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Jan 24, 2008 - RAG1/RAG2 · AID · DNA damage response · Non-homologous end-joining · Alternative ..... moter regions that drive germline transcription. ...... Pan-Hammarstrom Q, Dai S, Zhao Y, van Dijk-Hard IF, Gatti RA, Borresen-Dale AL ...
Immunol Res (2008) 41:103–122 DOI 10.1007/s12026-008-8015-3

DNA damage and repair during lymphoid development: antigen receptor diversity, genomic integrity and lymphomagenesis Nahum Puebla-Osorio · Chengming Zhu

Published online: 24 January 2008 © Humana Press Inc. 2008

Abstract Lymphocyte maturation requires generation of a large diversity of antigen receptors, which involves somatic rearrangements at the antigen receptor genes in a process termed V(D)J recombination. Upon encountering speciWc antigens, B-lymphocytes undergo rearrangements in the constant region of the immunoglobulin genes to optimize immune responses in a process called class switch recombination. Activated B-cells also undergo somatic hypermutation in the variable regions of the immunoglobulin genes to enhance their antigenic aYnity. These somatic events are initiated by the inXiction of DNA lesions within the antigen receptor genes that are strictly conWned to a speciWc developmental window and cell-cycle stage. DNA lesions are then repaired by one of the general DNA repair mechanisms, such as non-homologous end-joining. Mutations in key factors of these pathways lead to the interruption of these processes and immunodeWciency, making it possible to study the mechanisms of cellular response to DNA lesions and their repair. This review brieXy summarizes some of the recently developed animal models with focus on current advances in the understanding of the mechanism of DNA end-joining activities, and its role in the maintenance of genomic stability and the prevention of tumorigenesis. Keywords V(D)J recombination · Class switch recombination · Somatic hypermutation · RAG1/RAG2 · AID · DNA damage response · Non-homologous end-joining · Alternative end-joining · Genomic instability · Lymphoma

N. Puebla-Osorio · C. Zhu (&) Department of Immunology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77054, USA e-mail: [email protected] C. Zhu The University of Texas Graduate School of Biomedical Science at Houston, Houston, TX 77030, USA

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Introduction: DNA lesions during lymphocyte development Adaptive immunity requires a largely diversiWed repertoire of antigen receptors expressed on the surface of T- and B-lymphocytes to speciWcally recognize a wide spectrum of antigens the body might encounter. In order to generate such diversity, exons encoding variable regions of antigen receptors, critical for antigen recognition and binding are assembled via somatic rearrangement of the variable (V), diversity (D), and joining (J) gene segments in a process known as V(D)J recombination (Fig. 1a) [1]. This germline rearrangement event takes place during an early stage of lymphocyte development and could generate up to 106 diVerent types of combinations. In addition, nucleotide insertions and deletions often occur at V(D)J junctions. Thus, in theory, V(D)J recombination is capable of generating up to 1011 diVerent types of antigen receptors. V(D)J recombination is initiated by two lymphoid speciWc factors: recombination activating genes 1 and 2 (RAG1 and RAG2, Fig. 1a) [2, 3]. These two proteins recognize and bind the recombination signal sequences (RSSs), which Xank each coding segment. A RSS is comprised by a highly conserved heptamer and nonamer sequences separated by a spacer sequence of 12 or 23 base pairs in length. The RAG proteins form a synaptic complex between the two diVerent RSSs and mediate recombination only between antigen receptor gene segments containing a 12-RSS and a 23-RSS, referred to as the 12/23 rule (Fig. 1b) [1, 4, 5]. After forming a synaptic complex with two RSSs, RAG1/2 introduces a nick precisely at the border between an RSS and the coding segment, followed by a trans-esteriWcation reaction that produces two kinds of DNA double-strand breaks (DSBs): a blunt, 5⬘phosphorylated end that contains the entire RSS (signal end) and a covalently sealed hairpin end containing the coding sequences (coding end, Fig. 1c) [6, 7]. Both RAG1 and RAG2 are absolutely required for V(D)J recombination. Targeted inactivation or natural mutation of either one of the RAG proteins is translated into a severe combined immunodeWciency (SCID) and a complete abrogation of development in both T- and B-lymphocytes due to lack of initiation of V(D)J recombination [8–10]. RAG-liberated DSBs at RSSs are intermediates of V(D)J recombination. Signal ends are readily detectable in wild-type mouse thymocytes and bone marrow cells [6, 11–14]. These ends are slowly joined due to their continuous association with RAG1 and RAG2. In contrast, coding ends are quickly

RAGs

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D

J

NHEJ

OH

a

b

c

Fig. 1 V(D)J recombination. (a) Germline rearrangements at the V, D and J segments; (b) V(D)J recombination is initiated by RAG proteins binding a pair of 12- and 23-RSSs (empty and Wlled triagles) then cleaving DNA double-strands at the RSS; and completed by joining of DSBs by NHEJ; (c) Biochemistry of RAG cleavage: initiates with the introduction of a nick followed by nucleophilic attack of the opposite strand by the OH group to form a hairpin coding end and a blunt, 5-phosphorylate signal end

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processed and joined, as indicated by the absence of these ends in DNA from wild-type thymocytes and bone marrow cells. Upon encountering the corresponding antigen, B-lymphocytes proliferate extensively and undergo two types of antigen receptor modiWcations in the immunoglobulin (Ig) genes, known as class switch recombination (CSR) and somatic hypermutation (SHM). CSR allows activated B-lymphocytes to change the constant region of an Ig heavy chain (CH) from the initially expressed  heavy chain (C) to one of the several downstream constant gene segments (C, C, or C). CSR occurs between two switch regions (S region) that are 5⬘ of each CH exons. A typical S region contains 1–10 kb in length with short, repetitive, high G-C content sequences. The CSR process replaces the initial C segment with a downstream CH region by excising the interval sequences. Therefore, CSR changes the eVector functions of the antibodies without altering their antigenic speciWcity (Fig. 2a) [15–18], which is determined by V(D)J recombination at the variable region. In contrast to CSR, SHM mutates the variable regions of the antigen receptor genes to generate immunoglobulins with higher aYnity to the speciWc antigen after proliferation and selection [19, 20]. Both types of somatic modiWcations of genomic DNA are critical for the generation of an eYcient immune response against any speciWc antigen. In many ways reminiscent of the role of RAG proteins in the initiation of V(D)J recombination by generating DSBs at the RSS, activation-induced cytidine deaminase (AID) is required to initiate CSR and SHM [21, 22]. Targeted inactivation or mutation of AID leads to a complete absence of both CSR and SHM [23, 24]. AID is a member of the APOBEC family of cytidine deaminases, which are known RNA editing enzymes. However, genetic and biochemical evidence demonstrates that AID catalyzes the deamination at the cytidine residues on single-stranded DNA substrates [25–27]. A current model proposes that CSR is initiated with germline transcription starting from promoters upstream of an individual S region, followed by transcription through the S region and the downstream C regions. This germline transcript was originally proposed to render the S region accessible for recombination. Recent evidence suggests that germline transcriptions mechanistically participate in CSR by assisting the targeting of AID to the S region [18]. The transcription through the repetitive G-C rich S region leads to the generation of stable DNA–RNA hybrid structures termed R-loops. This unique high order structure exposes G-rich non-template single strand regions, which makes them a preferred

VDJ

Eµ SµCµ

Sγ

Sε Sα

5’Eα C G

G C

U G

G U

G G

G

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+ c

Fig. 2 Class switch recombination. (a) Germline rearrangements at the switch regions (to IgA in this case); (b) R-loops formation by transcription (curved lines), the AID deamination leads to nicks on both strands followed by the DNA repair machinery, DSBs are repaired by classic NHEJ to complete the rearrangements; (c) A current model for AID action: from deamination to double-strand breaks and end-joining

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substrate for AID mediated DNA deamination [27–29]. This model brings together three key elements: S region structure, germline transcription, and AID targeting [18] into one functional entity—initiation of CSR. Similarly, transcription through the variable region of the Ig heavy and light chains allows AID access to these regions in order to initiate SHM [20], but the stabilization of single strand DNA substrates is established via an AID-replication protein A (RPA) complex instead of R-loop formation [30]. Unlike V(D)J recombination, which is directed by the RSS, CSR and SHM are regionally directed as AID lacks a speciWc recognition site. However, a preferred target for AID is the “hotspot” sequence of RGYW (R = purine, Y = pyrimidine, W = T or A, [30], or its reverse complementary sequence WRCY or WRC [31]. The main function of AID is to convert the base cytosine (C) to uracil (U) after targeting into ssDNA substrates of S regions during the CSR initiation. This leads to a uracil to guanine mismatch recognizable by the base excision repair (BER) enzyme uracil DNA glycosylase (UNG) [32]. UNG catalyzes uracil base excision reaction followed by the apurine/apyrimidine endonuclease (APE1) cleavage to generate a nick on one DNA strand [25]. AID is also able to deaminate dC on the opposite strand [33, 34]. Two nicks on opposite strands and in close proximity result in a DSB within the S region (Fig. 2b). Alternatively, U-G mismatches may also be recognized by the mismatch repair (MMR) machinery, namely by the Msh2/Msh6 complex, followed by MMR proteins such as Pms2/Mlh1 and Exo 1 to generate nicks or gaps around the mismatch sites [25, 35]. Consistent with this model, deWciency of UNG or MMR components impairs CSR and SHM at the DNA cleavage step [33, 35–38]. CSR replaces the C exon with one of the downstream CH exons and juxtaposes the downstream CH to the rearranged V(D)J exons by deleting the intervallic sequences. In such DNA deletion processes, DSBs are expected intermediates. Indeed, DSBs were detected at the switch regions as intermediates of CSR at corresponding S regions by ligation-mediated PCR (LM-PCR) [39, 40] in an AIDdependent manner (Fig. 2c) [41]. In addition, phosphorylation of H2AX (H2AX) was detected at the S regions in cells actively undergoing CSR in an AID-dependent manner [42]. Finally, the role of AID in initiating CSR by introducing DSB was conWrmed in a recent observation of CSR products mediated by I-Sce1-induced DSBs in the absence of S regions or AID expression using a mutant mouse model with replaced S regions with I-Sec1 sites [43]. During SHM, the R-loop structure is not observed, but similar germline transcripts are found throughout the variable regions of the immunoglobulin genes. Transcription through the variable regions may generate short single-stranded regions that are in complex with other protein factors [34] like RPA. These factors stabilize the single-strand region as well as recruit AID to the complex [30]. Short ssDNA regions serve as substrates for AID deamination to convert dC to dU at the hotspot RGYW. This leads to transition mutations after one round of replication when dU can be recognized as dT by DNA polymerase [25]. In addition, BER or MMR may recognize and cleave at the dU-dG mismatch site and generate nicks and gaps [44]. The subsequent round of DNA replication randomly inserts a nucleotide at the damage site and leads to base transition and transversion mutations. Furthermore, strand gaps induced by dU-dG mismatch and repair machinery may be Wlled-in by an error-prone DNA polymerase, like DNA polymerase  [45], that leads to mutation at a position distal to the original deamination site. Unlike V(D)J recombination or CSR, which involve DSBs as intermediates, SHM does not require generation of DNA breaks at double-strands [46]. However, DNA single-strand lesions generated by AID or BER/MMR may introduce genomic instability and could be potentially dangerous to the B-lymphocytes.

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Synaptic complex formation before and after strand cleavage The function of RAG1 and RAG2 in V(D)J recombination goes beyond recognizing the RSS and nicking DNA double strands. Accumulated evidence has indicated that the RAG proteins contribute signiWcantly to the protection of DNA ends by preventing non-speciWc ligations and random nuclease degradation. Most importantly, the RAG complex guides the signal and coding ends to the correct repair pathway for end processing and joining to complete V(D)J recombination [47]. A current model describes that immediately upon strand cleavage, RAG1/2 is able to bind to the two nascent signal ends [48] and to a lesser extent, the two hairpin coding ends [49] to form a complex termed the post-cleavage complex. This complex serves at least three functions. First, it protects ends from degradation or nonspeciWc ligation; second, it “masks” ends from activating the extreme cellular damage response, such as the p53-dependent apoptosis; and third, it guides the ends to the proper repair machinery. Mutations in RAG-1 [50–52] and RAG-2 [53] make it possible to separate the cleavage function from the post-cleavage joining function of the RAG complex. Systematic mutations of both RAG proteins revealed a class of RAG mutants impaired only in the joining phase, leaving the cleavage phase relatively unaVected. These mutant RAGs are likely defective in the formation of the post-cleavage complex, which would result in the ability of the ends to “escape” from this complex and become accessible to other endjoining pathways, such as homologous recombination (HR) or an alternative non-homologous end-joining (NHEJ) pathway [54, further discussed below], these pathways are not normally involved in the end-joining during V(D)J recombination. A recent study from the D. Roth group further conWrmed the role of RAG in end-joining. A RAG2 frame-shift mutation, FS361, supports a substantial level of successful V(D)J recombination in DNAPKcs or XRCC4 deWcient cells, which are severely impaired during V(D)J recombination in the presence of wild-type RAGs [55]. These results lead to two conclusions. First, there is an alternative end-joining pathway active in the classic NHEJ deWcient background; second, the wild-type RAG complex “denies” V(D)J recombination-associated ends to be joined by this alternative pathway. Interestingly, this mutant RAG2 even “mislead” RAGgenerated DNA breaks into alternative NHEJ pathway in wild-type cells instead of the classic NHEJ pathway. This indicated that the alternative NHEJ co-exists with the classic NHEJ. These mutant RAG studies highlight the importance of RAG proteins in post strandcleavage: the RAG1/2 complex functions not only by holding ends in close proximity but also actively guiding the ends to the proper DNA repair machinery. This “guidance” is critical for protecting genomic integrity and suppressing oncogenic translocation. This property of RAG1/2 makes V(D)J recombination a distinctive process from general DNA end-joining mechanism, where randomly damaged DNA relies on the activation of a proper DNA damage response. CSR is an activation induced DNA rearrangement process that occurs at speciWc regions on the chromosome, which in many ways resembles V(D)J recombination. However, there is still an open question on whether there is formation of synaptic complex to promote coupled DNA cleavage and post-cleavage complex that holds DNA ends to facilitate joining during CSR. An equivalent of the 12–23 rule of V(D)J recombination does not exist in CSR. To successfully cleave and join the two S regions separated by a region of DNA up to 150 kb, some forms of communication must be established between the two S regions. Such communication was recently reported by using chromosome conformation capture (3C) technique. A. Kenter and colleagues were able to detect a long range interaction (LRI) between the 5⬘ enhancer (E) and 3⬘ downstream locus control region 3⬘E (Fig. 2a) [56]. This LRI, unique to B cells, leads to the formation of a loop structure on the chromosome.

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Upon activation of B cells, this E-to-3⬘E complex is able to further interact with promoter regions that drive germline transcription. This interaction brings downstream S regions to a closer proximity to the S, forming a complex similar to the V(D)J synaptic complex that brings two RSSs together. Interestingly, AID is able to stabilize such a complex and initiate CSR by deamination. However, unlike the ability of RAGs to initiate V(D)J recombination with coupled cleavage at the two RSSs, initiation of CSR by AID does not appear to require coordination between the two S regions. AID-dependent deletions and mutations are often observed within the S region [57, 58], but much less frequently in the downstream S regions. This suggests that cleavage might not be necessarily coupled at the two S regions. Consistent with this, analyses of targeted-mutant mice that had a single replacement at the downstream S region with I-Sce1 restriction sites revealed that a single I-Sce1-mediated DSB is capable of joining to an AID-mediated DSB [43]. This further supports the notion that strand cleavage in CSR is not necessarily coupled. However, the E-to-3⬘E LRI loop, assuming it is still formed in the B cells of these mutant mice with I-Sce1 replacement, may be suYcient to hold the two DSBs in a spatial proximity and support certain level of end-joining. Activated B-cells from mutant mice with both S and a downstream S region replaced by the I-Sce1 recognition sites showed a low but signiWcant level of CSR after I-Sce1 induction in the absence of AID [43]. This indicates that neither the AID nor S region is absolutely required for proper end-joining during CSR. Although 3C assay clearly demonstrated that AID expression is able to stabilize the interaction, which may be required for eYcient end-joining. SigniWcant amounts of CSR were observed in B cells with I-Sce1 replacement in the absence of any detectable transcripts in the region. This indicates that transcription is not absolutely required for the joining phase of CSR. Therefore, unlike those DSBs during V(D)J recombination, AID-mediated DSBs in the S regions might be held together after strand cleavage via DNA damage response factors or MMR/BER. These proteins may be recruited to the LRI loop to stabilize the complex and promote end joining. It is intriguing to observe that many DNA damage response genes, such as ATM, H2AX, and 53BP1, are not generally required for V(D)J recombination as demonstrated by the phenotypes of targeted mutant mice. However, these molecules play prominent roles in AID-mediated CSR. All of these deWcient mice exhibit diminished CSR albeit with diVerent severities. Furthermore, SHM and internal deletions within the S region are both AIDdependent events and mostly unaVected in these deWcient mice. This strongly suggests that these damage response proteins play a very important role in the joining of AID-mediated DSBs during CSR. These molecules are not essential during V(D)J recombination because the RAG complex and post-cleavage complex actively mediate the end-joining.

DNA damage response during lymphocyte maturation A rapid response to DNA damage that leads to DNA repair, cell-cycle arrest or apoptosis is essential for preventing genomic instability that may result in the activation of oncogenes and tumorigenesis [59]. One of the early responses to DNA damage includes the activation of the Mre11, RAD51, and Nbs1 (MRN) complex [60, 61], which is involved in many aspects of DSB detection, signaling, and repair [62–64]. Patients with hypomorphic mutations and mice with targeted mutations exhibit a phenotype similar to patients suVering from ataxia telangiectasia (A-T) and mice deWcient of the A-T mutated (ATM) gene [60, 65–68]. The role of MRN in V(D)J recombination is currently unknown. Mutant mice with hypomorphic Mre11 or Nbs1 are able to perform V(D)J recombination [66, 68], nevertheless, the

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MRN complex may still act as a potential player in this process. Other studies reveal Mre11 as a nuclease with speciWcity to single strands and unique structures, such as hairpin ends [69]. While this study initially implicated Mre11 and the MRN complex in hairpin coding ends processing, Artemis was later identiWed as the major player in opening of hairpin ends [70, 71]. The importance of the MRN complex is more evident in CSR. AID-mediated DNA breaks at the S regions induce Nbs1 foci formation [42]. CSR joints are abnormal in patients with hypomorphic mutations in Nbs1 or Mre11 [72, 73], and longer deletions are observed at the switch regions in these patients. These results link MRN complex to end protection or processing during CSR. B-cells with conditional disruption of Nbs1 exhibited defects in CSR independent of germline transcription and were capable of promoting cellular proliferation [74, 75]. Therefore, the MRN complex plays an essential role in sensing AID-induced DSBs, activating downstream damage responses, and possibly participating in end processing or joining. The central element to a DNA damage response is the family of the phosphatidylinositol 3-kinase-like protein kinases (PIKK), which includes ATM, ATR and DNA-PKcs, play important roles in transducing damage signals and DNA repair [76]. In response to DNA DSBs, ATM is recruited to the site by the MRN complex [61] and converted from its inactive dimer state to an active monomer form by autophosphoryation [77]. Activated ATM can phosphorylate many downstream substrates, including histone H2AX, p53, NBS1, 53BP1 and hundreds of other potential substrates [78]. All those molecules play speciWc roles in regulating the cellular response to DNA damage and are critical for the maintenance of genomic stability. In the case of V(D)J recombination during lymphoid development, ATM is localized on RAG-mediated DSBs at the antigen receptor genes [79], suggesting that it may play some roles during V(D)J recombination. ATM deWcient mice succumb to early thymic lymphomas with characteristic chromosomal translocation at the TCR  locus [80–83]. Although these results imply direct involvement of ATM in V(D)J recombination, ATM is not essential for V(D)J recombination as ATM deWcient mice are able to generate V(D)J rearrangements. In addition, ATM deWcient cells are able to rearrange transfected V(D)J recombination substrates [84–86]. Together, these results indicate that ATM may perform multiple but non-essential functions during V(D)J recombination. ATM may be directly involved in processing or protecting RAG-cleaved coding ends in the context of chromatin by stabilizing the post-cleavage complex [87, 88]. In addition, our observations reveal that ATM plays an essential role in containing those RAG mediated DSBs within the G0/G1 phase of the cell-cycle (Dujka et al, unpublished observation), preventing the conversion of these DSBs into chromosomal breaks [89]. These functions may not be essential for V(D)J rearrangement per se but are crucial to maintain genomic stability and prevent chromosomal translocation. Direct evidence links ATM to CSR. A-T patients have lower serum levels of secondary immunoglobulin isotypes [90], and ATM deWcient mice exhibit defect in CSR but not in SHM [88, 91]. Similar to its role in V(D)J recombination, ATM could be a key cell-cycle controller during CSR to contain DNA ends within the cell-cycle checkpoints and to prevent the persistence of these ends throughout the cell-cycle. However, the mechanism of cell-cycle control during CSR is still not fully understood. Additionally, ATM may be directly involved in the processing and joining of two broken ends, and mediating the repair of two distanced ends [92]. ATM could also function independently or in conjunction with the MRN complex to hold ends together and protect or process these ends. The phosphorylation of H2AX (H2AX) is another early event in the cellular response to DNA breaks [93], and its functional relevance is still under intense investigation. Mutation at the phosphorylation site S139A of H2AX reveals very little eVect [94]. However, the

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phosphorylation of H2AX along a megabase region surrounding DNA breaks represents a structural change of local chromatin that facilitates checkpoint and/or docking of DNA repair complexes [95]. H2AX foci are identiWed on the sites of V(D)J recombination undergoing active rearrangement [96]. H2AX is dispensable for V(D)J recombination since H2AX deWcient mice are able to normally rearrange their antigen receptor genes [97, 98]. One model argues H2AX may provide recognition and binding sites for other damage response proteins. A network of these proteins holds two distanced breaks in close proximity to facilitate end joining. Such function would be redundant of the RAG complex during V(D)J recombination, and therefore is not considered essential. H2AX deWcient mice show impaired CSR, indicating H2AX is required for the repair of AID-mediated DSBs [97, 98]. Phosphyorylation of H2AX is identiWed at the CSR site in an AID-dependent manner [42]. Similar to the general response to DNA DSBs, H2AX formation along the S region may be critical for the recruitment of other DNA damage response proteins. H2AX helps to stabilize the complex that holds the AID-generated DNA breaks together and to facilitate the appropriate end-joining [99]. Such stabilization is essential for the eYcient joining of distanced breaks. Alternatively, H2AX surrounding the damaged DNA functions to concentrate all the necessary proteins including damage response proteins and, more importantly, DNA repair proteins to perform end-joining. Therefore, H2AX is expected to be an important part to form and stabilize LRI in B cells, especially the interaction between S to the downstream S regions. Known as a mediator of DNA damage checkpoints, 53BP1 is an important player in sensing DNA damage by recognizing unique histone modiWcations and localizes at the sites of DNA damage and is phosphorylated by ATM. Although 53BP1 accumulates at the V(D)J recombination sites, its role in V(D)J recombination has not been elucidated. 53BP1 deWcient mice have relatively normal V(D)J recombination and lymphoid development [100, 101] therefore its role does not seem essential. However, 53BP1/p53 double deWcient mice succumb to lymphomas with chromosomal translocation involving the T-cell receptor loci. This indicates that 53BP1 may play a role in regulating V(D)J recombination in the context of chromatin. On the other hand, 53BP1 is clearly required for CSR as 53BP1 deWcient mice exhibit severely impaired CSR. This phenotype is not due to deWciencies in cell proliferation, AID-mediated cleavage or germline transcription [101, 102]. 53BP1 forms foci in response to AID-mediated DSBs at the IgH locus in an H2AX-dependent manner [93, 98, 101, 103]. The study of deWcient mice showed that 53BP1 is not essential for SHM [101, 102]. Interestingly, in the absence of 53BP1, AID-mediated breaks can be joined in a short distance (within one S region) that leads to a deletion within the donor and acceptor S regions [104]. This result directly implicates 53BP1 as a mediator of “long distance only” end-joining. The fact that among all the mouse models with deWcient DNA damage response proteins, 53BP1 deWciency impairs CSR most severely [101, 102], suggests additional roles of this protein in sensing DNA breaks and mediating joining. The unique ability of 53BP1 to recognize and bind modiWed chromatin structures [105] may provide some direction to further explore the role of 53BP1 upstream of AID activity. The mediator of DNA damage checkpoint protein 1 (MDC1) which regulates DNA damage response pathways, is required for the foci formation of MRN, 53BP1, and BRCA1 proteins upon DNA damage [106–108]. MDC1 can be phosphorylated by ATM upon DNA damage and is able to bind directly to phosphorylated H2AX. MDC1 deWcient mice have relatively normal T- and B-lymphocyte development, indicating that MDC1 is dispensable for V(D)J recombination [109]. CSR analysis of MDC1 deWcient mice showed a mild defect in surface IgG and secretion levels. The degree of CSR deWciency is similar

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to that in ATM deWcient mice but less severe compared to H2AX and 53BP1 deWcient mice [109]. Three recent studies have simultaneously reported a new player in DNA damage responses. A ubiquitin ligase, RNF8, is rapidly assembled at DNA damage sites and is required for foci formation of 53BP1 and BRCA1. RNF8 possesses an N-terminal FHA domain and a C-terminal ring tail and is able to bind speciWcally to MDC1 at the ATMdependent phosphorylation site via its FHA domain. RNF8 is also able to ubiquitylate histone H2A and H2AX upon DNA damage and plays a important role in inducing cell-cycle checkpoint. These studies placed RNF8 as a novel DNA damage response protein that may serve as a key link between protein phosphorylation and ubiquitylation signaling pathways, both are critical to the mediation of cellular response to DNA damage and to maintain the integrity of the genome [110–112]. Lymphocyte development and its accompanied DNA recombination activities may provide excellent models to further decipher its molecular mechanism. The DNA damage sensors, MRN complex, ATM, 53BP1, H2AX, and MDC1 are important molecules that regulate the cellular response to DNA damage and induce cellcycle checkpoints, DNA repair, or mediate cell death. Mice deWcient in these factors have relatively normal V(D)J recombination, because the RAG complex protects and guides the broken ends to the proper repair pathway. The RAG complex may also protect the cells from eliciting a massive cellular response and activating the apoptosis pathway in response to these DSBs. However, we cannot rule out some DNA damage proteins that may play non-essential roles during V(D)J recombination, in particular, the roles of ATM and H2AX which require further investigation. On the other hand, V(D)J recombination requires strict cell-cycle control and coordination between DNA repair and cell-cycle progression. Such coordination is essential for the maintenance of genomic integrity. ATM is implicated as one of the coordinators, although the molecular mechanism of this control is under investigation. The relatively normal V(D)J recombination in the developing lymphocytes of mice deWcient in each of the above mentioned factors also indicates that DNA end-joining activity is intact in these mice. Therefore, deWciency in CSR of these mice suggests the likely function of DNA damage response molecules is to facilitate DNA end-joining by holding distant breaks in close proximity in a synaptic complex. Other functions include sensing DNA damage, signaling, recruiting more response and repair factors, and Wnally to coordinate DNA repair with cell proliferation. Currently, the molecular mechanisms underlying these events during CSR are largely unknown.

Joining DNA breaks Homologous recombination (HR) and NHEJ are the two major DNA repair pathways for repairing DSBs in the eukaryotic cells [113]. These two distinct pathways work collaboratively, sometimes overlapping in the repair of spontaneous or physiologically relevant DSBs [114]. Both pathways are critical for the maintenance of genomic stability. HR is mostly active in the S/G2/M phases of the cell-cycle [115, 116], probably due to the availability of homologous chromatin. The NHEJ pathway is active in all phases of the cell-cycle but preferred in the G0/G1 phase [59, 116] and is responsible for repairing RAGliberated DSBs at the antigen receptor genes (Fig. 3) [114, 117]. Seven components of NHEJ were identiWed: the DNA-dependent protein kinase complex Ku70, Ku80 and its catalytic subunit DNA-PKcs, the repair factor Artemis, DNA ligase complex XRCC4, DNA

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AID

RAG RAG C-NHEJ DDR

?

C-NHEJ

A-NHEJ

Fig. 3 Non-homologous end-joining pathways. RAG proteins generate DSBs and guide the breaks to the classic NHEJ pathway; AID mediated DSBs induce general DNA damage response (DDR) proteins, which recruit classic NHEJ (C-NHEJ) factors to complete the joining; DSBs can also be joined by the alternative NHEJ (A-NHEJ) via yet unknown factors

ligase IV and the newly identiWed factor Cernunnus-XLF [118, 119]. Targeted inactivation of each of these seven factors renders a SCID phenotype in mice due to the inability to complete V(D)J recombination [13, 71, 120–123]. RAG-mediated DSBs induce p53dependent apoptosis in precursor lymphocytes of NHEJ deWcient mice. This indicates that in general, no other DNA repair activities are able to replace this “classic” NHEJ in mediating end-joining during V(D)J recombination with wild-type RAG proteins. However, some of these deWcient mice exhibit leaky phenotypes. A minor population of lymphocytes is able to partially rearrange at certain loci or complete few rearrangements in the NHEJ deWcient background, suggesting an alternative DNA repair pathway exists to join some portion of the stalled DSBs under the condition of NHEJ deWciency. The alternative DNA repair pathway will be further discussed below. After RAG cleavage at the RRS, the end-joining phase begins with the Ku70/Ku80 complex binding to DNA breaks, which commits the ends to be joined by the classic NHEJ pathway [124]. The Ku70/Ku80 heterodimer is capable of protecting DNA ends and both proteins are required for the joining of signal and coding ends [13, 122]. Binding of Ku70/ 80 heterodimer to the DNA ends activates another PIKK family member, DNA-PKcs [125]. Unlike ATM, DNA-PKcs is required for the joining of coding ends in V(D)J recombination. DNA-PKcs deWcient mice exhibit SCID phenotype due to defective V(D)J recombination [121, 126, 127]. The role of DNA-PKcs in V(D)J recombination is to phosphorylate and activate Artemis, a nuclease capable of opening hairpin coding ends generated through RAG1/2 cleavage [70]. In mice lacking DNA-PKcs or Artemis, hairpin coding ends accumulate in the developing lymphoid cells [71, 121, 127]. Joining of signal end is almost normal in these mice, indicating end-joining activity is nearly unaVected in DNA-PKcs deWciency [128–130]. This also suggests an additional role of Ku70/Ku80 heterodimer besides activating DNA-PKcs in end-joining. Once processed by the DNA-PKcs/ Artemis complex, coding ends are joined quickly by the DNA ligase complex, therefore no coding ends are accumulated in wild-type lymphoid cells. Signal ends, on the other hand, are readily detected at many TCR and IgH loci in wild-type thymocytes and bone marrow cells [12, 131]. The RAG complex likely binds these ends in a post-cleavage complex. Finally, joining of signal ends requires the presence of both the Ku heterodimer and DNA

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ligase IV complex. DiVerential processing of signal ends and coding ends marks the unique property of V(D)J recombination end-joining. In mammalian cells, the majority of DSBs are repaired by one of the two major pathways: HR or NHEJ. AID-generated DSBs are unlikely joined by HR because there are no extensive homologies between the two switching regions [132, 133]. In vivo studies suggest that classic NHEJ is required for CSR. Ku70 and Ku80 deWcient mice with pre-rearranged heavy and light chains of immunoglobulin transgenes (to bypass the requirement of V(D)J recombination) showed defect in CSR, suggesting the requirement of Ku proteins and NHEJ pathways in joining CSR DSBs [134, 135]. However, there was a concern in this study that CSR deWciency may have been caused by a poor proliferation capability of Ku deWcient cells [136]. The requirement of DNA-PKcs in CSR is still under debate. While a study using the SCID model indicated DNA-PKcs deWciency has very little eVect on CSR [137], a DNA-PKcs knockout mouse model reveals severe deWciency in all but Ig1 isotype switching [138]. Although one function of DNA-PKcs is clearly deWned in the activation of Artemis and processing of hairpin coding ends in V(D)J recombination, it is unlikely such function may be necessary in CSR since there is no evidence of the involvement of hairpin ends in CSR. However, it is believed that hairpin processing might not be the only function of DNA-PKcs. It is probable that DNA-PKCs is required for other activities such as end modiWcation, which might be crucial for joining some portion of CSR ends. Artemis in general is not required for CSR [139]. Hypomorphic DNA ligase IV mutant patients have a defect in end-joining and the CSR junction shows more characteristics of microhomology [140], indicating the involvement of DNA ligase IV complex in CSR end-joining. In line with this study, a recent report in mice with conditional inactivation reveals CSR is partially defective in the Ligase IV or XRCC4 deWcient background, indicating the “classic” NHEJ is the normal pathway to join CSR ends [141]. In addition to the classical NHEJ repair pathway, there is at least one “alternative” endjoining activity that is independent of all the “classic” NHEJ factors such as DNA-PKcs, Ku proteins and the DNA ligase IV complex. This end-joining activity is not easily observed in V(D)J recombination due to the protective role of RAG1/2. Almost no signal or coding joints were detected in XRCC4 or Ligase IV deWcient mice [120, 142]. This endprotecting role of RAG complex is further demonstrated by a recent study of RAG2 with a frameshift mutation [55]. This RAG2 mutant is able to cleave DNA strands in V(D)J recombination substrates, making these ends more accessible for joining in XRCC4 or DNA-PK deWcient cells. In comparison, such end-joining is rarely observed in wild-type RAG. Analysis of V(D)J recombination products reveals high percentage of microhomology at these junctions, indicating the usage of an alternative NHEJ. This Wnding leads to the conclusion that alternative NHEJ is active in the mammalian cells with defects in classic NHEJ. Interestingly, analysis of V(D)J recombination junctions in wild-type cells also revealed the use of this alternative NHEJ [55] under mutant RAG2, strongly suggesting a co-existence of both NHEJ pathways in wild-type cells. The alternative NHEJ activity is not normally observed in V(D)J recombination because the RAG complex exclusively guides the ends to the classic NHEJ. On the other hand, substantial amount of CSR joints (25–50% of wild-type level) were detected in the DNA ligase IV deWcient B-cells, indicating these AID-mediated ends are able to access the alternative repair pathway in the absence of the classic NHEJ [141]. A similar result was reported in conditional deletion of XRCC4 [143]. Therefore, AID-mediated DSBs can be joined rather eYciently by the alternative NHEJ when the classic NHEJ is defective. However, we still do not know if a portion of the joining is mediated by alternative NHEJ when the classic NHEJ is eYcient and how the AID-mediated DSBs are guided to the classic NHEJ.

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The alternative end-joining pathway was Wrst described in 1986 in an end-joining study using transfected DNA substrate, and was characterized by the appearance of 1–5 nucleotide homologues at the junction [144]. This property may be important to understand the biochemistry of this repair pathway. Most of the studies on this pathway use extra chromosomal DNA substrates with some microhomologies embedded near the DNA termini [145]. These studies reveal that alternative end-joining activity is capable of joining DNA ends at a rate similar to classic NHEJ [145]. However, this activity has not been well studied in vivo, mainly because of the dominant end-joining activities of classic NHEJ and HR. With more animal or cell models becoming available, molecular details of this pathway will begin to emerge. Some studies have indicated DNA ligase III may be the candidate ligase for alternative NHEJ activity, together with XRCC1 and PARP1 [146, 147].

Genome instability and tumorigenesis in lymphoid development Programmed DNA lesions generated during lymphocyte development, if not well contained, may lead to genomic instability and tumorigenesis [117, 148]. RAG1/2 plays an important role in protecting ends and maintaining the integrity of the genome. However, RAG activity may also be associated with chromosome translocation at certain oncogene regions and hot spots [149] since it is able to recognize and cleave these regions that contain unique DNA structures in vitro. In addition, AID, a potent mutator, may directly target some non-Ig genes that are actively transcribing [150, 151]. These events render developing lymphoid cells vulnerable to oncogenic transformation. It is well established that V(D)J recombination is initiated and completed during the G0/ G1 phase of the cell-cycle [152]. RAG expression is strictly regulated during this phase in developing lymphoid cells and is rapidly degraded during G1-S transition [153, 154]. The signal end intermediates of V(D)J recombination are only observed in the G0/G1 phase of the cell cycle [12]. Furthermore, the NHEJ repair pathway is preferentially active during G0/G1 phase. It is crucial to contain DSBs within the G0/G1 phase of the cell-cycle to maintain genomic integrity because initiation of DNA replication may disturb the postcleavage complex and liberate DNA ends. Consistent with this notion, deWciency in the cell-cycle controller ATM leads to the persistence of DSBs (Dujka et al, unpublished observation) and chromosomal breaks [89] throughout the cell-cycle. This leads to chromosomal translocation and tumorigenesis in the developing lymphoid cells [155, 156]. In NHEJ deWcient mice, accumulation of RAG-mediated DNA breaks induces p53-dependent apoptosis [157] to prevent massive genomic instability. NHEJ/p53 double deWcient mice succumb to early pro-B lymphoma with co-ampliWcation of the IgH and c-MYC genes [158, 159]. Therefore, the ability of p53 to induce apoptosis is the main gate-keeping mechanism to suppress lymphomagenesis under the condition of NHEJ deWciency during lymphoid development. Interestingly, analyses of translocated junctions revealed 1–5 microhomologies in each of the junctions, indicating the alternative NHEJ is able to mediate oncogenic interchromosomal end-joining [159]. Chromosomal translocation is a hallmark of lymphomas and plays an essential role in tumorigenesis. The majority of human lymphomas are of B-cell origin containing translocations involving the IgH locus [148]. These translocations juxtapose the IgH locus with certain proto-oncogenes so that the transcription elements at the IgH locus control the expression and often lead to the over expression of the oncogenes. AID-dependent DNA lesions generated during CSR and SHM are associated with chromosomal translocations and tumorigenesis. A study using an IL-6 transgene plasmacytoma mouse model indicated

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IgH to c-MYC translocation was AID-dependent [160]. Likewise, activated B cells from H2AX, 53BP1, ATM, or MDC1 deWcient mice exhibit chromosomal breaks and translocations at the IgH site [161, 162] in an AID-dependent manner. These results suggest that these DNA damage response proteins play essential roles in protecting ends or facilitating end-joining to prevent chromosomal breaks and translocations. Remarkably, AID associated translocations in activated B-cells are also Ku-independent, indicating chromosomal translocation is not mediated by classic NHEJ [162]. These results clearly suggest AIDmediated DSBs are more accessible for translocation than the DSBs generated by RAG complex during V(D)J recombination. This once again highlights the role of RAG and the post-cleavage complex in protecting DNA ends and preventing genomic instability.

Conclusions and perspective RAG-dependent V(D)J recombination and AID-dependent CSR and SHM are site/ regional-directed somatic events that are involved in the generation and repair of DNA lesions. These processes are essential to build a proper immune system to eYciently Wght against infections. DNA damages generated during these physiological events induce cellular responses that lead to cell-cycle checkpoint activation and DNA repair activity. Mice deWcient for each of the responding or repairing factors are excellent models to understand how the DNA damage response, cell-cycle checkpoint and DNA repair activities work in concert to repair DNA lesions and to prevent genomic instability. It is clear that RAG proteins play more important roles than simply initiating V(D)J recombination and breaking DNA strands. RAG complex functions essentially in the endjoining phase of V(D)J recombination. However, it is still poorly understood how RAG proteins communicate with classic NHEJ DNA repair factors such as Ku. Questions regarding the role of RAG proteins in promoting aberrant end-joining and chromosomal translocation in vivo remain unanswered. More importantly, we do not completely understand how the potentially harmful activity of the RAG complex is contained in vivo and how RAG activity is controlled during the cell-cycle. Several recent studies on AID have shed some light in the understanding of the biochemical events in AID initiation of CSR and SHM. Yet, many questions remain unanswered. We still do not completely understand how AID targets to the designated regions, especially to the variable regions of immunoglobulins, but not other non-Ig regions. How DSBs are produced in the S regions remains unknown. We know very little about how AID activity is controlled during the cell-cycle. Since cell proliferation is a requirement for successful CSR, the communication between DNA end-joining and the cell-cycle is absolutely critical. The role of DNA damage response proteins during CSR is evident but not completely understood, and their roles in facilitating end-joining need to be deWned biochemically. Accumulating evidence points to the existence of the robust activity of the alternative NHEJ pathway, most likely simultaneous to the classic NHEJ. Several mouse lymphoma studies showed the involvement of this activity in mediating end-joining during chromosomal translocation. Many aspects of this pathway are yet to be discovered. Currently we do not know any participating factor in this pathway, or how this activity is regulated beyond the cell-cycle. In addition, we still do not understand how critical microhomology is in mediating this alternative end-joining, and how this pathway is activated. Somatic DNA recombination processes in developing lymphocytes are essential events for the generation and maturation of adaptive immunity. They also have provided an excellent system to enhance our understanding of how DNA lesions are introduced under a

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physiological condition and how cells respond to DNA damage and activate DNA repair machinery. More importantly, mouse models with deWciencies in these processes are ideal in vivo systems for us to understand tumorigenesis in the lymphoid system. These models will continue to provide us with basic understanding of the molecular mechanism of immune development, DNA damage response and repair. Further advancement of these models will also create an avenue to identify potential tumor precursors and to test potential tumor antigens and targets for therapy. Acknowledgements We would like to thank Melanie Dujka, Omid Tavana and Drs. Phillip Carpenter and Cara Benjamin for their critical review of this manuscript. Our work is supported by the American Cancer Society and the National Cancer Institute (to C.Z.).

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