Genetic interactions between ATM and thenonhomologous end-joining factors ingenomic stability and developmentJoAnn Sekiguchi*†, David O. Ferguson*‡, Hua Tang Chen§, Eva Mary Yang¶, John Earle¶, Karen Frank†, Scott Whitlow*,Yansong Gu*, Yang Xu¶, Andre Nussenzweig§, and Frederick W. Alt*†i**

*The Center for Blood Research, †The Children’s Hospital, iHoward Hughes Medical Institute, Harvard Medical School, Boston, MA 02115; ‡Department ofPathology, Brigham and Women’s Hospital, Boston, MA 02115; ¶Department of Biology, University of California at San Diego, La Jolla, CA 92093; and§Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892

Contributed by Frederick W. Alt, December 29, 2000

DNA ligase IV (Lig4) and the DNA-dependent protein kinase (DNA-PK)function in nonhomologous end joining (NHEJ). However, althoughLig4 deficiency causes late embryonic lethality, deficiency in DNA-PKsubunits (Ku70, Ku80, and DNA-PKcs) does not. Here we demonstratethat, similar to p53 deficiency, ataxia-telangiectasia-mutated (ATM)gene deficiency rescues the embryonic lethality and neuronal apo-ptosis, but not impaired lymphocyte development, associated withLig4 deficiency. However, in contrast to p53 deficiency, ATM defi-ciency enhances deleterious effects of Lig4 deficiency on growthpotential of embryonic fibroblasts (MEFs) and genomic instability inboth MEFs and cultured progenitor lymphocytes, demonstratingsignificant differences in the interplay of p53 vs. ATM withrespect to NHEJ. Finally, in dramatic contrast to effects on Lig4deficiency, ATM deficiency causes early embryonic lethality inKu- or DNA-PKcs-deficient mice, providing evidence for an NHEJ-independent role for the DNA-PK holoenzyme.

The stability of the mammalian genome relies on efficient repairof DNA double-strand breaks (DSBs), which can arise from

normal metabolism, exogenous DNA-damaging agents, or endo-nuclease activity (1). In mammalian cells, the nonhomologousend-joining (NHEJ) and homologous recombination DSB-repairpathways serve as genomic caretakers (1, 2). Cellular gatekeepersregulate progression through the cell cycle in response to DNAdamage and induce either apoptosis or cell-cycle arrest to providesufficient time for repair by caretaker functions (3). The knownNHEJ factors include Ku70, Ku80, and the DNA-dependent pro-tein kinase catalytic subunit (DNA-PKcs), which comprise theDNA PK holoenzyme, plus XRCC4 and Lig4, which function inligation. These factors also are required for V(D)J recombination,the process by which antigen-receptor variable-region genes areassembled during lymphocyte development. Initially, the recombi-nation-activating gene 1 and 2 (RAG1y2) endonuclease introducesDSBs at component V, D, and J recombination signal sequences;then, ubiquitously expressed NHEJ factors perform the subsequentjoining steps (4).

Mice deficient in Lig4 (or XRCC4) exhibit a pleiotropic pheno-type including late embryonic lethality, cellular growth defects andionizing-radiation sensitivity, severely impaired lymphocyte devel-opment caused by inability to form V(D)J joins, and massiveapoptosis of newly generated neurons (5–7). Ku-deficient micedisplay most of these defects and are smaller than littermates, butthey are born in normal numbers and survive into adulthood;DNA-PKcs-deficient mice appear relatively normal except for someionizing-radiation sensitivity and impaired lymphocyte develop-ment caused by defective V(D)J joining (8). The Ku, XRCC4, andLig4 proteins are conserved in yeast, where they also function inNHEJ (9). To date, there is no compelling evidence for any in vivofunction of the DNA-PK or Lig4–XRCC4 protein complexesbeyond their common role in NHEJ.

The embryonic lethality and increased neuronal apoptosis asso-ciated with Lig4 and XRCC4 deficiency were rescued dramatically

by p53 deficiency, indicating that these phenotypes result from ap53-dependent response to DNA damage (10, 11). In contrast, p53deficiency did not rescue either the B or T cell developmentaldefects of Lig4- or XRCC4-deficient animals, consistent withinability of p53 deficiency to restore the V(D)J recombinationyNHEJ defects (10, 11). Likewise, p53 deficiency did not rescue theV(D)J recombination defects associated with Ku80 or DNA-PKcsdeficiency and also did not affect ability of these genotypes togenerate normal numbers of viable progeny (12–16). However, inthe p53-deficient background, absence of any NHEJ factor resultedin nearly universal development of aggressive pro-B cell lymphomasthat harbored translocations between IgH and c-myc loci. NHEJ-factor deficiencies also led to increased spontaneous genomicinstability in cultured embryonic fibroblasts (MEFs; refs. 10, 12, and17–19), consistent with a critical role as a genomic caretaker.

The ATM protein, a serineythreonine protein kinase, is a mem-ber of a family of large proteins, including DNA-PK, which containsa phosphatidylinositol 3-kinase domain (20). ATM, like p53, func-tions as a cellular gatekeeper and is a key initiating factor in thecascade of events leading to activation of multiple DNA damage-responsive signaling pathways and cell-cycle checkpoints (2). Inresponse to DSBs, ATM acts upstream of p53 and controls itsactivity through phosphorylation and stabilization of the protein(2). Cells deficient in ATM exhibit defective cell-cycle checkpointsat the G1yS transition, during S phase, and at the G2yM boundary;whereas p53 mutation predominantly effects the G1yS transition(2). In addition to impaired cell-cycle-checkpoint activation, ATM-deficient cells also possess distinct DNA-repair defects (20). Thiscombination of defects may lead to the complex phenotypesobserved in ataxia telangiectasia patients and cell lines, includingionizing-radiation sensitivity, genomic instability, neurological andvascular abnormalities, infertility, and predisposition to lymphoidmalignancy (20).

MethodsMice. All mice were housed in a pathogen-free facility. The Lig4(5), Ku70 (21), Ku80 (22), DNA-PKcs (23), and ATM (24–26)knockout mice used in this study were generated and charac-terized previously. The severe combined immunodeficient (scid)mice were obtained from The Jackson Laboratories. The Pvalues for rescue of embryonic lethality were calculated based onthe total number of Lig42y2ATM1y1 (5, 11) and Lig42y2ATM2y2

or Lig42y2ATM1y2 pups born (this study) by using the Fisher’s

Abbreviations: DSB, double-strand break; DNA-PKcs, DNA-dependent protein kinase cat-alytic subunit; NHEJ, nonhomologous end-joining; PK, protein kinase; MEF, embryonicfibroblast; scid, severe combined immunodeficient; En, embryonic day n.

**To whom reprint requests should be addressed at: The Children’s Hospital, 300 Long-wood Avenue, Enders 861, Boston, MA 02115. E-mail: alt@rascal.med.harvard.edu.

The publication costs of this article were defrayed in part by page charge payment. Thisarticle must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.§1734 solely to indicate this fact.

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exact probability test. The P values for rescue of Lig42y2 embryoniclethality by ATM2y2 and ATM1y2 are P , 0.01 and P , 0.1,respectively.

Histological Analysis. Whole embryos were fixed in either Bouins’solution or in 4% (wtyvol) paraformaldehyde (in PBS), pro-cessed, embedded in paraffin, sectioned serially (5 mm), andstained with hematoxylinyeosin. The terminal deoxynucleotidyl-transferase-mediated UTP end labeling (TUNEL) assay wasperformed on paraformaldehyde-fixed sections by using theDeadEnd colorimetric apoptosis detection kit (Promega).

Growth Assays. Passage 3 MEFs (2 3 105) were plated in duplicateonto gelatinized 6-cm dishes. The cells were trypsinized, stainedwith trypan blue, and counted every 48 h. Cell number (3105)is plotted as a function of time (days). The results represent theaverage of three independent experiments by using MEFsderived from at least two different embryos. Senescence-associated b-galactosidase activity was measured by assaying forb-galactosidase activity as described (11).

Lymphocyte Development. Fetal liver cultures were established asdescribed (5). The cells were harvested at day 10 in culture andthen stained with anti-IgM-phycoerythrin and anti-B220-FITCantibodies (PharMingen) and propidium iodide. Data werecollected on a FACSCalibur flow cytometer (Becton Dickinson)and analyzed by using FLOWJO software (TreeStar, San Carlos,CA). Single-cell suspensions of splenocytes from postnatal day-1(P1) littermates were stained with anti-IgM and anti-B220antibodies and analyzed as above. Single cell suspensions ofthymocytes from P1 littermates were stained with anti-CD25,anti-CD4, and anti-CD8 fluorescence-conjugated antibodies(PharMingen and Southern Biotechnology Associates).

Spectral Karyotyping Analyses. Passage 3 MEFs (1 3 106) wereplated onto gelatinized 10-cm dishes and cultured for 16 h.Colcemid (GIBCOyBRL; KaryoMAX solution) was added (100ngyml), and the cultures were incubated for 3 h. Fetal liver cells(day 10 in culture) were incubated with colcemid (10 ngyml) for12 h. Chromosomal aberrations were quantified by using a NikonEclipse microscope equipped with an Applied Spectral Imaginginterferometer (Carlsbad, CA) and 340 and 363 objectives.

ResultsGiven the role of ATM in regulating p53 activity, it seemed likelythat ATM could modulate the cellular and organismal pheno-types associated with NHEJ deficiency in a manner similar top53. Therefore, to elucidate potential genetic interactions be-tween ATM and NHEJ, we bred the ATM-deficient backgroundinto various NHEJ-deficient backgrounds. First, we bredLig41y2 mice (5) with ATM1y2 mice (26), then bred Lig41y2

ATM1y2 progeny to generate Lig42y2 mice with backgrounds ofall ATM genotypes. Mendelian numbers of developing embryosof all genotypes were observed between embryonic days (E) 11.5and 16 (Table 1). However, by E15 the Lig42y2ATM2y2 andLig42y2ATM1y2 embryos were significantly smaller than litter-mate controls (20–25% of controls), although they were ofsimilar size to Lig42y2ATM1y1 embryos of the same age. Asexpected, no Lig42y2ATM1y1 pups were born; however, we didobserve live-born Lig42y2ATM2y2 pups (Fig. 1a), demonstrat-ing that ATM deficiency can rescue Lig42y2 embryonic lethality,albeit at numbers below the predicted Mendelian frequency.ATM haplosufficiency also rescued Lig42y2 embryonic lethality,although to an even lesser extent (Fig. 1a). In all cases, therescued pups were growth retarded, and most did not survivebeyond postnatal day P1 or P2 (see Fig. 1a, legend). Thus,although clearly significant, neither the extent of postnatalrescue nor the duration of postnatal survival was as great as that

observed in the context of rescue by p53 deficiency (11).Although differences in the genetic background of the ATM- vs.p53-deficient strains could contribute theoretically to thesesurvival differences, we favor the possibility that more funda-mental differences in the role of ATM vs. p53 in maintenance ofgenomic stability may be a major factor (see Discussion).

ATM has been suggested to play an important role duringneuronal development as a survival checkpoint that eliminatesneurons with excessive DNA damage (27, 28). We performed ahistologic analysis of the developing central nervous system (CNS)at E13.5 in Lig42y2ATM2y2 embryos and found that ATM defi-ciency, like p53 deficiency (11), substantially alleviated the in-creased neuronal apoptosis in this NHEJ-deficient background(Fig. 1b). Similar conclusions were reached by a parallel study (29).However, levels of pyknosis remained substantial in Lig42y2

ATM1y2 embryos (Fig. 1c, data not shown), which is in contrast tothe significant level of rescue observed in the Lig42y2p531y2

genotype (11). To confirm these observations, we assayed forcellular DNA fragmentation, a hallmark of apoptosis, by TUNEL.Significantly, we observed few TUNEL-positive cells in Lig42y2

ATM2y2 embryos, as opposed to the high numbers in Lig42y2

ATM1y2 embryos (Fig. 1c). These findings, together with ourearlier findings of essentially complete rescue by p53 deficiency(11), suggest that most of the increased neuronal apoptosis in thedeveloping Lig42y2 CNS is signaled by ATM via p53.

In Lig4-deficient animals, B cell development is blocked at theB2201IgM2 progenitor stage and T cell development is blocked atthe CD42 CD82 progenitor stage because of inability to completeV(D)J recombination via the NHEJ pathway (11). Notably, p53deficiency does not rescue either B or T cell development in theLig4-deficient background because of its inability to rescue NHEJ(11). A parallel study to ours found that ATM deficiency also failedto restore Lig4-deficient T cell development (29). Surprisinglyhowever, the latter study also reported that development of neo-natal Lig42y2ATM2y2 splenic B cells was similar qualitatively tothat of wild-type controls, with Lig42y2ATM2y2 B cells apparentlyprogressing to the B2201IgM1 B cell stage (29). Such differentially‘‘rescued’’ B cell development would imply substantial rescue ofV(D)J recombination by ATM deficiency in Lig4-deficient B cellsbut not Lig4-deficient T cells. However, in preliminary studies, wefound that both B and T cell development remained severelyimpaired at the progenitor stage in an analysis of thymocytes andspleen cells of a neonatal Lig42y2ATM2y2 mouse (postnatal-dayP1; data not shown).

Because of poor survival and poor health, it is difficult toobtain large numbers of viable postnatal Lig42y2ATM2y2 an-imals for analyses of lymphocyte development. Moreover, it isimpossible to obtain a Lig42y2 control mouse at this postnatalstage. Therefore, to assess the effects of ATM deficiency on Lig4

Table 1. ATM-deficiency causes early embryonic lethality in micedeficient in the components of the DNA-PK holoenzyme

Age Genotype ATM1y1 ATM1y2 ATM2y2

Live-born pups Ku702y2 16 (10) 12 (20) 0 (10)Ku802y2 13 (13) 24 (25) 0 (13)scid 21 (16) 44 (33) 0 (16)

E13–16 Lig42y2 9 (10) 18 (20) 16 (10)E11.5–13.5 Ku802y2 8 (11) 23 (22) 0 (11)

The number of live-born pups and embryos at the indicated developmentalstages of the relevant genotypes (as indicated) is shown with the expectedMendelian numbers in parentheses. No scid2y2 ATM2y2 pups were observedin over 360 pups born from scid1y2 ATM1y2 intercrosses (data not shown). Inpreliminary results, no live-born DNA-PKcs2y2 ATM2y2 pups were observed inbreedings between DNA-PKcs1y2 ATM1y2 mice, and no scid2y2 ATM2y2 em-bryos (E12) from breedings between scid2y2 ATM1y2 and scid1y2 ATM1y2

mice were observed.

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deficient B cell development more thoroughly, we used an in vitrodifferentiation system to determine the ability of fetal liverprogenitor B cells from the various backgrounds to differentiateinto IgM1 B cells (5). As a positive control, we observedprogression of B2201IgM2 progenitors to the B2201IgM1 B cellstage in all of 45 independent (each from a different embryo)fetal liver cultures from the various ATM genotypes on either a

Lig41y1 or Lig41y2 background (Fig. 1d, representative data areshown). However, we found no obvious rescue of B cell devel-opment beyond the IgM2 pro-B stage in eight independentLig42y2ATM2y2 cultures, which appeared similar to sevenLig42y2ATM1y2 and four Lig42y2ATM1y1 cultures that servedas negative controls (Fig. 1d, representative data are shown). Weconclude that in the strains we have studied, ATM deficiency,

Fig. 1. Effects of ATM deficiency on Lig42y2 phenotypes. (a) ATM deficiency rescues embryonic lethality of Lig4 deficiency. The numbers of actual pups bornand the Mendelian expected number of pups of the relevant genotypes are shown. The ATM-rescued pups, which were runted in comparison to littermates, didnot appear to have nursed and died shortly after birth; however, the cause of the perinatal lethality is not known yet. (b) Histological analysis ofhematoxylinyeosin-stained coronal sections of the diencephalon (E13.5) of the indicated genotypes. Arrows indicate pyknotic nuclei. VZ, ventricular zone; ML,mantle layer. Original magnification was 3400. (c) TUNEL staining of transverse sections of the developing cerebral cortex (E13.5) of the indicated genotypes.Arrows point to TUNEL-positive nuclei. V, ventricle; IZ, intermediate zone; CP, cortical plate. Original magnification was 3400. (d) Flow-cytometric analysis oflymphocytes from E15 fetal liver cells of the indicated genotypes showing B220 and IgM profile and the percentages of B2201 and IgM1 cells.

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similar to p53 deficiency, does not lead to significant rescue ofLig4-deficient B cell development beyond the IgM2 pro-B stage.Potential reasons for this apparent discrepancy with the parallelstudy (29) will be considered in the Discussion.

Increased doubling times and premature senescence are charac-teristics of Lig4-deficient MEFs, and p53 deficiency rescues thesephenotypes (5, 11). In striking contrast, ATM deficiency furtherimpaired the ability of Lig42y2 cells to proliferate in culture (Fig.2a). The Lig42y2ATM2y2 MEFs showed nearly complete growtharrest under normal culture conditions, even at the earliest passageanalyzed (passage 2–3). This severe growth defect correlates witha premature senescence phenotype as assayed by the senescence-associated b-galactosidase activity assay (data not shown). Underthe same culture conditions, Lig4- and ATM-deficient MEFsexhibited significantly reduced growth rates but still retained mea-

surable proliferative capacity (Fig. 2a; refs. 5 and 30). It seems likelythat the dramatically impaired growth exhibited by Lig42y2

ATM2y2 MEFs may result from overwhelming genomic instabilityin the absence of NHEJ factors and ATM (see below).

We used spectral karyotyping (SKY), a chromosome-paintingtechnique, to assay for chromosomal abnormalities in Lig42y2

ATM2y2 MEFs and developing lymphocytes. Lig4 or ATMdeficiency alone significantly increased the frequency of chro-mosomal aberrations in early-passage MEFs (19). However, p53deficiency, either alone or in combination with Lig4 deficiency,had no observable effect on genomic stability as assayed by SKY(19). In contrast, the combined Lig4 and ATM deficienciesresulted in an additive phenotype in which 100% of the met-aphases examined contained at least one chromosomal abnor-mality (Table 2). In addition to the quantitative effect, the

Fig. 2. Growth analysis and cytogenetic studies of MEFs. (a) Growth curves of Lig42y2ATM2y2 MEFs with controls. Genotypes are as indicated in the graph. (b–d)Metaphase chromosomes from primary MEF cultures. Arrows indicate single chromatid fusions; arrowheads indicate single chromatid breaks. (b) Robertsoniantranslocation (chromosome fusion) in an ATM2y2 metaphase involving chromosomes 4 (dark blue) and 1 (gray). (c and d) Partial translocations in Lig42y2ATM2y2

metaphases. (c) Chromosome 2 (red) contains a broken chromatid in which one end remains free and the other is fused to a chromatid on chromosome 13 (bright blue).(d) Chromosome 1 (gray) contains a broken chromatid with two free ends, whereas the other chromatid is fused to a single chromatid of chromosome 8 (green).

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combined Lig4 and ATM deficiencies led to frequent appear-ance of complex aberrations in which single chromatids fromdifferent chromosomes were fused, whereas the sister chroma-tids of each either remained intact or were broken (Fig. 2 b–d).These unusual interchromosomal structures may represent par-tial translocations in which intermediates are trapped because ofimpaired ability of cells to complete the generation of translo-cations in the absence of both Lig4 and ATM functions. SKYanalysis of fetal liver progenitor B cell cultures revealed furtherdifferences between the effects of p53 and ATM deficiencies(Table 2). Surprisingly, of the metaphases examined, neitherLig4 nor p53 deficiency, either as single or double mutants, ledto genomic instability in developing B lineage cells. However,ATM deficiency caused observable levels of genomic instabilityin the developing B cell cultures, and in Lig42y2ATM2y2

populations instability was further increased. Overall, our find-ings indicate that ATM and the NHEJ pathway play separate butcomplementary roles in maintaining genomic stability and sup-port the notion that ATM plays a more significant role than p53in suppressing chromosomal aberrations.

To explore the interplay between ATM and the NHEJ pathwayfurther, Ku701y2 (21), Ku801y2 (22), and mice homozygous for thescid mutation (which inactivates DNA-PKcs) were bred withATM1y2 mice. The resulting Ku701y2ATM1y2, Ku801y2

ATM1y2, and scid2y2ATM1y2 progeny were intercrossed to gen-erate NHEJ2y2 offspring in ATM1y1, ATM1y2, and ATM2y2

backgrounds. We note that Ku702y2, Ku802y2, and scid2y2 miceare born in expected Mendelian ratios after crosses of theirrespective heterozygous parents (8). In addition, these ratios werenot influenced significantly by p53 deficiency (12–16). Surprisinglyhowever, we observed no live-born Ku702y2ATM2y2, Ku802y2

ATM2y2, or scid2y2ATM2y2 pups from these intercrosses, indi-cating that ATM deficiency induces embryonic lethality in aDNA-PK-deficient background (P , 0.001 for each intercross;Table 1). Moreover, Ku80yATM double-deficient embryos werenot represented at E11.5, indicating that death occurred early indevelopment (P , 0.001; Table 1). This embryonic lethality was notassociated with a specific ATM2y2 background, because we testeddifferent ATM-deficient strains in the context of the Ku70 (26),Ku80 (25), and scid (24) breedings (Table 1). Therefore, these

observations coupled with the contrasting results of ATM defi-ciency in rescuing Lig42y2 embryonic lethality point to a uniqueand vital role for the DNA-PK holoenzyme during embryonicdevelopment, which is distinct from its role in Lig4-dependentNHEJ.

DiscussionATM deficiency significantly rescues embryonic lethality and in-creased neuronal apoptosis associated with Lig4 deficiency. On thebasis of our previous findings of similar rescue by p53 deficiency (10,11), our current findings indicate that most neuronal apoptosisresults from an ATM-signaled p53 response to unrepaired DSBs inthe NHEJ deficient background. This interpretation is consistentwith the observation that ionizing-radiation-induced apoptosis ofneuronal cells is an ATM-dependent response (27). Likewise, ourfindings indicate that the ATM pathway is a major contributor tothe embryonic lethality associated with Lig4 deficiency. Becausethe ATM-dependent cellular response is specific to DSBs, thesefindings further support the notion that DSBs, as opposed to othertypes of damage, are the major offending lesions in developingNHEJ-deficient neurons. Finally, we note that ATM deficiency, likep53 deficiency, failed to substantially rescue impaired B lymphocytedevelopment that occurs in a Lig42y2 background. Our preliminaryanalyses also indicate that ATM deficiency similarly failed to rescueT cell development. The latter observations further suggest thatabsence of ATM, similar to absence of p53 (10, 11), does not rescueLig4-deficient lymphocyte development, likely because of failure torestore NHEJ required for normal joining of RAG-liberated V, D,and J ends.

Our findings of severely impaired development of ATM2y2

Lig42y2 B lineage cells beyond the IgM2 progenitor stage contrastswith those of a parallel study that reports development ofATM2y2Lig42y2 B cells to the IgM1 stage (29), which wouldrequire V(D)J recombination to allow expression of both Ig heavy-and light-chain genes. One possibility for this apparent discrepancywould be strain differences. In this regard, there are no reports ofthe effects of the homozygous Lig4 mutation (7) used in the parallelstudy on lymphocyte development in the absence of ATM defi-ciency (29); conceivably, this mutation could be leaky. However,more detailed molecular analyses still may reveal subtle differences,

Table 2. Combined effects of ATM and Lig4 deficiencies result in increased genomic stability

Genomic instability in MEFs

Lig41y2ATM1y2 Lig42y2 ATM2y2 Lig42y2ATM2y2

Metaphases karyotyped 24 22 22 21Fragmented chromatids and

chromosomes1 26 21 34

Translocations 0 1 1 0Partial translocations 0 0 1 7Robertsonian translocations 0 1 2 4Metaphases with structural

abnormality4% 54% 61% 100%

Genomic instability in developing lymphocytes

Wild type Lig42y2 p532y2 Lig42y2p532y2 ATM2y2 Lig42y2ATM2y2

Metaphases karyotyped 34 26 29 16 38 35Fragmented chromatids and

chromosomes0 0 0 0 8 12

Robertsonian translocations 0 0 0 0 0 2Metaphases with structural

abnormality0% 0% 0% 0% 16% 42%

The number of events observed in each of the categories of chromosomal aberrations was scored for metaphases from MEFs (Upper) and developinglymphocytes from fetal liver cultures (Lower) of the indicated genotypes. The percentage of metaphases with any structural abnormalities is shown on the bottomrow. Some metaphases contain multiple anomalies.

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perhaps strain dependent, in the repair of DSBs between the p53-and ATM-deficient backgrounds; for example, because of theabsence of additional cell-cycle checkpoints in the latter thattheoretically lead to ability to generate functional V(D)J ends.Finally, we note that the apparent rescue of B cell developmentobserved in the parallel study may have resulted from an overes-timate of the numbers of IgM1 cells in all samples. In this regard,essentially no B2201IgM2 B lineage cells (that should be residenteven in wild-type neonatal spleen) were noted in any reportedsample (29). In any case, we conclude that in our lines ATMdeficiency does not result in a substantial rescue of Lig4-deficientB cell development.

Despite the qualitatively similar effects of p53 and ATM defi-ciencies on the rescue of Lig42y2 embryonic lethality and neuronalapoptosis, our studies demonstrate significant differences in thefunction of these cellular gatekeepers. Thus, ATM deficiencysignificantly augments the premature senescence and growth de-fects exhibited by Lig4-deficient MEFs and enhances the sponta-neous genomic instability in these cells. In contrast, p53 deficiencyhas been shown to rescue the growth defects of Lig42y2 MEFs andhas no significant impact on genomic instability. These differenceslikely result from loss of the more pleiotropic functions of ATM incomparison to those of p53. ATM-deficient cells exhibit multipledefects in cell-cycle checkpoints (G1yS transition as well as S phaseand G2yM boundary), which may decrease the potential for accu-mulated DSBs to be repaired efficiently by NHEJ-independentrepair pathways in the Lig42y2ATM2y2 background. In addition,ATM plays a distinct role in controlling DSB repair (20). This rolemay be mediated through the regulation of DNA-repair proteinssuch as NBS1 (31–34) and BRCA1 (35, 36). The combined effectsof loss of these ATM-dependent functions may contribute to theearly postnatal death of Lig42y2ATM2y2 mice, which contrastswith the survival of Lig42y2p532y2 mice into young adulthood (11).In this regard, p53 deficiency allows cells normally arrested at G1 toprogress into other phases of the cell cycle in which functional

checkpoints may permit damage repair by alternative pathways (i.e.,homologous recombination; ref. 37).

In contrast to Lig4 (or XRCC4) deficiency, which causes lateembryonic lethality, mice deficient in Ku- or DNA-PKcs surviveinto adulthood and have a relatively normal lifespan. Although p53deficiency dramatically rescued the embryonic lethality of Lig4 (orXRCC4) deficiency, it had no impact on development in Ku- orDNA-PKcs-deficient mice. Strikingly, despite the ability of ATMdeficiency to rescue embryonic lethality of Lig4 deficiency, it causedearly embryonic lethality in Ku- and DNA-PKcs-deficient back-grounds. This synthetic lethal effect is distinct from the embryoniclethality caused by mutation in Lig4 or XRCC4, which leads todeath of the mutant embryos at a significantly later stage ofdevelopment. On the basis of our findings, we conclude that theDNA-PK holoenzyme must have a function that is distinct from itsrole in the NHEJ pathway of DNA repair. Based on the knownfunction of the evolutionarily conserved Ku heterodimer as a DNAend-binding factor, this additional DNA-PK function likely involvesDSB recognition. Thus, it is possible that such a function mayoverlap with an essential ATM function involved in recognition ofDNA lesions and initiation of subsequent cellular responses,leadingto early death of embryos deficient for both ATM and DNA-PKactivities. Alternatively, this DNA-PK function may be distinct fromthat of ATM but additive in the impact of its loss. For example, thetwo proteins may have different functions in maintenance oftelomere length (18, 38, 39). Further resolution of such issues maybe provided by analyses of cells and mice containing conditionalmutations that inactivate ATM andyor DNA-PK function.

We thank Dr. A. Wynshaw-Boris for providing the ATM1y2 mice usedin the Ku80 breedings. We also thank Dr. Ronald DePinho for criticallyreading this manuscript. This work was supported by National Institutesof Health Grants AI35714 and AI20047 (F.W.A.), CA77563 (Y.X.), andAI01428 (K.M.F.). J.S. is the Richard D. Frisbee III Foundation Fellowof the Leukemia and Lymphoma Society, and Y.G. was an associate andF.W.A is an Investigator of the Howard Hughes Medical Institute.

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