By
From the * Department of Medical Physics and Department of Radiation Oncology, Memorial
Sloan-Kettering Cancer Center, New York, 10021; Los Alamos National Laboratory, Los Alamos,
New Mexico 87545; § Thomas Jefferson University, Philadelphia, Pennsylvania 19107;
Rockefeller
University, New York, 10021
Ku is a complex of two proteins, Ku70 and Ku80, and functions as a heterodimer to bind
DNA double-strand breaks (DSB) and activate DNA-dependent protein kinase. The role of
the Ku70 subunit in DNA DSB repair, hypersensitivity to ionizing radiation, and V(D)J recombination was examined in mice that lack Ku70 (Ku70/
). Like Ku80
/
mice, Ku70
/
mice showed a profound deficiency in DNA DSB repair and were proportional dwarfs. Surprisingly, in contrast to Ku80
/
mice in which both T and B lymphocyte development were
arrested at an early stage, lack of Ku70 was compatible with T cell receptor gene recombination and the development of mature CD4+CD8
and CD4
CD8+ T cells. Our data shows,
for the first time, that Ku70 plays an essential role in DNA DSB repair, but is not required for
TCR V(D)J recombination. These results suggest that distinct but overlapping repair pathways
may mediate DNA DSB repair and V(D)J recombination.
Two distinct processes involving DNA double-strand
breaks (DSB)1 have been identified in mammalian
cells: the repair of DNA damage induced by ionizing radiation and V(D)J recombination during T and B cell development. So far, all mammalian cell mutants defective in
DNA DSB repair share the common phenotype of hypersensitivity to radiation and impaired ability to undergo
V(D)J recombination (1). Cell fusion studies using DSB
repair mutants of human-rodent somatic hybrids have defined four ionizing radiation (IR) complementation groups:
IR4, IR5, IR6, and IR7. Genetic and biochemical analyses have revealed that cells of IR5 (e.g., xrs-6) and IR7 (e.g.,
scid) are defective in components of the DNA-dependent
protein kinase (DNA-PK) (2, 7). DNA-PK is a serine/
threonine kinase comprised of a large catalytic subunit and
a DNA-targeting component termed Ku, which itself is a
heterodimer of a 70- (Ku70) and a 86-(Ku80) kD polypeptide (10). Recently, the DNA-PK catalytic subunit has
been shown to be the gene responsible for the murine scid defect (13), and Ku80 has been identified to be
XRCC5 (16), the x-ray repair cross-complementing
gene for IR5. Ku80 knockout mice were found to exhibit
scid, defective processing of V(D)J recombination intermediates, and growth retardation (19, 20).
Although Ku70 has been designated as XRCC6 (7, 8)
and is an important component of the DNA-PK complex,
the function of Ku70 in vivo is hitherto unknown. To define the role of Ku70 in DNA repair and V(D)J recombination, we targeted the Ku70 gene in mice. Ku70 homozygotes exhibit proportional dwarfism, a phenotype of
Ku80 Target Disruption of Ku70 and Generation of Ku70
/
, but not of scid mice. Absence of Ku70 confers
hypersensitivity to ionizing radiation and deficiency in
DNA DSB repair, which are characteristics of both Ku80
/
and scid mice. Surprisingly, in contrast to Ku80
/
and scid
mice in which both T and B lymphocyte development are arrested at early stage, lack of Ku70 is compatible with T
cell receptor gene recombination and the development of
mature CD4+CD8
and CD4
CD8+ T cells. Our data, for
the first time, provide direct evidence supporting that Ku70
plays an essential role in DNA DSB repair, but is not required for TCR gene recombination. These results suggest
that distinct but overlapping repair pathways may mediate DSB repair and V(D)J rejoining; furthermore, it suggests
the presence of a Ku70-independent rescue pathway in
TCR V(D)J recombination. The distinct phenotype of
Ku70
/
mice should make them valuable tools for unraveling the mechanism(s) of DNA repair and recombination.
/
Mice.
Mouse genomic Ku70 gene was isolated from a sCos-I cosmid library constructed from a mouse strain 129 embryonic stem (ES) cell lines (21). The replacement vector was constructed using a
1.5 kb 5
-fragment that contains the promoter locus with four GC boxes and exon 1, and an 8-kb EcoRV-EcoRI fragment extending from intron 2 to intron 5 as indicated in Fig. 1 A. Homologous replacement results in a deletion of 336-bp exon 2 including the translational initiation codon.
Fig. 1.
Inactivation of Ku70 by homologous recombination. (A)
Diagrammatic representation of the Ku70 locus (top), the targeting construct (middle), and the targeted allele and hybridization probe (bottom). EcoRI (E) restriction sites used to detect the targeted gene are indicated
(21). (B) Southern blot of EcoRI-digested tail DNA from control wild-type (WT), heterozygous (+/), and homozygous (
/
) Ku70-targeted
mice. The wild-type and mutant fragments are 13 and 5.7 kb, respectively. (C) Western blot analysis showing that Ku70 protein is not expressed in Ku70
/
cells. Whole cell lysates prepared from mouse ear fibroblasts (50 µg) and mouse embryo fibroblasts (100 µg) were separated
by 10% SDS-PAGE, transferred to a nitrocellulose membrane, and
probed with polyclonal antibodies against full-length rodent Ku80 (top) and Ku70 (bottom), respectively. (D) Gel mobility shift assay (22) showing
the lack of DNA-end-binding activity in Ku70
/
cells. Ku-DNA-binding complex is indicated by arrow on the right.
[View Larger Version of this Image (27K GIF file)]
/
mice
were generated by crossing Ku70+/
heterozygotes.
Western Blot Analysis and Gel Mobility Shift Assay.
To confirm
that the disruption of Ku70 produces a null mutation, Ku70 protein expression was measured by Western blotting using polyclonal
antisera against intact mouse Ku70. The lack of Ku70 was also
verified by a Ku-DNA-end-binding assay (gel mobility shift analysis). Cell extracts were prepared and gel mobility shift assays were
performed as described (22). Equal amounts of cellular protein (50 µg) from Ku70+/+ (wild type), Ku70+/, and Ku70
/
mouse
embryo fibroblasts were incubated with a 32P-labeled double-stranded oligonucleotide, 5
-GGGCCAAGAATCTTCCAGCAGTTTCGGG-3
. The protein-bound and free oligonucleotides were
electrophoretically separated on a 4.5% native polyacrylamide gel. Gel
slabs are dried and autoradiographed with X-Omat film (Kodak,
Rochester, NY).
Immunohistochemistry.
To determine the pathological changes,
histological sections of various organs of Ku70/
, Ku80
/
, and
wild-type littermate mice were prepared and examined as previously described (23). Lymph nodes, spleens, and thymuses from
4-5-wk-old mice were fixed in 10% buffered formalin and embedded in paraffin, or embedded in (optimal cutting temperature)
compound (Sakura Finetek, USA, Incorp., Torrance, CA) and
frozen in liquid nitrogen at
70°C. Sections (5 µm) were stained
with hematoxylin and eosin, and representative samples were selected for immunohistochemical analysis. Immunophenotyping
was performed using an avidin-biotin immunoperoxidase technique (24). Primary antibodies included anti-CD3 (purified rabbit
serum, 1:1,000; Dako Corp., Carpinteria, CA), anti-B220 (rat
monoclonal, 1:1,000; PharMingen, San Diego, CA), and anti-CD19 (rat monoclonal, 1:1,000; PharMingen), and were incubated overnight at 4°C. Samples were subsequently incubated with
biotinylated secondary antibodies (Vector Labs., Burlingame, CA)
for 30 min (goat anti-rabbit, 1:100; rabbit anti-rat, 1:100), and
then with avidin-biotin peroxidase (1:25 dilution; Vector Labs.)
for 30 min. Diaminobenzadine was used as the chromogen and
hematoxylin as the counter stain. Wild-type lymphoid organs including thymus, spleen, and lymph nodes from different mice were
used for titration of the antibodies and positive controls. Anti-CD3
and anti-CD19 antibodies were tested in both frozen and paraffin
sections of wild-type lymphoid organs and showed the expected
specific patterns of staining (data not shown). For negative controls, primary antibodies were substituted with class-matched but
unrelated antibodies at the same final working dilutions.
Cell Preparation and Flow Cytometric Analysis. For flow cytometry, single cell suspensions from lymphoid organs of 4-6-wk-old mutant and littermate control mice were prepared for staining as described previously (19) and analyzed on a FACScan® with Cell Quest software (Becton Dickinson, San Jose, CA). Cells were stained with combinations of PE-labeled anti-CD4 and FITC-labeled anti-CD8, or PE-labeled anti-B220 and FITC-labeled anti-CD43, or FITC-anti-IgM and PE-anti-B220 (PharMingen), as needed. Bone marrow cells were harvested from femurs by syringe lavage, and cells from thymus and spleen were prepared by homogenization. Cells were collected and washed in PBS plus 5% FCS and counted using a hemacytometer. Samples from individual mice were analyzed separately. Dead cells were gated out by forward and side scatter properties. Experiments were performed at least three times and yielded consistent results.
DNA Preparation and Analysis of V(D)J Recombination Products.
To determine whether a null mutation in Ku70 affects the
recombination of antigen-receptor genes in T and B lymphocytes
in vivo, we measured the immunoglobulin and T cell antigen
receptor (TCR) rearrangements by PCR. DNA from bone
marrow was amplified with primers specific to immunoglobulin
D-JH and V-DJH rearrangements, and DNA from thymus was
amplified with primers that detect V-DJ and D
-J
rearrangement (20, 25).
Cell Survival Determination.
8-10-wk-old Ku70/
and Ku80
/
mice and wild-type littermates were used for our studies. Bone
marrow cell suspensions were prepared by flushing the femur with
MEM supplemented with 15% FCS. The cell suspension was then
counted using a hemacytometer and centrifuged at 1,000 rpm for
12 min. The resulting pellet was resuspended and diluted to ~106
cells/ml in MEM plus 15% FCS for further experiments.
Asymmetric Field Inversion Gel Electrophoresis.
To determine the
rate and extent of DNA DSB repair in Ku-deficient cells after exposure to ionizing radiation, primary embryo fibroblasts derived
from Ku70/
, Ku80
/
and wild-type littermate mice were
used. Mouse embryo fibroblasts from day 13.5 embryos growing
in replicate cultures for 3 d in the presence of 0.01 µCi/ml
[l4C]thymidine (New England Nuclear, Boston, MA) and 2.5 µM cold thymidine were exposed to 40 Gray (Gy) of x-rays and
returned to 37°C. At various times thereafter, one dish was removed and trypsinized on ice; single cell suspensions were made
and embedded in an agarose plug at a final concentration of 3 × 106 cells/ml. Asymmetric field inversion gel electrophoresis
(AFIGE) was carried out in 0.5% Seakem agarose (FMC Bioproducts, Rockland, ME; cast in the presence of 0.5 µg/ml
ethidium bromide) in 0.5 × TBE (45 mM Tris, pH 8.2, 45 mM
boric acid, 1 mM EDTA) at 10°C for 40 h by applying cycles of
1.25 V/cm for 900 s in the direction of DNA migration, and 5.0 V/cm for 75 s in the reverse direction as described (31).
To study the role of
Ku70 in vivo, we generated mice containing a germline
disruption of the Ku70 gene. Murine genomic Ku70 gene
was isolated and a targeting vector was constructed (Fig. 1
A). Homologous replacement results in a deletion of 336-bp exon 2, including the translational initiation codon.
Two targeted ES clones carrying the mutation in Ku70
were injected into C57BL/6 blastocysts to generate chimeric mice. One clone was successfully transmitted through
the germline after chimeras were crossed with C57BL/6
females. No obvious defects were observed in Ku70+/
heterozygotes, and these Ku70+/
mice were subsequently
used to generate Ku70
/
mice (Fig. 1 B). 25% of the offspring born from Ku70+/
× Ku70+/
crosses were
Ku70
/
. Adult Ku70
/
mice are fertile, but give reduced
litter size (two to four pups) as compared to the Ku70+/
or Ku70+/+ mice (about eight pups).
To confirm that the disruption produced a null mutation,
Ku70 protein expression was analyzed by both Western
blotting (Fig. 1 C) and a DNA end binding assay (Fig. 1 D).
Ku70 immunoreactivity was undetectable (Fig. 1 C), and
there was no Ku-DNA-end-binding activity in Ku70/
fibroblasts (Fig. 1 D). The Ku80 subunit of the Ku heterodimer was found, but at much reduced levels (Fig. 1 C),
suggesting that the stability of Ku80 is compromised by the
absence of Ku70. These observations are consistent with
the finding that the level of Ku70 was significantly reduced
in Ku80
/
fibroblasts and Ku80
/
ES cells (19). Taken
together, these data suggest that the stability of either component of Ku is compromised by the absence of the other.
Newborn Ku70/
mice were 40-60% smaller than
their Ku70+/
and Ku70+/+ littermates. During a 5-mo
observation period, Ku70
/
mice grew and maintained
body weight at 40-60% of controls. Thus, Ku70
/
mice,
like Ku80
/
mice, are proportional dwarfs (19).
Examination of various organs from Ku70/
mice showed abnormalities only
in the lymphoid system (Fig. 2 A). Spleen and lymph nodes
were disproportionately smaller by 5-10-fold relative to controls. In particular, splenic white pulp nodules were significantly reduced. Immunohistochemistry on deparaffinized
tissue sections revealed that the splenic white pulp contained
cells that stained with anti-CD3 (i.e., CD3-positive T cells),
but there were no CD19-positive B cells (Fig. 2 A, k and n).
The Ku70
/
thymus was also disproportionately smaller
and contained 50-100-fold fewer lymphocytes than Ku70+/+
littermates (3 × 106 in the former versus 2 × 108 in the latter; measured in three mice of each genotype). In contrast to
the Ku80
/
mice, the Ku70
/
thymus displayed normal
appearing cortical-medullary junctions (Fig. 2 A, g and j).
Overall, the lymphoid tissues and organs of Ku70
/
mice
are somewhat disorganized and much smaller than Ku70+/+
mice (Table 1); yet, they are relatively more developed and slightly larger than in Ku80
/
mice.
To further examine the immunological defect in Ku70/
mice, cells from thymus, bone marrow, and spleen were
analyzed using monoclonal antibodies specific for lymphocyte surface markers and flow cytometry (19). Consistent
with the immunohistological data, there was a complete
block in B cell development at the B220+CD43+ stage in
the bone marrow, and there were no mature B cells in the
spleen (Fig. 2 B). In contrast, thymocytes developed
through the CD4+CD8+ double-positive stage and matured into CD4+CD8
and CD4
CD8+ single-positive (SP),
TCR-
-positive cells (Fig. 2, B and C). In six 4-wk-old
Ku70
/
mice analyzed, the percentage of CD4
CD8
double-negative thymocytes ranged from 11 to 62%, and
the CD4+CD8+ double-positive cells varied from 35 to
73%. CD4
CD8+ (1-11%) and CD4+CD8
(1-3%) SP
cells were also detected in the thymus. Furthermore, CD4+
CD8
or CD4
CD8+ SP T cells were found in the spleen
in 67% of the mice studied (Fig. 2 B), which expressed surface TCR-
(Fig. 2 C) and CD3 (data not shown). Thus,
in contrast to the early arrest of both T and B cell development in Ku80
/
mice (Fig. 2 B), lack of Ku70 is compatible with the maturation of T cells.
To determine whether a null mutation in Ku70 affects antigen-receptor gene recombination, DNA from bone marrow was amplified with primers specific to immunoglobulin D-JH and V-DJH rearrangements and DNA from
thymus was amplified with primers that detected V-DJ and D
-J
rearrangements (20, 25). Fig. 3 A shows that
Ku70
/
B cells do undergo D-JH recombination at a level
which is similar to Ku80
/
B cells, but is 2-3-fold lower
than the level found in scid mice, and 10-50-fold lower
than wild-type littermates. It is possible that some, but not
all, of the decrease in D-JH rearrangement is due to a lower
fraction of B lineage cells in the mutant sample, since the
wild-type littermate mice have only approximately 5-fold more B220+ cells than the Ku70
/
mice (see Table 1).
V-DJH rearrangements were not detected in either Ku70
/
,
Ku80
/
, or scid bone marrow samples, possibly accounting for the absence of mature B cells in these mutant mice
(Fig. 3 A).
In contrast to the immunoglobulin heavy chain gene recombination, semiquantitative PCR analysis of thymocyte
DNA for V-DJ joints showed normal levels of TCR-
rearrangements on a per cell basis (Fig. 3 B). Similarly, D
2
and J
1 coding joints were found in Ku70
/
thymocytes
at levels that resembled the wild type. To determine the
molecular nature of the amplified coding joints, cloned
V
8-DJ
2.6 joints were sequenced. We found normal
numbers of N and P nucleotides, as well as normal levels of
coding end deletions (Fig. 4). Thus, coding joints in
Ku70
/
thymocytes differ from coding joints produced in
xrs6 Ku80-deficient cells in that there were no large aberrant deletions (4, 18). We conclude that TCR V(D)J recombination in vivo does not require Ku70.
Absence of Ku70 Confers Radiation Hypersensitivity and Deficiency in DNA DSB Repair.
To assess radiation sensitivity in the absence of Ku70, cells from the bone marrow
were exposed to ionizing radiation and were assayed for
colony formation (30, 32). Fig. 5 A shows the survival
curves of the CFU-GM from Ku70/
, Ku80
/
, and
wild-type control mice. CFU-GM from Ku70-deficient
mice were more sensitive to ionizing radiation than those
from Ku-proficient control mice (Fig. 5 A). Similar hypersensitivity to radiation was seen for Ku80
/
CFU-GM
(Fig. 5 A).
The rate and extent of rejoining of x-ray-induced DNA
DSB in Ku70/
, Ku80
/
, and Ku70+/+ cells were measured using AFIGE (31). Fibroblasts derived from day 13.5 embryos were exposed to 40 Gy of x-rays and returned to
37°C for repair. At various times thereafter, cells were prepared for AFIGE to quantitate DNA DSB (Fig. 5 B, top).
DNA DSB were nearly completely rejoined in wild-type
cells within ~2 h after radiation exposure. However, fibroblasts derived from Ku70
/
mice showed a drastically reduced ability to rejoin DNA DSB. A similar deficiency in
DNA DSB rejoining was also observed in fibroblasts derived from Ku80
/
embryos. Despite the large differences
observed in rejoining of DNA DSB between wild-type fibroblasts and fibroblasts derived from Ku70
/
or Ku80
/
mouse embryos, dose-response experiments showed that
Ku70
/
, Ku80
/
, and wild-type fibroblasts were equally
susceptible to x-ray-induced damage (Fig. 5 B, bottom).
Thus, Ku deficiency primarily affects the ability of cells to
rejoin radiation-induced DNA DSB without significantly
affecting the induction of DNA damage.
Absence of Ku70 results in radiation hypersensitivity and
proportional dwarfism, as well as deficiencies in DNA DSB
repair and V(D)J recombination. Thus, Ku70/
mice resemble Ku80
/
mice in several respects, but the two mutations differ in their effects on T and B cell development.
Lack of Ku70 was compatible with TCR gene rearrangement and development of mature CD4+CD8
and
CD4
CD8+ T cells, whereas mature T cells were absent in
Ku80
/
mice. In contrast, B cells failed to complete antigen receptor gene rearrangement and did not mature in either Ku70
/
or Ku80
/
mice.
What could account for the differences we find in TCR
and immunoglobulin gene rearrangements in the Ku70/
mice? One implication of our findings is that there are alternative Ku70-independent rescue pathways that are compatible with completion of V(D)J recombination in T cells.
It is likely at the critical phase of T cell maturation, other
DNA repair activity may be stimulated (33, 34) and can
functionally complement the Ku70 gene in T cell-specific
V(D)J recombination. Since Ku80
/
mice are deficient in
both T and B lymphocyte development, it is plausible that
these yet to be identified alternative DNA repair pathways
include Ku80. The much reduced level of Ku80 protein in
Ku70
/
cells may in part account for the hypocellularity
of Ku70
/
thymuses.
Although the role of Ku in V(D)J recombination is not molecularly defined, Ku has been proposed to protect DNA ends from degradation (18, 35), to activate DNA-PK (10, 11), and to dissociate the recombination-activating protein RAG-DNA complex to facilitate the joining reaction (20). These functions are not mutually exclusive, and they are all dependent on the interaction of Ku with DNA. Thus, the finding that Ku70 is not required for TCR gene rearrangement is particularly unexpected because the Ku70 subunit is believed to be the DNA-binding subunit of the Ku complex (36), and DNA-end-binding activity was not detected in Ku70-deficient cells (Fig. 1 D).
In summary, our studies provide direct evidence supporting the involvement of Ku70 in the repair of DNA
DSB and V(D)J recombination and the presence of a
Ku70-independent rescue pathway(s) in TCR V(D)J rearrangement. The distinct phenotype of Ku70/
mice
should make them valuable tools for unraveling the mechanism(s) of DNA repair and recombination.
Address correspondence to G.C. Li, Department of Medical Physics and Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave./Box 72, New York, NY 10021. Phone: 212-639-6028; FAX: 212-639-2611; E-mail: g-li{at}ski.mskcc.org
Received for publication 23 May 1997 and in revised form 14 July 1997.
The work was supported in part by National Institutes of Health grants CA-31397 and CA-56909 (to G.C. Li), CA-42026 (to G. Iliakis), CA-50519 (to D.J. Chen), and Department of Energy Office of Health and Environmental Research (to D.J. Chen). A. Nussenzweig is a research fellow supported by National Institutes of Health training grant CA61801 and M. Nussenzweig is an associate investigator in the Howard Hughes Medical Institute.We thank D. Roth for PCR primers, D. Kim and L. Wu for Ku antiserum, T. Deloherey for FACS® analysis, P. Krechmer for word processing, A. Haimovitz-Friedman and Hatsumi Nagasawa for valuable suggestions, and C.C. Ling and Z. Fuks for advice and support.
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