Role for the BRCA1 C-terminal Repeats (BRCT) Protein 53BP1 in Maintaining Genomic Stability*

p53-binding protein-1 (53BP1) is phosphorylated in response to DNA damage and rapidly relocalizes to pre-sumptive sites of DNA damage along with Mre11 and the phosphorylated histone 2A variant, (cid:1) -H2AX. 53BP1 associates with the BRCA1 tumor suppressor, and knock-down experiments with small interfering RNA have revealed a role for the protein in the checkpoint response to DNA damage. By generating mice defective in m53BP1 ( m53BP1 tr/tr ), we have created an animal model to further explore its biochemical and genetic roles in vivo . We find that m53BP1 tr/tr animals are growth-re-tarded and show various immune deficiencies including a specific reduction in thymus size and T cell count. Consistent with a role in responding to DNA damage, we find that m53BP1 tr/tr mice are sensitive to ionizing radiation ( (cid:1) -IR), and cells from these animals exhibit chromosomal

DNA damage-response mechanisms ensure the fidelity of chromosomal transmission, and their failure may lead to the development of diseases such as cancer (1). In response to ␥-IR, 1 phosphoinositide-like kinases (PIKs) such as ATM (mutated in ataxia-telangiectasia) transduce damage signals to kinases, transcription factors, and DNA repair proteins by targeting (S/T)Q motifs (2). A second PIK, ATR (ATM-and Rad3-related), also responds to ␥-IR, but it appears to respond primarily to agents that create replicational stress (i.e. hydroxyurea and aphidicolin) (2). ATM and ATR have distinct but overlapping substrate specificities including the ability of both enzymes to target p53 serine residue 15 (Ser-15) as well as the product of breast cancer susceptibility gene 1, BRCA1, at Ser-1423 (3,4). BRCA1 is a major target of the DNA damage response, and mutations in BRCA1 contribute to nearly 50% of familial forms of breast and ovarian cancer (5). BRCA1 had been found associated with RNA polymerase II (6), chromatinremodeling factors (7), and a variety of DNA repair and replication factors (8 -10). Indeed, BRCA1 has been shown to function in genomic stability by controlling homologous recombination, transcription-coupled repair of oxidative DNA damage, and cell cycle checkpoints (11)(12)(13)(14).
One protein that contains numerous (S/T)Q motifs and two C-terminal BRCT repeats is p53-binding protein 1 (53BP1). 53BP1 was discovered as a p53-interacting factor in a twohybrid screen (15) and was subsequently proposed to function as a transcriptional co-activator of p53 (16). Although the relationship between 53BP1 and p53 has not been fully established, 53BP1 and p53 from both Xenopus and humans have been shown to interact either directly or indirectly in experimental settings that express high levels of 53BP1 protein from plasmids or that naturally occur in eggs (15,17). We, as well as others, have demonstrated previously that 53BP1 is involved in the DNA damage-response network (17)(18)(19)(20). 53BP1 proteins are phosphorylated in response to ␥-IR, and this is likely governed by the action of PIKs like ATM (17,19,20). ␥-IR also induces 53BP1 to rapidly relocalize to DNA repair foci, and this response is delayed or inhibited by treatment with the PIK inhibitors caffeine and wortmannin. 53BP1 foci also overlap with those formed by the Mre11 complex, BRCA1, and the phosphorylated form of the histone variant H2AX (␥-H2AX; see Refs. 18 -20). As both the Mre11 complex and ␥-H2AX are believed to localize to physical sites of DNA damage (21)(22)(23) and to recruit various DNA repair factors to these sites, 53BP1 has been inferred to localize to these sites as well. This notion is further supported by the fact that ␥-H2AX recruits 53BP1 to nuclear foci and physically interacts with 53BP1 (20,24). Recent studies have revealed a role for 53BP1 in cell cycle checkpoints (25)(26)(27) as well as in maintaining p53 levels in response to ␥-IR (27). Here we show that a 380-amino-acid region of 53BP1 that includes a recently described kinetochore-binding domain (28) is necessary for the formation of irradiation-in-duced foci. We further deciphered the role of 53BP1 in the DNA damage response by generating mice defective in m53BP1. We report that murine animals expressing a truncated form of m53BP1 (m53BP1 tr/tr ) exhibit a pleiotropic phenotype that includes growth retardation, immune deficiencies including defects in T cell maturation, sensitivity to ␥-IR, as well as increased chromosomal aberrations. Taken together, these results reveal that 53BP1 is an integral component of the DNA damage-response network and indicate that the protein plays an important role in maintaining genomic stability.

EXPERIMENTAL PROCEDURES
Antibodies and Indirect Immunofluorescence-Three antibodies that recognize both the human and murine 53BP1 proteins were generated for this study. We found that our 53BP1 antibodies recognize both the murine and human proteins. Polyclonal antibodies raised against glutathione S-transferase fusion proteins encoding the first 524 amino acids of human 53BP1 (␣53BP1) or the last 200 residues of the protein (␣53BP1-C) were affinity-purified by established procedures and used as described in the text. ␣53BP1-N is a polyclonal, anti-peptide antibody that was raised against an N-terminal sequence GVLELSQSQD-VEE that is conserved between human and murine 53BP1 proteins. Polyclonal antibodies were affinity-purified by standard methods. Anti-HA antibodies were purchased from Covance, and anti-ATR antibodies were obtained from Oncogene Research Products.
Mouse Genetics and Genotyping of m53BP1 tr/tr Animals-Murine animals defective in m53BP1 (m53BP1 tr/tr ) were generated with a random retrovirus as described previously (29). Genomic DNA was isolated from mouse tail snips by standard methods. Insertion of VICTR54 introduces new XbaI sites into the intron preceding exon 14 of the wild-type allele. Therefore, a disrupted m53BP1 allele will be broken into multiple XbaI fragments including a 5Ј-proximal, 1.5-kb fragment. To detect this fragment, we PCR-amplified and labeled with 32 P a probe downstream of the 5Ј naturally occurring XbaI site but upstream of the one introduced by VICTR54, as shown in Fig. 2A. The primers used to amplify the 700-base pair probe for Southern analysis were 5Ј-CTCAG-CATCCATGCTGGGC-3Ј and 5Ј-TACTTAATGAGGCTAGAGCACAGC-3Ј. The sequences of the primers used for RT-PCR analysis were as follows: A, 5Ј-CCTCAGGCAGAGTGAACA-3Ј; B, 5Ј-CTCTGTGTCGTC-CACGGGAGCACT-3Ј; C, 5Ј-GTGGCGATGCAAGACATGGCCA-3Ј; D, 5Ј-GCCAAGAACAGATGGAACAGCTGA-3Ј. Either poly(A) ϩ or total RNA was isolated by standard methods and used to prepare cDNA with the Superscript one-step PCR system (Invitrogen).
Immune System Analysis-Bone marrow, thymus, and spleen tissue were analyzed in 8-week-old male and female mice. Bone marrow was flushed with Hank's balanced salt solution (Invitrogen) from two femurs per animal. Cells were counted in 3.0% acetic acid with a hemocytomter. Bone marrow cells were stained in Hank's balanced salt solution/2.0% fetal bovine serum, with Fc block, CD11b (Mac-1), Gr-1, ter119, CD19, anti-IgM, CD45R/B220, and CD43 (all from Pharmingen). Flow cytometric analysis was performed on a BD Biosciences FACScan, with CellQuest software, and appropriate negative isotype control antibodies (Pharmingen) were used in all analyses. Spleens and thymuses were excised and gently crushed through 100-m cell strainers (Falcon) in Hank's balanced salt solution/fetal bovine serum. Red blood cells were lysed with ACK buffer (0.15 M NH 4 Cl, 1.0 mM KHCO 3 , 0.1 mM EDTA, pH 7.2) (5 ml/spleen or thymus, 5 min at room temperature). After centrifugation and washing with phosphate-buffered saline/2.0% fetal bovine serum, the cells were counted as above and stained for flow cytometric analysis. Thymic cells were stained with CD4, CD8, CD25, CD44, and CD3 (Pharmingen). Flow cytometry was performed as above.
Chromosome Aberration Studies-Exponentially growing passage 2 MEFs were irradiated at room temperature with 0, 0.5, or 1.5 Gy of ␥-IR using a Nasatron irradiator, returned to 37°C for 30 min to allow cells irradiated in mitosis to exit, and then incubated with 1 g/ml colcemid for 2 h prior to cell harvest, hypotonic (0.075 M KCl) fixation (3:1 methanol:glacial acetic acid), and metaphase spread preparation. Dried slide preparations were stained with Giemsa and examined for the presence of chromatid gaps, breaks, and exchanges by light microscopy. Between 50 and 100 metaphases were scored for each treatment.

RESULTS AND DISCUSSION
Dynamic Nuclear Localization of 53BP1 in the Absence and Presence of DNA Damage-It has been recently shown that 53BP1 localizes to the kinetochore during mitosis (28). How-ever, the behavior of 53BP1 during interphase in the absence of extrinsic DNA damage has not been fully investigated. To examine the interphase behavior of 53BP1 during the course of normal, unperturbed MCF-7 cell cycles, we used a laser scanning cytometer to determine the nuclear localization of 53BP1 and the cellular DNA content for any given cell. In G 1 , 53BP1 exists in a diffuse nuclear pattern as well as in large nuclear "dots" (Fig. 1A). In S-phase, 53BP1 can be found in a discrete, punctate pattern (Fig. 1A). The nuclear distribution pattern of 53BP1 in G 2 cells appeared in two types, one similar to S-phase but with fewer foci (Fig. 1A) and one that exhibited few, if any, large dots (not shown). It is well established that 53BP1 relocalizes to nuclear foci in response to DNA damage (17)(18)(19)(20). We found that 53BP1 and ATR co-localized to nuclear foci in response to hydroxyurea (Fig. 1B). We also found that 53BP1 physically associates with ATR in nuclear extracts derived from K562 cells (Fig. 1C). 53BP1 can be detected in ATR immunoprecipitates and ATR is present in 53BP1 immunoprecipitates, and the association occurs independently of DNA damage (Fig. 1C). Moreover, ATR phosphorylates 53BP1 in vitro (not shown). Thus, 53BP1 interacts with various factors implicated in genomic stability including ATR, p53, H2AX, BRCA1, and Chk2. To address which structural elements of 53BP1 are required for the formation of irradiation-induced foci, we created a series of mutant constructs in the 53BP1expression vector pCMH6K53BP1 (16). We generated mutant forms of 53BP1 that deleted the C-terminal BRCT motifs (⌬BRCT), the kinetochore-binding region (⌬KINET), the Nterminal 1,234 residues except for the initiation codon (⌬NH3), and a protein mutated at 15 potential phosphorylation sites (15AQ), some of which are known to be targeted during the DNA damage response. 2 All constructs maintained the nuclear localization signal. Transient tranfections with these various constructs into MCF-7 cells revealed that the mutant proteins were being expressed (not shown). We confirmed that the wildtype, HA-tagged version of 53BP1 encoded by pCMH6K53BP1 generated nuclear foci in response to DNA damage when immunostained with an antibody specific for the HA tag (Fig. 1D). Untransfected cells were found to stain negative with anti-HA antibodies (not shown). Our results indicate that the majority of 53BP1 appears dispensable for DNA damage-inducible focus formation, including the N-terminal 1,234 residues (which includes numerous (S/T)Q motifs) as well as the C-terminal BRCT motifs (Fig. 1D). Surprisingly, ⌬KINET, a 380-aminoacid deletion (residues 1,236 -1,615) that removes the kinetochore binding region (28) of 53BP1, failed to form irradiationinduced foci as the protein persisted in a diffuse nuclear pattern after irradiation (Fig. 1D).
Generation of Mice Defective in m53BP1 (m53BP1 tr/tr )-To begin to decipher the functional role for 53BP1 in the DNA damage response, we identified embryonic stem cells (OST94324) from Omnibank (29) containing a single, ϳ5.0-kb retroviral insertion (VICTR54; Fig. 2A) in murine 53BP1 (m53BP1; 1,957 amino acids; 80% identity to human 53BP1; see Ref. 28). VICTR54 was found inserted within a 4.9-kb intron located between exons 13 and 14 ( Fig. 2A). VICTR54, and its related vectors, are usually found within introns and contain splice acceptor (SA) and donor (SD) sequences such that a neomycin (NEO) resistance gene and flanking sequences are spliced into the mature transcript as an exon ( Fig. 2A) (29). These transcriptional fusions disrupt the coding sequence through the introduction of premature stop codons. Such gene trapping methodologies have been applied previously to understanding gene function (29). OST94324 cells were used to gen-erate transgenic animals heterozygous in m53BP1 (m53BP1ϩ/ tr) as described previously (29). Southern blotting with DNA isolated from tail biopsies confirmed the disruption in m53BP1 and was used to genotype the animals ( Fig. 2B; see "Experimental Procedures"). Crosses between heterozygous animals produced m53BP1 tr/tr progeny born at the expected frequencies. The m53BP1 tr/tr animals were found to be fertile, but we did observe that crosses between mutant animals produced smaller litters as some embryos spontaneously aborted and were reabsorbed by the mother (data not shown). RT-PCR analysis with various primers 5Ј and 3Ј to the insertion demonstrated that exon 13 failed to properly splice next to exon 14 in the m53BP1 tr/tr mice (Fig. 2C). Rather, the "artificial" exon containing neomycin from VICTR54 was spliced adjacent to exon 13 as verified with primers specific for exon 13 and the neomycin gene (primer set D/A; Fig. 2C). Sequencing of a cloned RT-PCR product spanning the insertion event revealed that the natural coding sequence of m53BP1 had stopped after residue 1,205, where it then fused to 21 residues derived from VICTR54 before terminating (Fig. 2D). Therefore the disrupted allele of m53BP1 encodes a truncated 1,226 residue protein (m53BP1 tr ), and notably, m53BP1 tr is missing over 700 residues including its functional nuclear localization signal, kinetochore binding domain (KINET), and two BRCT motifs (Fig.  2D). To determine whether m53BP1 tr was expressed, we performed immunoprecipitation/Western blotting (IP/WB) analysis from brain extracts derived from m53BP1 ϩ/ϩ , m53BP1 ϩ/tr , and m53BP tr/tr animals. By using antibodies specific for the N and C termini of 53BP1 (␣53BP1-N and ␣53BP1-C, respectively; see "Experimental Procedures"), we determined that a truncated m53BP1 protein corresponding to m53BP1 tr appeared in heterozygous and mutant extracts but not in wildtype ones (Fig. 2E). The levels of m53BP1 tr appeared much lower than the full-length protein and, in some cases, we observed an apparent isoform of m53BP1 in wild-type and heterozygous animals (Fig. 2E). The disappearance of full-length m53BP1 in the mutant samples was accompanied by the appearance of a smaller protein corresponding to m53BP1 tr (Fig.  2E). We observed that ␣53BP1-N cross-reacted with m53BP1 tr but not with ␣53BP1-C, demonstrating that m53BP1 is indeed truncated in m53BP1 tr/tr animals (Fig. 2, E and F).
Immune Deficiencies in m53BP1 tr/tr Mice-We observed that m53BP1 tr/tr animals were growth-retarded as the males and females were found on average to weigh 25 and 15% less, respectively, than their wild-type littermates (Fig. 3A). We found that thymuses derived from m53BP1 tr/tr animals were significantly smaller and possessed fewer cells than those from m53BP1 ϩ/ϩ animals (Fig. 3B). This suggests that defects in m53BP1 may contribute to immune deficiencies, a result that has been observed for various DNA damage-response factors, including H2AX (24). We found that the lymphoid organ architecture of thymuses, as assayed by hematoxylin and eosin staining of tissue sections, appeared normal in m53BP1 tr/tr mice (data not shown). In addition, flow cytometric analysis FIG. 1. Dynamic nuclear localization of human 53BP1 during interphase, association with ATR, and structural requirements for DNA damage inducible focus formation. As shown in A, immunofluorescence analysis with an antibody specific for 53BP1 (␣53BP1; see "Experimental Procedures") reveals the nuclear staining pattern for cells unambiguously assigned to the G 1 -phase (left), S-phase (middle), and G 2 -phase (right) of the cell cycle as determined by DNA content with a lasing scanning cytometer. Black and white images were captured with a ϫ100 objective on a Zeiss Axiophot and pseudocolored in Adobe PhotoShop. As shown in B, ATR and 53BP1 co-localize to nuclear foci in response to hydroxyurea (2.0 mM). Left panel, immunostaining for 53BP1. Middle, immunostaining with an antibody specific for ATR (see "Experimental Procedures"). Right, merged images to show co-localization between 53BP1 and ATR. As shown in C, ATR and 53BP1 physically interact before and after DNA damage. K562 cells were grown and either left untreated (lanes 1 and 2) or treated with 2.0 mM hydroxyurea (HU; lane 3) for 18 h prior to the preparation of nuclear extracts for immunoprecipitation with an antibody against 53BP1 (left panel) or ATR (right panel). Immunoblotting was then performed with the reciprocal antibodies as shown. D, schematic representation of primary structure of 53BP1 (not drawn to scale). Hatched lines represent locations of (S/T)Q sites mutated in 15AQ. KINET, kinetochore-binding region (28); NLS, nuclear localization signal (28). E, identification of a region of 53BP1 required for irradiation-induced focus formation. Wild-type, HA-tagged 53BP1 and various mutant derivatives were transfected into MCF7 cells and treated with 10 Gy of ionizing radiation prior to fixation and immunostaining with an antibody specific for the HA tag (Covance). The following constructs expressing in-frame 53BP1 deletions or mutations were made: ⌬BRCT (deletes amino acid residues 1,786 -1,964), ⌬KINET (deletes amino acid residues 1,236 -1,615), ⌬NH3 (deletes the first 1,234 amino acids residues except for the initiation codon), and 15AQ, a construct with mutations in 15 (S/T)Q sites. The following serine or threonine residues were mutated to alanines in 15AQ: Ser-6, Ser-13, Ser-25, Ser-29, Ser-105, Ser-166, Ser-176, Ser-178, Thr-302, Ser-452, Ser-523, Ser-543, Ser-625, Ser-784, Ser-892. All constructs were verified by DNA sequencing and expressed in either 293T or MCF7 cells (not shown).

FIG. 2. Generation and characterization of mice defective in m53BP1 (m53BP1 tr/tr ). A, schematic diagram of insertion event in m53BP1
(not drawn to scale). The thick horizontal lines represent positions of probes for Southern blotting as described in B. Arrows represent the position and orientation of PCR primers used in C. The insertion of VICTR54 was determined by DNA sequencing to reside within the intron preceding exon 14 at nucleotide position 1,730 (marked by *). Splicing of the neomycin gene and flanking DNA produces a transcript that potentially disrupts the proper splicing of exons 13 and 14. LTR, long-terminal repeat; NEO, neomycin resistance gene; PGK, phosphoglycerate kinase-1; BTK, Bruton's tyrosine kinase; SA and SD, splice acceptor and donor, respectively. B, top, Southern blotting to determine the genotype of m53BP1-defective animals. 10 g of genomic DNA was digested with XbaI and was probed with a radiolabeled fragment (see "Experimental Procedures") capable of discerning wild-type (WT) and mutant alleles as discussed under "Experimental Procedures." Bottom, a 700-bp probe derived from the neomycin gene was used to help genotype the animals. ϩ/ϩ, wild type; ϩ/tr, heterozygous; tr/tr, homozygous. As shown in C, RT-PCR analysis indicates that improper splicing occurs between exons 13 and 14 in m53BP1 tr/tr mice. Positions and orientation of primers for PCR are indicated in A. Control reactions without reverse transcriptase showed essentially no amplified products (not shown). As shown in D, m53BP1 tr/tr encodes a truncated protein of 1,226 amino acids. RT-PCR products derived from primer set A/D (as shown in C) using RNA isolated from m53BP1 tr/tr animals as template were cloned into the TA vector (Invitrogen). DNA sequencing and conceptual translation indicated that m53BP1 tr/tr animals potentially encode a truncated m53BP1 protein (m53BP1 tr ) of 1,205 natural residues along with an additional 21 residues derived from the VICTR54 vector. m53BP1 tr is missing over 700 C-terminal residues, including those that specify the kinetochore binding domain (KINET; amino acids 1,220 -1,601), the nuclear localization signal (NLS; mapped to amino acids 1,601-1,703; ref), and the C-terminal BRCT repeats (amino acids, 1,665-1,957). The small, vertical rectangle in m53BP1 tr represents the additional vector-derived 21 residues. E and F, detection of m53BP1 tr protein by IP/WB analysis. 1.0 mg of total brain protein extracts derived from ϩ/ϩ, ϩ/tr, and tr/tr animals was immunoprecipitated with 5 g of affinity-purified antibody (␣53BP1-N) raised against an N-terminal peptide sequence (see "Experimental Procedures") and split into two equal parts. One part (E) was blotted with ␣53BP1-N, and the other (F) was immunoblotted with an affinity-purified antibody specific for the C terminus (␣53BP1-C). E, lane 1, IP from 100 g of MCF-7 nuclear extract. Lane 2, IP with nonspecific IgG control. Lanes 3-5, IP with ϩ/ϩ, ϩ/tr, and tr/tr animals as determined in B. IP/WB analysis shows the presence of m53BP1 tr in m53BP1 ϩ/tr and m53BP1 tr/tr animals but not of m53BP ϩ/ϩ ones. In some cases, we observed an apparent isoform of m53BP1 in brain tissue, as designated by the asterisk. F, WB with ␣53BP1-C, an affinity-purified antibody against the C-terminal 200 residues of human 53BP1. As shown, ␣53BP1-C recognizes full-length m53BP1 but fails to immunoreact with m53BP1 tr , indicating that the protein is indeed missing C-terminal residues of m53BP1. with a variety of markers (e.g. B220, CD43, Gr-1, CD11a, and Ter119) revealed that bone marrow pro-B, pre-B, myeloid, and erythroid progenitor populations were normal in m53BP1 tr/tr mice (not shown). Although CD4 and CD8 T cell populations were proportionately similar in m53BP1 tr/tr and m53BP1 ϩ/ϩ thymuses, we observed that progression out of the DNIII stage of early thymocyte development was impaired in m53BP1 tr/tr animals (Fig. 3C), the stage at which ␤-gene rearrangement occurs. This indicates that m53BP1 participates in proper T cell development, a process known to require various DNA repair factors (30). We also found that spleens derived from m53BP1 tr/tr animals were similar in size and organ architecture to those from m53BP1 tr/tr animals and that the lack of functional m53BP1 did not affect the proportions of B and T lymphocytes (data not shown). We did observe, however, that m53BP1 tr/tr spleens were deficient in mature B cells (IgM lo IgD hi ; Fig. 4D), suggesting that deficiencies in m53BP1 may also result in defective B lymphocyte development.
Genomic Instability in m53BP1 tr/tr Mice-Mice with defects in double-stranded break repair are highly sensitive to ␥-IR. To evaluate whether m53BP1 contributes to increased sensitivity to DNA damage, we treated m53BP1 tr/tr or wild-type animals with 7 Gy of ␥-IR. After this whole body irradiation treatment, we found that 100% of the mutant animals died between 9 and 15 days post-irradiation in contrast to only 16% of the control littermates (Fig. 4A). This shows that animals defective in m53BP1 are highly sensitive to ␥-IR, a result that parallels previous observations with H2AX-deficient mice (24). Despite this, we found that m53BP1 tr/tr animals treated with lower doses of ␥-IR (1.5 Gy) remained viable (Fig. 4B). To further explore m53BP1 function, we generated embryonic fibroblasts (MEFs) from wild-type and m53BP1 tr/tr animals. m53BP1 tr/tr MEFs proliferated more slowly than their wild-type counterparts (Fig. 5A). Immunofluorescence analysis indicated that the truncated m53BP1 protein expressed in m53BP1 tr/tr animals failed to form foci in response to DNA damage as it was essentially absent from the nucleus (data not shown). This result is consistent with our transfection studies, which have shown that C-terminal determinants (⌬KINET) are necessary for focus formation. The relative growth of the mutant and the wild-type MEFs was reminiscent of what has been recently described for H2AX (24). To further characterize cells defective in m53BP1, we examined the cytological consequences of impaired m53BP1 function in early passage MEFs derived from m53BP1 tr/tr and m53BP1 ϩ/ϩ animals. For this, exponentially growing MEFs (passage 2) were treated with 0, 0.5, or 1.5 Gy of ␥-IR, and metaphase preparations were examined 2.5 h postirradiation. Untreated MEFs derived from m53BP1 tr/tr animals showed increased levels of chromatid gaps, breaks, and, to a lesser extent, exchanges when compared with those derived from m53BP1 ϩ/ϩ mice, suggesting an intrinsic genomic stability defect in the mutant cells (Fig. 5, B and C). More strikingly, irradiated MEFs derived from m53BP1 tr/tr animals showed an ϳ2-fold increase in levels of chromatid breaks and gaps when compared with MEFs derived from wild-type mice (Fig. 5, B and C). Although MEFS from m53BP1 tr/tr animals showed relatively high chromatid exchange rates at 0.5 Gy when compared with those from m53BP1 ϩ/ϩ animals, this difference was less apparent at 1.5 Gy, perhaps due to the limited progression to mitosis of the most damaged cells from both populations during this time frame. One possible explanation for the increased frequencies of chromosomal aberrations observed in the m53BP1 tr/tr MEFs following irradiation might be a deficiency in a G 2 checkpoint response whereby more damaged cells would still be permitted to enter mitosis and would be available for chromosome analysis. In fact, recent reports have implicated 53BP1 in the G 2 /M checkpoint (25)(26)(27). To examine this in our MEFs, either m53BP1 tr/tr or wild-type MEFs were treated with 0, 1.5, or 10 Gy of ␥-IR, and cultures were analyzed for the fraction of cells showing phospho-histone H3 immunostaining (mitotic cells) either after 1 or 16 h post-irradiation (in the presence of colcemid). Although all cell types showed evidence of a partial G 2 /M block following irradiation, MEFs derived from m53BP1 tr/tr mice showed only a slight decrease, if any, in the G 2 block when compared with MEFs derived from wild-type mice (data not shown). The minimal effects on the G 2 /M checkpoint observed in our m53BP1 tr/tr MEFs may be due to the nature of the truncated protein produced from the m53BP1 tr/tr allele that is expressed in our mutant animals described here.
53BP1 interacts with a variety of factors known to be involved in the maintenance of genomic stability including ATR, p53, H2AX, BRCA1, Chk2, and ATM (15,20,25,27). The generation of murine animals defective in m53BP1 provides a valuable tool to further understand the role of the protein in the DNA damage response. The m53BP1 tr/tr allele expresses a truncated version of m53BP1, and this likely represents a  (closed circles). B, metaphase preparation of mutant MEF following 1.5 Gy of ␥-IR. Note the presence of a chromatid gap, two chromatid breaks, and one chromatid exchange in the metaphase sample. C, relative frequencies of chromatid gaps, breaks, and exchanges in metaphases of wild-type and mutant MEFs following 0, 0.5, and 1.5 Gy of ionizing radiation. significant impairment in some aspects of its function. m53BP1 tr/tr is missing over 700 amino acids including the nuclear localization signal, the C-terminal BRCT motifs, and a kinetochore-binding domain. We have observed that this domain is also necessary for forming irradiation-induced nuclear foci. Indeed, the lack of detectable, irradiation-induced foci in mutant MEFs suggests that the protein cannot fully perform its functions as a DNA damage-response element. Moreover, the lethality observed for m53BP1 tr/tr mice at higher doses of radiation (7 Gy) suggests that there are no other factors acting redundantly with m53BP1 with respect to this aspect of radiation resistance and indicates that m53BP1 is a critical element for double-stranded break repair. Therefore, the C-terminal 700 amino acids of m53BP1 encode important, functional determinants of the protein.
We observed that m53BP1 tr/tr animals are growth-retarded as the males weigh, on average, 25% less than their wild-type littermates. The decreased thymus size, reduced T cell count, immature B cell population, and lack of progression out of DNIII for thymus T cells reveal that m53BP1 tr/tr animals are immune-deficient. How m53BP1 contributes to this process remains to be established, but one possibility is that the protein participates in the maturation of T-cell receptors and immunoglobulins during V(D)J recombination, a process known to utilize DNA repair proteins (30). This is particularly interesting given the involvement of m53BP1 in double-stranded break repair as revealed by several factors including, most notably, the sensitivity of m53BP1 tr/tr animals after exposure to 7 Gy of ionizing radiation. Indeed, sensitivity to ionizing radiation often correlates with impaired V(D)J joining (30). Moreover, H2AX defective-animals are also immune-deficient (24). As H2AX is required for the formation of 53BP1 foci and because it physically associates with 53BP1 (20), it is possible that an ordered pathway of assembly of DNA damage-response proteins at these programmed breaks may facilitate V(D)J recombination and maximize antibody diversity.
Our results show that genetic defects in m53BP1 result in a pleiotropic phenotype consistent with defects in DNA repair and checkpoint control. The phenotype of 53BP1-defective animals is quite similar to H2AX-deficient ones, consistent with the notion that H2AX operates upstream of 53BP1 in a DNA damage-response pathway. When such pathways are defective, cells cannot properly repair damaged DNA, a situation that may lead to increased genomic instability and the development of diseases such as cancer. For example, given the immune deficiencies in m53BP1 tr/tr mice, one may anticipate the generation of lymphomas. In light of this, we have not observed the development of any cancerous phenotypes in our m53BP1defective mice. Although there are a variety of possible reasons for this (i.e. genetic background, allele, etc.), it is interesting to note that mice nullizygous for H2AX also apparently fail to generate cancers. 3 As H2AX and 53BP1 are not required for viability, it is possible that mutations in 53BP1, when combined with other mutations in critical DNA damage-response elements (i.e. H2AX, ATM, and p53) will lead to more severe defects in genomic stability, a process that may then lead to the development of cancer. The analysis of cells derived from these crosses is likely to provide more insight into how 53BP1 functions in the DNA damage response in concert with its various interacting partners.