Artemis-independent functions of DNA-dependent kinase in Ig heavy chain class switch recombination and development

Sean Rooney*, Frederick W. Alt†, JoAnn Sekiguchi‡, and John P. Manis§

Howard Hughes Medical Institute, Children’s Hospital, CBR Institute for Biomedical Research, and Department of Genetics, Harvard Medical School, Boston, MA 02115

Contributed by Frederick W. Alt, December 30, 2004 Assembly of Ig in B lineage cells involves two distinct DNA V(D)J coding hairpins and also appears to function similarly rearrangements. In early development, site-specific double in end-processing in general DNA DSB repair (4). strand breaks (DSBs) at germ-line V, D, and J segments are Ig heavy chain (IgH) class switch recombination (CSR) allows joined via nonhomologous end-joining (NHEJ) to form variable mature IgMϩ B cells to change the class (isotype) and effector region exons. Activated mature B cells can change expressed Ig function of their IgH through replacement of the C␮ constant heavy chain constant region exons by class switch recombination region with one of several downstream IgH constant region (CH) (CSR), which also involves DSB intermediates. Absence of any exons (5, 6). CSR employs a recombination͞deletion event that known NHEJ factor severely impairs joining of cleaved V, D, and J links two participating switch (S) regions, which are repetitive segments. In NHEJ, DNA-dependent protein kinase (DNA-PK), sequences that lie 5Ј of individual sets of CH exons. Like V(D)J which is comprised of the Ku70͞80 end-binding heterodimer and recombination, CSR appears to proceed through DSB interme- the catalytic subunit (DNA-PKcs), activates Artemis to generate a diates (reviewed in ref. 5). CSR requires activation-induced nuclease that processes DSBs before ligation. Because inactivation cytidine deaminase (AID) (7, 8), which deaminates cytidines to of DNA-PKcs components also severely affects CSR, we tested uridines within S regions in a transcription-dependent fashion, whether DNA-PK also functions in CSR via activation of Artemis. To which can lead to DSBs (reviewed in ref. 5). AID-induced obviate the requirement for V(D)J recombination, we generated S-region DSBs include staggered-end intermediates (9, 10), DNA-PKcs- and Artemis-deficient B cells that harbored preas- which might require end-processing activities for resolution (11). sembled Ig heavy chain and ␬-light chain ‘‘knock-in’’ (HL) alleles. NHEJ has been implicated in the joining phase of CSR based ϩ We found that Artemis-deficient HL B cells undergo robust CSR, on lack of CSR in Ku-deficient mice in which the mature IgM indicating that DNA-PKcs functions in CSR via an Artemis-indepen- B cell population was reconstituted with knock-in IgH and Ig dent mechanism. To further elucidate potential Artemis-indepen- light chain alleles with preassembled variable region exons [IgH ␬ dent functions of DNA-PKcs, we asked whether the embryonic and -light chain knock-in (HL)-reconstituted] (12–14). How- ͞ lethality associated with double-deficiency for DNA-PKcs and the ever, HL-reconstituted DNA-PKcs-null (DP-T HL) B cells are related ataxia-telangiectasia-mutated (ATM) kinase was also defective for CSR to all IgH isotypes except IgG1, despite a lack observed in mice doubly deficient for ATM and Artemis. We found of growth defects (15), whereas HL-reconstituted severe com- that ATM͞Artemis double-deficient mice were viable and born bined immunodeficiency (DNA-PKcs kinase-inactive) B cells in normal Mendelian numbers. Therefore, we conclude that also show CSR defects, albeit more modest ones (16, 17). Based DNA-PKcs has Artemis-independent functions in CSR and normal on these findings, one might speculate that the DNA-PK ho- development. loenzyme plays a similar role in CSR as it does in V(D)J recombination. In this scenario, some, but not all, CSR DSBs ͞ ATM ͉ DNA-PKcs might require the DNA-PKcs Artemis processing activities, leading to less severe CSR defects in DNA-PKcs-deficient mice than in Ku-deficient mice. On the other hand, DNA-PKcs may recursor B and T cells assemble antigen receptor genes have Artemis-independent functions in NHEJ, CSR, and other Pfrom component, germ-line V, D, and J segments via processes. In this regard, DNA-PKcs-deficiency has a modest V(D)J recombination (1). V(D)J recombination is initiated by impact on the fidelity of RS joins, whereas Artemis deficiency ͞ the lymphoid-specific RAG1 2 endonuclease (RAG), which does not (18, 19). Although the nature of this DNA-PKcs RS introduces DNA double strand breaks (DSBs) between V(D)J joining function is unknown, it might reflect in vitro ability of coding sequences and flanking recombination signal (RS) DNA-PKcs to synapse or protect DNA ends (20–22). sequences. RAG cleavage generates two distinct intermedi- DNA-PKcs belongs to a family of known as PI3- ates: hairpin coding ends and blunt RS ends, which are then kinase-like kinases, which include ataxia-telangiectasia-mutated fused via the nonhomologous end-joining (NHEJ) pathway of (ATM) protein (23). ATM is activated rapidly upon generation DSB repair (2). There are six known mammalian NHEJ factors (2). XRCC4 and DNA-ligase IV function as a ligation com- plex, whereas Ku70 and Ku80 form the Ku DNA end-binding Abbreviations: ATM, ataxia-telangiectasia-mutated; CSR, class switch recombination; DNA- heterodimer, which associates with DNA-dependent protein PK, DNA-dependent protein kinase; DNA-PKcs, DNA-PK catalytic subunit; DP-T͞HL, HL- reconstituted DNA-PKcs-null; DSB, double strand break; HL, IgH and ␬-light chain knock-in; kinase catalytic subunit (DNA-PKcs) to form the DNA- IgH, Ig heavy chain; NHEJ, nonhomologous end-joining; RS, recombination signal; S, switch. dependent protein kinase (DNA-PK). DNA-PK, in turn, as- *Present address: Laboratory for Cell Biology and Genetics, The Rockefeller University, New sociates with Artemis and activates its endonuclease activity York, NY 10021. (3). XRCC4, ligase IV, Ku70, and Ku80 are evolutionarily †To whom correspondence should be addressed. E-mail: [email protected]. conserved and are required to repair all DSBs joined by NHEJ, ‡Present address: Department of Internal Medicine, University of Michigan, Ann Arbor, MI including coding and RS ends. In contrast, DNA-PKcs and 48109. Artemis are relatively dispensable for RS joining but required §Present address: Joint Program in Transfusion Medicine, Children’s Hospital, Department

for coding joining. In this context, the DNA-PKcs-activated of Pathology, Harvard Medical School, Boston, MA 02115. IMMUNOLOGY Artemis endonuclease activity is required for processing © 2005 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0409857102 PNAS ͉ February 15, 2005 ͉ vol. 102 ͉ no. 7 ͉ 2471–2475 Downloaded by guest on September 24, 2021 Table 1. Mice doubly-deficient for ATM and Artemis are viable Genotype ATMϩ͞ϩ ATMϩ͞y ATMy͞y

Artϩ͞ϩ 8 (4) 7 (5) 2 (4) Artϩ͞N 8 (9) 22 (17) 9 (9) ArtN͞N 3 (8) 13 (10) 5 (8)

The progeny yielded from Artϩ͞N ATMϩ͞y ϫ Artϩ͞N ATMϩ͞y and ArtN͞N ATMϩ͞y ϫ ArtN͞N ATMϩ͞y breedings. The numbers listed represent the actual number of pups observed, and the numbers in parentheses represent the numbers theoretically expected for Mendelian segregation of the various genotypes. Fig. 1. Reconstituted B cells in ArtN/NHL mice. Single-cell suspensions from 8-week-old ArtN/NHL, DP-T͞HL, and WT͞HL mice were stained for surface of a DSB and then functions to activate downstream DSB expression of B220 and IgM, and analyzed by FACS. Percentage of B220͞IgM response proteins that effect cell-cycle checkpoints and DNA double-positive lymphocytes is depicted in the upper right corner of the FACS plot. repair (24). Several DSB response proteins that are ATM substrates, including H2AX and 53BP1, are required for CSR and may function at the level of S-region synapsis (13, 25–27). In mice. Cells from day-4 cultures were washed twice in PBS͞2% this regard, ATM-deficient mice (29, 30) and humans (28) also FCS and stained with various conjugated with fluo- have a defect in CSR, but these defects appear more modest than rescein (IgM, IgG1, IgG2b, and Ig␬), phycoerythrin (IgM, IgG3, that of H2AX- or 53BP1-deficient mice, perhaps reflecting the and Ig␭), or CyChrome (B220) (Pharmingen). The cells were redundancy of other PI3-kinase-like kinases in the DSB response analyzed by using a FACSCalibur (BD Biosciences), and data (25, 29). Inactivation of ATM is synergistically lethal with were interpreted by using FLOWJO software (Tree Star, Ashland, DNA-PKcs-deficiency (31, 32) but not with DNA ligase IV OR). The FACS profiles representing 10,000 events were gated deficiency (31), arguing that overlapping developmental func- for live, lymphoid cells as determined by forward scatter versus tions of ATM and DNA-PKcs may lie outside of NHEJ per se. To further define the role of DNA-PKcs in CSR and normal side scatter. development, we now compare the requirement for Artemis ELISA. ELISA to detect IgM, the various IgG isotypes, and IgA versus DNA-PKcs in CSR in HL-reconstituted mice. To provide ͞ further insight into DNA-PKcs functions, we test whether Ar- in serum and or in culture supernatants was done as described temis also has synergistic function with ATM in normal devel- in ref. 15. For all analyses, we used isotype-specific antibodies opment. Our findings demonstrate Artemis-independent DNA- purchased from Southern Biotechnology Associates. Mice PKcs functions in CSR and normal development. ranged in age from 6–12 weeks, and culture supernatants were assayed after 5 days of stimulation. Methods Generation of ArtN/NHL, DP-T͞HL, and ArtN//N͞ATMy/y Mice. The Nucleotide Sequence Analyses. Splenic B cells from homozygous ArtN/NHL and DP-T͞HL mice were made by a three-way cross ArtN/NHL mice were activated with anti-CD40 plus IL-4 for 4 of mice containing a productively rearranged B1–8 VH(D)JH days, and genomic DNA was prepared by using standard tech- variable region exon inserted (‘‘knocked-in’’) into the endoge- niques. S␮–S␥1 junctions were PCR-amplified as described in nous JH (34), with mice containing a ␬3–83 V␬J␬ variable ref. 37. At least two independent reactions were used to obtain region exon knocked-in to the J␬ region of the Igk locus (33), and reproducible products that hybridized with the S␥1 probe. The with ArtN/N (18) or DP-T mice (33). The various genotypes were products were cloned into the Topo-TA (Invitrogen), sequenced, identified by Southern blotting and PCR analyses of tail-derived and analyzed by using SEQUENCHER (Gene Codes, Ann Arbor, genomic DNA as described in refs. 34 and 35. ArtN/NHL and MI) software and the NCBI database. DP-T͞HL mice were crossed on a 129SvEvTac background for at least six generations to allow comparison in the same genetic Results N/N background. Analyses of the Art HL mice were performed on Knock-In IgH and Ig Light Chain Alleles Rescue B Cell Development in progeny derived from Artemis-deficient mothers to exclude Artemis-Deficient Mice. To assay for potential roles of Artemis in transplacental transfer of lymphocytes or Ig. To elucidate po- CSR, we crossed ArtN/N mice with B1–8-HC (H) (34) and tential synergistic effects of Artemis- and ATM-deficiency, we N/N y 3–83k-LC (L) (33) knock-in mice to generate Art HL mice on used the previously described mice harboring the ATM knock- a 129S6͞SvEvTac mouse strain background. The H and L alleles ϩ/N͞ ϩ/Y out allele (36). For the breedings, either Art ATM or contain preassembled IgH and ␬ Ig light chain variable region ArtN/NATMϩ/Y mice were intercrossed and progeny were ana- gene exons, which obviate the need for V(D)J recombination to lyzed for Artemis and ATM genotypes (see Table 1). generate mature B lymphocytes. To exclude any transplacental transfer of maternal Ig or mature lymphocytes, mouse breeding Splenic B Cell Cultures. Single-cell suspensions of spleen cells were N/N N/N ϫ 5 ϫ 6 strategies with Art HL mothers or Art mothers were used cultured at 5 10 or 1 10 cells per ml in RPMI medium ͞ supplemented with 10% FCS and either 25 ng͞ml LPS (to assay for to generate all of the Artemis-deficient HL mice for these switching to IgG2a, IgG3, and IgG2b) or anti-CD40 (Pharmingen) studies. As expected, the knock-in H and L alleles did not rescue ͞ ͞ T cell development in ArtN/N mice, because we detected no using 500 ng ml with 25 ng ml mouse recombinant IL-4 ϩ ϩ (R & D Systems) (for CSR to IgG1 and IgE) as described in ref. 15. substantial numbers of CD4 or CD8 cells in the spleens of six N/N Cultured cells were maintained daily at a density of 1 ϫ 106 per ml. independent Art HL mice (data not shown). In contrast to Cells were sampled on various days for flow cytometry analyses, ArtN/N mice, which lack mature B cells because of their V(D)J DNA was prepared, and culture supernatants were harvested for recombination defect (18), 5- to 8-week-old ArtN/NHL mice had ϩ ϩ ELISA analysis as described in ref. 15. substantial populations of B220 ͞IgM mature B cells in their spleens as detected by FACS analyses (Fig. 1). The absolute Flow Cytometry Analysis. Single-cell suspensions from spleen were numbers of splenic B cells in ArtN/NHL mice approached those prepared according to standard methods from 6- to 10-week-old of Artϩ/ϩHL or Artϩ/NHL mice (WT͞HL), with equivalent

2472 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0409857102 Rooney et al. Downloaded by guest on September 24, 2021 surface IgM expression on Artϩ/NHL and ArtN/NHL B cells (Fig. 1 and data not shown).

Normal CSR in ArtN/NHL B Cells. To determine whether ArtN/NHL B cells were capable of undergoing CSR in vivo, we used ELISA to measure the concentrations of the various IgH isotypes in sera from mice ranging in age from 6 to 8 weeks of age. All IgG isotypes and IgA were detected in serum isolated from ArtN/NHL mice, although we observed variability in the actual amount of antibody among individual mice (data not shown). Both the variability and the reduced concentration of serum IgH isotypes has been observed previously in other models in which Ig knock-in alleles were used to rescue V(D)J recombination defects (12, 16, 17, 38) and may reflect a lack of substantial numbers of peripheral T cells. In addition, serum levels of IgH isotypes are influenced by additional factors including mouse age and infection status (6). Therefore, to more directly look for potential CSR defects, we assayed purified B cells from the various lines for ability to undergo CSR in vitro. To directly compare CSR in the context of Artemis-deficiency versus DNA-PKcs-deficiency, we backcrossed our original DP- T͞HL mice (which were on a mixed C57B6͞129͞OLA back- ground) onto the same 129S6͞SvEvTac mice (DP-T͞HL͞129) as the ArtN/NHL mice. For in vitro studies, B220ϩ͞CD43Ϫ mature splenic B cells were purified from ArtN/NHL and DP-T͞HL͞129 mice, as well as from ArtN/ϩHL and DPϩ͞T HL control mice, and stimulated in vitro either with LPS to stimulate CSR to IgG3 and IgG2b or anti-CD40 plus IL-4 to stimulate CSR to IgG1 and IgE. Trypan-blue-excluded live cells were counted on days 2, 3, and 4 of culture, with no significant differences in the total number of live cells observed for any of the three genotypes, either in LPS or anti-CD40 plus IL-4 conditions (LPS, P ϭ 0.23; Fig. 2. IgH class switching to IgG1 or IgG2b is unaffected by Artemis- anti-CD40 plus IL-4, P ϭ 0.18; for comparison of WT and deficiency. Purified splenic B cells were activated in vitro for 4.5 days with ArtN/NHL). These findings indicate lack of a major proliferation anti-CD40 plus IL-4 (A) to assay switching to IgG1 (Upper) or with LPS (L) to defect in Artemis- and DNA-PKcs-deficient B cells after stim- assay switching to IgG2b (Lower). The percentage of IgG1- or IgG2b-positive ulation for CSR. cells that also stained for B220 is shown in the upper right corner of each FACS In-vitro-activated B cells were harvested on day 4 or 5 of plot. culture and analyzed by FACS for surface IgH isotype expres- sion. Robust switching was observed in ArtN/NHL and ArtN/ϩHL N/N B cells, with no obvious difference from WT-HL with respect to Art HL, and the resultant sequences were compared with the percentage of B cells that switched to IgG1, IgG3, or IgG2b germ-line sequences deposited in the NCBI public database, as (Fig. 2 and data not shown). Preliminary assays also indicated well as those obtained from previously published studies (15). switching to IgE (data not shown). By comparison, DP-T͞HL͞ Most junctions were joined either directly (6͞13) or with 1 or 2 129SvEv B cells switched only to IgG1 (Fig. 2 and data not nucleotide homology overlap (average overlap ϭ 1.2 nt; Fig. 4, shown), consistent with our previous studies of C57B6͞129͞ which is published as supporting information on the PNAS web OLA DP-T͞HL mice (15). We also measured secreted IgH- isotype concentrations in supernatants from each splenic B cell culture. No substantial difference was observed in the amount of each IgH isotype produced by ArtN/NHL compared with WT͞HL control cultures (Fig. 3). On the other hand, no IgH isotype except IgG1 was detected in supernatants from DP-T͞HL͞129 B cells cultures, consistent with our earlier findings of CSR in DP-T͞HL mice on a mixed background (Fig. 3) (15). Together, these findings indicate that there is no substantial block for CSR in vitro in ArtN/NHL B cells, in contrast to the major defect of DP-T͞HL B cells to switch to any IgH isotypes except IgG1.

Normal Processing of S␮–S␥1 Switch Junctions in IgG1؉ ArtN/NHL B Cells. Analysis of the sequence composition of S-region junctions can provide insights into the mechanistic pathways used to resolve CSR-associated breaks. For example, CSR junctions from B cells deficient for the mismatch-repair factors Mlh1 or Pms2 have longer stretches of sequence microhomology (37, 39–41). To evaluate the sequence at CSR junctions formed in Fig. 3. Normal secretion of different IgG isotypes in activated ArtN/N͞HL B the absence of Artemis, DNA was isolated from day-4 cultures cells. Supernatants from cultures of stimulated splenic B cells from the indi- stimulated with anti-CD40 plus IL-4, and S␮–S␥1 junctions were cated sources were analyzed by ELISA for secreted IgM and IgG isotypes at day amplified by PCR, cloned into a plasmid vector without size 5 after treatment in vitro. Circles, WT͞HL; triangles, ArtN/N͞HL; squares, IMMUNOLOGY selection, and sequenced. Thirteen junctions were obtained from DP-T͞HL.

Rooney et al. PNAS ͉ February 15, 2005 ͉ vol. 102 ͉ no. 7 ͉ 2473 Downloaded by guest on September 24, 2021 site), similar to the junctions in normal mice (15). Moreover, have an Artemis-independent NHEJ function in CSR and other there was no obvious alteration of nucleotide substitutions, processes, for example, by acting as scaffold for the XRCC4͞ mutation load, or general location of S-region junctions relative ligase IV complex (20, 48–52). In addition, DNA-PK also may to the constant region genes (data not shown). These findings have functions outside of NHEJ in CSR (15, 16). indicate that Artemis-deficiency does not alter substantially the How might DNA-PK function outside of NHEJ in CSR? CSR nucleotide sequence pattern of S-region junctions. relies on cellular DSB-response factors, such as H2AX and 53BP1, for its joining phase (13, 25). In this regard, DNA-PKcs, and related Artemis and ATM Double-Deficient Mice Are Born in a Mendelian PI3-kinase-like kinases, initiate the DSB response by phosphory- Fashion. The profound defect in CSR in DNA-PKcs-deficient B lating these factors (26, 53). Thus, a potential DNA-PKcs function cells, as compared with Artemis-deficient B cells, indicates that in CSR, not directly related to NHEJ, may be in the activation of DNA-PKcs has functions in CSR that are independent of the DSB response. However, DNA-PKcs kinase activity is not Artemis. To further elucidate potential Artemis-independent absolutely required for CSR, as evidenced by CSR (at reduced functions of DNA-PKcs, we asked whether DNA-PKcs- and levels) to all isotypes in mice homozygous for the scid mutation, Artemis-deficiency were similar in the context of ATM- which generates a kinase-null form of DNA-PKcs (16, 17, 54). One deficiency. For this purpose, we crossed ArtN/N mice to ATM- interpretation of the latter finding, when compared with the more deficient mice (ATMy/y mice) (36). In contrast to the embryonic dramatic effects of the complete DNA-PKcs knockout on CSR, is lethality associated with DNA-PKcs͞ATM-deficiency, ArtN/N͞ that DNA-PKcs may have a CSR function independent of kinase ATMy/y mice were born alive, and (based on limited numbers of activity. In this regard, DNA-PKcs has been suggested to synapse breedings) at close to the expected frequency (Table 1). Fur- DNA ends based on in vitro studies (20–22), although there is no thermore, the ArtN/N͞ATMy/y mice appeared healthy, and some direct in vivo evidence for such a role. However, indirect support for lived for up to 9 months with no gross phenotypic defects, other a DNA-PKcs role in the synapsis portion of the CSR reaction came than immunodeficiency due to impaired NHEJ and smaller size from the finding that intra-S␮-region deletions occur normally in associated with ATM deficiency (data not shown). These find- DP-T͞HL B cells (15). Finally, Ku and DNA-PKcs may have ings indicate that the synergistic lethality of the ATM- and additional functions in regulation of apoptosis and telomere main- DNA-PKcs-deficiencies does not involve Artemis-related func- tenance (55, 56). Thus, until potential CSR roles of XRCC4 or tions of DNA-PKcs. ligase IV, which are not known to have roles outside of NHEJ, are assessed, evidence that classical NHEJ is fully responsible for repair Discussion of DSBs during CSR remains indirect. IgH CSR has been assumed to involve DNA DSBs as interme- diates and to employ the NHEJ pathway for their resolution. For Differential Effects of Artemis-Deficiency Versus DNA-PKcs-Deficiency NHEJ, in general, and V(D)J recombination, in particular, in the Context of ATM-Deficiency. DSBs activate cell-cycle check- Artemis functions downstream of DNA-PK (4). Because all points to prevent unrepaired lesions from being carried into the three DNA-PK components are required for normal CSR (5), it next phase of the cell cycle (23), and ATM is central to activation was reasonable to assume that Artemis may also play a major role ͞ of the DSB response. Notably, inactivation of any of the in the generation and or processing of CSR-associated DNA DNA-PK holoenzyme components in mice results in synergistic breaks via its DNA-PK-activated endonuclease and exonuclease lethality with ATM-deficiency (32). In contrast, we have found activities (42, 43). Yet, we found that Artemis is not required for that mice with combined deficiency for Artemis and ATM are efficient CSR. Thus, if end-processing is required for CSR, the viable, with some living up to at least 9 months. In this regard, dispensability of Artemis for the process suggests that such recent studies suggest that Artemis may, in fact, be epistatic to activities may be provided by alternative factors or pathways (44, ATM in the IR-induced ATM signaling pathway (47, 57, 58). The 45). On the other hand, Artemis is not required for the NHEJ- viability of Artemis͞ATM double-deficient mice suggests that mediated ligation of DNA ends that do not require processing, synergistic function of DNA-PKcs with ATM in preventing such as blunt RS ends in V(D)J recombination (46) or noncom- embryonic lethality does not involve Artemis. Moreover, the plex ends generated by etoposide (47). Therefore, a major role finding that DNA-PKcs deficiency, but not Artemis-deficiency, for NHEJ in CSR still could be rationalized if most S-region synergizes with ATM-deficiency in generating embryonic lethal- DSBs do not require processing. In this regard, our studies do not ity provides additional evidence that DNA-PKcs has functions rule out some role for Artemis in CSR. For example, if the blunt separable from those of Artemis in processes other than CSR. DNA ends that constitute a subset of S-region DNA breaks (10) Some of these synergistic functions may lie outside of NHEJ. are efficiently joined, they might contribute to relatively normal Thus, ATM-deficiency actually rescues the neuronal apoptosis cellular CSR levels. Finally, although classic NHEJ has been strongly implicated in CSR, as discussed in more detail below, it and embryonic lethality associated with ligase-IV-deficiency remains possible that other repair pathways function in CSR and (32), indicating that it is not loss of NHEJ per se that leads to the that DNA-PK may have roles other than, or in addition to, synergistic lethality of ATM and DNA-PK deficiencies. The potential roles in NHEJ. finding that this synergism does not appear to involve Artemis supports this notion. As speculated previously (32), synergistic Potential Roles of DNA-PKcs in CSR. In contrast to Artemis, all developmental effects of ATM and DNA-PKcs may involve DNA-PK components (Ku70͞80 and DNA-PKcs) are required various functions, ranging from activation of the DSB response for normal CSR. Ku70- and Ku80-deficient B cells fail to switch to potential roles in telomere maintenance. It will be of interest detectably to any IgH isotype (12, 14), whereas DP-T͞HL B cells to determine whether such DNA-PKcs functions overlap with are defective in CSR to all but IgG1 (15). To be certain that the the role of DNA-PKcs in CSR. differential impairment of CSR observed for DNA-PKcs- We thank Y. Fujiwara, T. Borjeson, S. Whitlow, and L. Kaylor for deficient versus Artemis-deficient mice was not due to a strain- ␬ specific modifier, we backcrossed DP-T͞HL mice into the same technical assistance, K. Rajewsky for providing the B1–8 and 3–83 N/N ͞ knock-in mouse lines, and, especially, Drs. M. Lieber, A. Nussenzweig, genetic background as the Art HL mice (129 SvEv). Our and K. Rajewsky for critical review of the manuscript. This work was analyses of these mice confirmed that DNA-PKcs-deficiency still supported by National Institutes of Health Grants AI31541 and AI35714 resulted in a severely impaired CSR to all IgH isotypes except (to F.W.A.). J.P.M. was supported by a Lymphoma Research Foundation IgG1. Therefore, we conclude that DNA-PKcs functions inde- Fellowship. J.S. was a Special Fellow of the Leukemia and Lymphoma pendently of Artemis in CSR. In this regard, DNA-PK might Society. F.W.A. is an Investigator of Howard Hughes Medical Institute.

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