 as a stabilizing factor for DNA-PK complexes.
The evidence that XRCC4 interacts with DNA-PK in vitro is 2-fold: 1) XRCC4 is phosphorylated by DNA-PK; and 2) XRCC4 facilitates Ku binding to DNA and DNA-PK assembly and alters the electrophoretic mobility of DNA-PK bound to DNA. These findings coupled with the fact that XRCC4 interacts with DNA ligase IV, suggests a role for XRCC4 in targeting cellular ligase activities to damaged DNA which is bound by DNA-PK in a multiprotein complex. Leu and Schatz (47) have shown that RAG 1 and RAG 2 are part of a large multiprotein complex, anchored to the nuclear matrix and that V(D)J recombination may be restricted to certain nuclear compartments. Experimental evidence suggests that V(D)J coding intermediates have extremely short half-lives in vivo, suggesting that factors involved both in initiation and resolution of V(D)J recombination are tightly linked (33, 34). In fact, very recently, Agrawal and Schatz (48) demonstrated that DNA-PK is associated with V(D)J recombination intermediates after the cleavage step of V(D)J recombination. Thus, it will be of considerable interest to ascertain whether XRCC4 is a component of this complex.
In summary, this report provides the first biochemical characterization of murine XRCC4. XRCC4 is a disulfide-linked homodimer which can also multimerize. XRCC4 is phosphorylated during expression in insect cells and is also phosphorylated in vitro on serine and threonine residues by the DNA-dependent protein kinase and other unidentified kinases. Recombinant XRCC4 facilitates the association of DNA-PKCS with DNA bound Ku possibly through its ability to facilitate DNA-protein interactions; furthermore, XRCC4 and DNA-PK appear to form stable complexes in vitro. Thus, data presented here are consistent with a model invoking XRCC4 as a stabilizer of the DNA-PK complex.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. Donald Capra for the Ku baculoviruses and for anti-Ku antisera. We thank Dr. Clive Slaughter and the HHMI biopolymer core facility at University of Texas Southwestern Medical Center for performing amino acid sequence analyses and mass spectrometric analyses on XRCC4. We especially thank Dr. Gary Rathbun and Dr. Charles Hasemann for critical review of the manuscript and for numerous helpful discussions. We thank Dr. David Roth for many stimulating dialogues concerning this project.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI31229 and AI32600, the Harold C. Simmons Research Center, and the Arthritis Foundation (to K. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Contributed equally to the results of this work.
** To whom correspondence should be addressed: Harold C. Simmons Arthritis Research Center, Dept. of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-8884. Tel.: 214-648-3411; Fax: 214-648-7995; E-mail: kmeek{at}mednet.swmed.edu.
1 The abbreviations used are: RAG, recombination activating gene; SCID, severe combined immunodeficiency; DNA-PK, DNA dependent protein kinase; DNA-PKCS, DNA dependent protein kinase, catalytic subunit; XRCC, x-ray cross-complementation group; V(D)J, variable, diversity, and joining immune receptor gene segments; DSB, double strand break repair; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assays.
2 K. Meek, unpublished data.
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