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How to make ends meet in V(D)J recombination Ulf Grawunder and Eva Harfst In most vertebrate species analyzed so far, the diversity of soluble or membrane-bound antigen-receptors expressed by B and T lymphocytes is generated by V(D)J recombination. During this process, the coding regions for the variable domains of antigen-receptors are created by the joining of subexons that are randomly selected from arrays of tandemly repeated V, D (sometimes) and J gene segments. This involves the site-specific cleavage of chromosomal DNA by the lymphocyte-specific recombination-activating gene (RAG)-1/2 proteins, which appear to have originated from an ancient transposable element. The DNA double-strand breaks created by RAG proteins are subsequently processed and rejoined by components of the nonhomologous DNA end-joining pathway, which is conserved in all eukaryotic organisms — from unicellular yeast up to highly complex mammalian species. Addresses Universitaetsklinikum Ulm, Department of Immunology, Albert-Einstein-Allee 11, D-89081 Ulm, Germany Correspondence: Ulf Grawunder; e-mail: [email protected] Current Opinion in Immunology 2001, 13:186–194 0952-7915/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations bp CJ DNA-PKcs DSB NHEJ N-region P-region RAG RSS SCID SJ TdT XRCC

basepairs coding joint DNA-dependent protein-kinase catalytic subunit DNA double-strand break nonhomologous DNA end-joining nontemplated nucleotide region palindromic nucleotide region recombination-activating gene recombination signal sequence severe combined immunodeficiency signal joint terminal deoxynucleotidyl transferase X-ray cross-complementation group

RSSs of different spacer lengths, which is referred to as the ‘12/23 bp rule’. During V(D)J recombination, gene segments, which can be located several hundred kilobases apart, are brought into a synaptic complex. The selection of gene segments depends on the accessibility of the gene loci, which correlates with the differential expression of transcription factors and their interaction with enhancer and promoter elements during lymphopoiesis [2]. The differential accessibility of gene segments for V(D)J recombination involves the remodeling of local chromatin structure and appears to be modulated by histone acetylation and deacetylation [3••,4•]. In addition to accessibility control, recent studies suggest that the order of rearrangements within a given locus can also be controlled by the type of RSS and apparently, therefore, by its spacer sequence [5••]. Following synapsis of gene segments, DNA double-strand breaks (DSBs) are generated precisely at the border of the RSS heptamers and the adjacent coding regions of each gene segment. The signal-ends are then rejoined into a signal joint (SJ) that retains two head-to-head-ligated RSSs, usually without major modifications. In contrast, resealing of the two coding-ends into a coding joint (CJ) is often accompanied by major modifications, including nucleotide loss and addition (see below), which contribute significantly to antigen-receptor diversity. Depending on the orientiation of the rearranging gene segments, V(D)J recombination results either in inversions of large regions of genomic DNA or in their excision from the locus in form of closed, circular DNA containing one SJ. This review focuses on recent progress in understanding the intricate interplay between the DNA end-joining factors in the context of V(D)J recombination.

Introduction

Factors mediating V(D)J recombination

During the evolution of vertebrates, the development of a specific and adaptive immune system depended on the ability to generate a diverse repertoire of antigen-receptors that are able to recognize a myriad of potential pathogens. The coding regions for the variable domains of antigen-receptors are generated, during early B and T lymphopoiesis, by the site-specific rearrangement of V, D and J gene segments; for each antigen-receptor specificity, one V, one J and (sometimes) one D segment are randomly selected from clusters of up to several hundred V/D/J segments by the process of V(D)J recombination [1]. The site-specificity of the reaction is mediated by conserved recombination signal sequences (RSSs) flanking all coding regions and consisting of a palindromic heptamer separated from an A/T-rich nonamer by a spacer sequence of either 12 or 23 basepairs (bp). A pair of gene segments is only rearranged if they are flanked by

It is now well established that the initial DSBs at RSSs are generated by a complex of the recombination-activating gene (RAG)-1 and RAG-2 proteins [6]. Both RAG genes had been cloned on the basis of their ability to mediate V(D)J recombination on artificial substrates in nonlymphoid cells [7,8]. Hence, mice with targeted mutations in either of the RAG genes suffer from severe combined immunodeficiency (SCID), resulting from a block of B and T lymphocyte differentiation due to the inability to initiate recombination within endogenous antigen-receptor gene loci [9,10]. Many of the known components involved in later stages of V(D)J recombination are ubiquitously expressed DNArepair factors that are also important for the repair of DSBs by the process of nonhomologous DNA end-joining

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(NHEJ). One exception is the lymphocyte-specific terminal deoxynucleotidyl transferase (TdT), a nonessential component that catalyzes the occasional addition of nontemplated nucleotide region (N-region) sequences at CJs [11]. The functional overlap between general DSB repair and V(D)J recombination was already realized some time ago, when a series of X-ray-sensitive rodent cell lines was characterized and used to define different X-ray cross-complementation groups (XRCC4−XRCC7); the groups of cells were not only impaired in the efficiency of repairing irradiation-induced DSBs but also deficient in mediating V(D)J recombination. These cell lines were crucial for the identification of genes required for DNA end-joining in V(D)J recombination, including those encoding XRCC4, Ku86 (XRCC5), Ku70 (XRCC6) and DNA-PKcs (DNA-dependent protein-kinase catalytic subunit; XRCC7). The Ku70 and Ku86 proteins are abundant nuclear proteins constituting the major DNA-end-binding activity in nuclear extracts from mammalian cells [12]. A complex of Ku70 and Ku86 (from now on referred to as Ku) can associate with DNA-PKcs, forming the DNA-PK holoenzyme [13]. Ku is thought to recruit DNA-PKcs to DNA ends, upon which its kinase activity is stimulated, although DNA-PKcs also exhibits some DNA-end-binding and kinase activity independently of Ku [14,15]. The gene encoding DNA-PKcs is mutated in scid mice [16], where the mutation results in the expression of a truncated and catalytically inactive DNA-PKcs protein [17,18]. Either the scid mutation or complete null mutations of DNA-PKcs in mice [19,20] lead to the inhibition of CJ formation whereas SJs can still be formed to some extent (see below). In contrast, targeted deletions of either Ku70 or Ku86 genes in mice [21–24] lead to deficiencies in both SJ and CJ formation. The mutation in XRCC4 was identified and cloned using a single X-ray-sensitive chinese hamster ovary cell line, defining this complementation group [25]. It was later shown that the XRCC4 protein associates with DNA ligase IV, resulting in the stimulation of the in vitro ligation activity [26,27]. As in the case of Ku mutations, cells deficient for either XRCC4 or DNA ligase IV are unable to catalyze SJ or CJ formation. However, in contrast to Ku70 and Ku86 mutant mice — which are viable — a targeted deletion of either XRCC4 or DNA ligase IV results in embryonic lethality caused by neuronal cell death [28,29].

initiation stage involves formation of a cleavage complex and generation of DSBs; secondly, there is a processing stage for the newly generated DNA ends; and, thirdly, a DNA end-joining stage results in the formation of SJs and CJs. We would like to concentrate our review on recent advances dealing with the postcleavage stages of V(D)J recombination. However, this requires a brief summary of the initiation of V(D)J recombination (reviewed recently in detail [31]) because initiation and end-joining in V(D)J recombination appear to be functionally and mechanistically linked [32]. In addition, the DNA-ends generated by RAG cleavage are unique to V(D)J recombination and may determine V(D)J-recombination-specific functions of some NHEJ components. Initiation of V(D)J recombination

V(D)J recombination is initiated by binding of RAG proteins to a pair of RSSs. This results in the synapsis of the signals with their adjacent coding regions in a precleavage complex. The DNA is then cleaved in a coordinated fashion by RAG proteins at both RSSs by a two-step reaction [33]. Firstly, at each RSS a single-stranded nick is generated in one DNA strand (Figure 1a). Following this, the 3′OH group of the nick attacks the phosphodiester bond of the complementary DNA strand in a direct transesterification reaction, resulting in a DSB with a covalently sealed, hairpinned coding end and a 5′-phosphorylated, blunt signal end (Figure 1a). Coordinated cleavage at a pair of RSSs can be catalyzed by RAG proteins alone [34,35] but high mobility group (HMG) proteins increase the degree of concerted cleavage [36]. The newly generated DNA-ends are retained in a RAG postcleavage complex [37] that is able to catalyze the formation of hybrid joints [38]. Such joints are the result of ‘cross-over’ ligations of signal ends and coding ends by a RAG-mediated transesterification involving the 3′OH group of the signal ends with the phosphodiester groups of the ‘opposite’ coding-end hairpins (Figure 1b). RAG proteins are even able to mediate transposition of DNA ends containing RSSs [39,40] (Figure 1c). This latter finding adds weight to the hypothesis that the RAGs and the RSSs are derived from an ancient transposable element. This had already been hypothesized on the basis of the compact genomic organization of the RAG genes and the biochemistry of the RAG cleavage reaction, which is related to retroviral integration and transposition [41]. Processing of DNA-ends

With the exception of DNA-PKcs, all mammalian NHEJ components involved in V(D)J recombination are evolutionarily conserved down to unicellular Saccharomyces cerevisiae, where the genes are called HDF1 (a Ku70 homologue), HDF2 (a Ku86 homologue), DNL4 (a DNA ligase IV homologue) and LIF (ligase interacting factor; an XRCC4 homologue) [30].

Following the generation of DSBs by RAG proteins, the postcleavage complex contains blunt-ended signal ends and hairpinned coding ends (Figure 2). Whereas signal ends can potentially be directly ligated, coding ends need to be processed before CJ formation can occur. This requires the opening of the coding-end hairpin by an activity that generates a single-stranded nick at, or close to, the hairpin tip (Figure 3a).

Stages of V(D)J recombination The chronology of V(D)J recombination makes it reasonable to subdivide the reaction into three stages: firstly, an

Recent data suggest that coding-end hairpin opening can be performed by a complex of RAG proteins itself [42,43].

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Figure 1 (a)

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Current Opinion in Immunology

The types of reactions catalyzed by complexes of RAG-1 and RAG-2 proteins. (a) RAG proteins recognize a pair of RSSs (depicted here as open and closed triangles for a 12 bp and a 23 bp spacer RSS, respectively) and catalyse two DSBs by a two-step reaction. Firstly, following synapsis of the signals (which for simplicity is not depicted here), single-stranded nicks are generated in a coordinated fashion at the border between the coding regions (shaded boxes) and the heptamers of the RSSs. The 3′OH groups generated at the nicks attack the phosphodiester bond of the complementary DNA strands and cleave the DNA by a direct transesterification reaction. This creates hairpinned coding-ends (CEs) and blunt signal-ends (SEs). (b) CEs and SEs held in a synaptic complex can be ligated to hybrid

joints by RAG proteins. For this, RAGs catalyze the transesterification of the 3′OH groups at the SE to the phosphodiester bonds of the opposite CE hairpin, resulting in a ‘cross-over’ ligation. (c) SEs bound by RAG proteins are able to transpose into DNA by attacking staggered phosphodiester bonds on both strands of a target site with their 3′OH groups. The phosphodiester bonds involved in the transesterifications with the signal ends are 5 bp apart, which results in 5 nucleotides of singlestranded, complementary regions flanking the transposed DNA (as indicated). If the transposition occurred in vivo, the single-stranded regions would be filled in by DNA polymerase, leading to 5-bp duplications at the integration site.

A role for RAG proteins in the processing of coding ends would be compatible with the requirement of RAG proteins for the postcleavage stages of V(D)J recombination [32]. Hairpin opening by RAG proteins was demonstrated biochemically in vitro, with artificial DNA-oligomer substrates and also with intermediates from a RAG-mediated cleavage reaction [42,43]. High mobility group proteins appeared to stimulate the efficiency of hairpin-nicking and shifted the nick position from 3′ of the hairpin tip to positions 5′ of the hairpin end [43], which is more compatible with the direct generation of palindromic nucleotide region (P-region) sequences (Figure 3).

to open hairpinned coding ends in vitro, why can they not be resolved in the different NHEJ-deficient genetic backgrounds? One possibility is that assembly of other NHEJ components to the RAG postcleavage complex, as has been shown biochemically [46], is required to allow the remodeling of the complex and thereby the activation of RAG proteins for hairpin opening under physiologic conditions. Alternatively, it could be the case that, in vivo, different activities that depend on the function of DNA-PK and XRCC4 are involved in coding-end-hairpin nicking.

Despite these in vitro findings, hairpin opening in vivo requires the expression of catalytically active DNA-PKcs, as it has been shown that hairpinned-coding-end intermediates accumulate in mice carrying a homozygous scid mutation [44]. In fact, unresolved hairpinned-coding-end intermediates have been detected in cells with mutations in any of the DNA-PK components and even in XRCC4deficient cells [22,45]. If RAG proteins alone are sufficient

A possible alternative candidate for a coding-end-hairpinnicking activity could be the Nbs1−Mre11−Rad50 protein complex. Like RAG proteins, the complex of these mammalian DNA-repair proteins is able to open hairpinned DNA ends endonucleolytically in vitro [47••]. The homologue of mammalian Nbs1−Mre11−Rad50 in S. cerevisiae (sc-XRS2−sc-MRE11−sc-RAD50) is known to be involved in DSB repair by homologous recombination and NHEJ. A role of the mammalian Nbs1/Mre11/Rad50 for V(D)J recombination is therefore not unlikely although patients

How to make ends meet in V(D)J recombination Grawunder and Harfst 189

with Nbs1 mutations — who suffer from the rare autosomal recessive genetic instability disorder, Nijmegen breakage syndrome (NBS) [48,49] — develop mature B and T lymphocytes. However, a detailed analysis V(D)J recombination in NBS patients has not been reported yet.

Figure 2

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Hairpin nicking can occur at the tip or off-center, generating either a 5′ or a 3′ overhang (Figure 3). 5′ overhanging ends can directly be filled-in by DNA polymerase, which leads to the generation of short P-region sequences that are often found in CJs. 3′ extensions need to be resected in order to generate DNA ends that can be directly ligated, unless compatible 3′ extensions are present at both coding ends. In most cases, however, it can be expected that coding-end processing, for example following exonuclease digestion or N-region addition by TdT, results in noncompatible DNA ends that can only be joined after annealing and trimming of flap structures (see below).

One function of Ku proteins in V(D)J recombination is most likely the recruitment of other NHEJ components to the postcleavage complex. DNA-PKcs is one of these factors and TdT appears to be another. The recruitment of TdT to the postcleavage complex by Ku was already suggested by the absence of N-region sequences in rare CJs isolated from Ku-deficient lymphocytes [51]. This notion was further substantiated by the recent finding that TdT is able to interact with Ku proteins in a DNA-independent fashion [52•]. Preparation for DNA end-joining

Opening of hairpinned coding-ends and their subsequent modification by nucleases, polymerases and TdT might still not result in ligatable DNA ends (Figure 3b). CJ formation in these cases probably has to involve annealing of the DNA ends using short microhomologies close to both

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As stated above, opening of hairpinned coding-ends in vivo depends on the expression of DNA-PK components and even on the presence of XRCC4. If RAG proteins catalyze the hairpin opening in vivo, it can therefore be expected that all NHEJ components have to join the postcleavage complex before processing commences. Because of their high affinity for DNA ends, it can be assumed that Ku proteins are among the first factors that associate with signal and coding ends generated by RAG proteins. Ku proteins have been implicated in protecting DNA ends from exonucleolytic degradation in mammalian cells [50]. However, in the context of V(D)J recombination this function might only play a minor role. This is indicated by the presence of full-length signal ends and hairpinned codingends at practically wild-type levels in Ku-deficient animals [22], suggesting that DNA-ends are protected from nonspecific nuclease digestion in the RAG-containing postcleavage complex. Furthermore, hybrid joints and rare CJs isolated from Ku86-deficient animals display normal length in comparison with wild-type controls [51].

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Current Opinion in Immunology

Schematic representation of V(D)J recombination. A pair of RSSs is recognized and synapsed by a complex of RAG-1 and RAG-2 proteins, and DSBs are generated in a coordinated fashion at the heptamer side of the RSSs (open and black triangles) and the coding regions (shaded boxes). The coding ends (CEs) are processed by nicking at, or close to, the hairpin tip, followed by the optional addition of nucleotides by TdT, which leads to the generation of P- and N-region diversity (as indicated; see also Figure 3). Blunt signal-ends are usually ligated without further modification to generate an SJ. In contrast, CEs may be subjected to trimming of the ends, alignment via microhomologies and fill-in of singlestranded gaps, before ligation into a CJ can occur.

coding ends (Figure 3b). Joining by microhomologous annealing of 1–3 nucleotides is often found at CJs, particularly in the absence of TdT [53]. In contrast, SJ formation in wild-type cells usually occurs with >95% fidelity by a precise and blunt head-to-head ligation of the ends, indicating that sequence homologies at the signals are not required for SJ formation. Following alignment via microhomologies, 5′ and 3′ flap structures need to be trimmed. 3′ flap structures might be removed by either RAG proteins, which display a 3′ flap endonuclease activity in vitro [54••], or by the Mre11 nuclease, which exhibits a 3′→5′ exonuclease activity in vitro [55] (Figure 3b). Interestingly, it has recently been shown that the Mre11 nuclease pauses hydrolysis of single-stranded overhangs exactly at regions of microhomology pairing [56••].

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in the efficiency of NHEJ events that would require 5′ flap removal [58•], suggesting that mammalian FEN-1 could have a role in the processing of 5′ flap structures in V(D)J recombination as well. After removal of 5′ and 3′ flap structures, single-stranded gaps have to be filled in by DNA polymerase(s), before ligation of the ends into SJs and CJs can proceed (Figure 3b).

Current Opinion in Immunology

Possible scenarios for coding-end processing. (a) Hairpinned (HP) coding ends can be nicked 5′ of the tip, at the tip or 3′ thereof (indicated by encircled 1, 2 and 3, respectively), which may be catalyzed by a complex of RAG-1 and RAG-2 proteins (or possibly by a complex of Nbs1−Mre11− Rad50). Nicking up- or down-stream of the tip results in the generation (by DNA polymerase) of short P-region sequences. Coding end hairpin opening can therefore result in 5′ overhangs, blunt ends or 3′ overhangs that may further be modified by TdT-catalyzed N-region addition (as indicated), DNA-polymerase fill-in (as indicated), or nuclease digestion (not shown). (b) Processing of coding ends often results in noncompatible ends that cannot be directly ligated. In these cases, annealing via short 1–3 bp microhomologies close to the coding ends will most probably occur, resulting in 5′ and 3′ flap structures (as indicated). 3′ flap structures might be a substrate for the 3′ flap endonuclease activity of the RAG proteins (R) or the 3′→5′ exonuclease activity of Mre11 (M). 5′ flaps could be removed by the 5′ flap endonuclease FEN-1 (F), and single-stranded gaps can be filled-in by DNA polymerases (Pol). Single-stranded nicks are then a substrate for a complex of XRCC4 and DNA ligase IV (X4−L4), catalyzing the ligation into a CJ.

5′ overhangs might be a substrate for FEN-1, which in vitro is able to endonucleolytically hydrolyze 5′ flaps [57]. In S. cerevisiae, it was recently shown that mutations in the yeast homologue of FEN-1, sc-RAD27, result in a decrease

The final ligation of SJs and CJs is catalyzed by DNA ligase IV in complex with its binding partner, XRCC4 [28,29,59]. A recent study suggests that Ku proteins are required to recruit the DNA-ligase-IV−XRCC4 complex to DNA ends, leading to the stimulation of end ligation [60•]. This recruitment was shown to be mediated by a physical interaction between Ku proteins and the DNA-ligase-IV–XRCC4 complex, but protein−protein interactions were enhanced by the addition of DNA. In this study a DNA-ligase-IV−XRCC4 complex was not found to bind DNA on its own. However, another study demonstrated exactly the opposite, namely that the DNA-ligase-IV−XRCC4 was able to bind to DNA-ends without the cooperation of Ku proteins and that the presence of Ku inhibited the ligation of DNA ends by DNA-ligase-IV−XRCC4 [61••]. Nevertheless, both studies agree that a physical interaction between Ku proteins and DNA-ligase-IV−XRCC4 occurs at DNA ends. Interestingly, the latter study also demonstrated a physical interaction of DNA-ligase-IV−XRCC4 and DNA-PKcs on DNA-ends, detected by atomic force microscopy (AFM). This interaction did not interfere with the ligation activity of XRCC4−DNA-ligase-IV but instead changed the outcome of the ligation reactions. XRCC4–DNA-ligase-IV alone mainly catalyzed intramolecular ligations of linearized plasmid substrates whereas addition of Ku proteins resulted in 50% dimeric and multimeric intermolecular ligation products [61••]. Interestingly, addition of the DNA-PK holoenzyme to XRCC4−DNA-ligase-IV ligation reactions resulted in almost 100% dimeric intermolecular ligation products [61••]. In order to reconcile some of the apparently contradictory conclusions of these two studies, one could envision that Ku proteins or the entire DNA-PK holoenzyme interact with DNA-ligase-IV/XRCC4 on DNA-ends, leading to the stimulation of some (intermolecular) ligation events but inhibiting others (intramolecular ligations). The difference in the effect of Ku proteins for the activity of DNA-ligase-IV−XRCC4 reported in these studies [60•,61••] might therefore simply reflect differences in the quality of the substrates. XRCC4 has previously been shown to bind to DNA [62••] and it has been demonstrated that the S. cerevisiae XRCC4 homologue, LIF, is required to target yeast DNA ligase IV to sites of DSBs [63•]. These data, together with the demonstration of XRCC4−DNA-ligase-IV binding to DNA-ends by AFM and EMSA (electrophoretic mobility

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shift assay) [61••], clearly show that XRCC4−DNA-ligaseIV can bind to DNA ends independently of Ku.

Effects of different mutations in NHEJ components There are different relative requirements of individual NHEJ components for SJ and CJ formation in V(D)J recombination. Mutations in either Ku70/86 or DNA-ligase-IV−XRCC4 affect the capability of cells to perform either kind of joining reaction. However, in contrast to a targeted deletion of Ku86, which completely abolishes B and T lymphopoiesis [21,22], mature T lymphocytes develop in Ku70-deficient animals although at levels reduced by roughly 100-fold [23,24]. Because a significant fraction of the CJs derived from the TCRβ locus of these T lymphocytes is comparable to CJs from wild-type T lymphocytes, it appears that normal coding-joining can occur at reduced levels in Ku70-deficient mice, which still express low amounts of Ku86 protein [64]. These findings suggest that Ku86 retains some partial activity in the absence of Ku70 protein but not vice versa. Mutations in the DNA-PKcs gene in mice affect SJ and CJ formation differentially. Whereas coding-joining is severly impaired, cells from scid mice carrying a truncating mutation of DNA-PKcs are still capable of forming SJs at almost wildtype levels [65]. This finding has been confirmed in mice that have complete null mutations of DNA-PKcs [19,20,66] and in other rodent (V-3, irs-20) and human (M059J) cell lines carrying different mutations in DNA-PKcs [67,68]. This is in contrast to the phenotype for V(D)J recombination in equine scid [69] and in two other rodent DNA-PKcs-mutant cell lines (XR-C1 and SX9) [70,71] that were found to have severely impaired SJ and CJ formation. The generation of complete null mutants of DNA-PKcs has demonstrated that the capability of scid mice to perform SJ formation is not related to a potential residual activity of a truncated DNA-PKcs protein. Furthermore, it was recently shown that the equine scid mutation can be complemented by expression of a wild-type DNA-PKcs cDNA [72•], and complementation of the DNA-PKcs deficiencies has also been demonstrated for the mutations in the XR-C1 and SX9 cell lines [70,71]. These findings exclude the possibility that the different phenotypes of DNA-PKcs mutants result from a dominant negative mutation in DNA-PKcs mutants. A solution to this paradox comes from the analysis of the fine structure of SJs formed in different DNA-PKcs-deficient backgrounds. Most studies analyzing signal-joining in scid and DNA-PKcs-null mice agree that there is an approximately 2–10-fold reduction in SJ formation and that only 15–50% of these SJs are precisely joined [20,66,68,71,73•]. This already suggests that DNA-PKcs plays a role in SJ formation. It further indicates that some, or all, of the signal-joining in DNA-PKcs-deficient cells can be rescued by an alternative, DNA-PKcs-independent, DNA repair pathway, which is less efficient and less precise. The

reported differences in the fidelity of signal-joining in various DNA-PKcs-deficient backgrounds [19,20,66,68,71,73•] might therefore merely reflect different activities of this rescue pathway. Thus, the activity of an alternative, and DNA-PKcs-independent, signal-joining pathway appears to vary between species, explaining why a DNA-PKcs deficiency in some cases still allows SJ formation whereas it severely impairs signal joining in others.

Novel factors for DNA end-joining in V(D)J recombination? Our knowledge about the enzymology of DNA end-joining in V(D)J recombination has greatly improved during the past couple of years because of the detailed characterization and analysis of mutant cell lines and the generation of mice carrying targeted deletions in individual NHEJ components. A recently described in vitro NHEJ assay [74] might be helpful to identify novel NHEJ components. This assay was shown to depend on Ku proteins and XRCC4−DNA-ligase-IV, which were detectable in two out of three extract subfractions, but the activity could only be reconstituted when a third fraction, with unknown components, was included in the assay. It can be expected that these unidentified NHEJ components may play a role in DNA end-joining of V(D)J recombination as well. The existence of as-yet-unidentified factors required for V(D)J recombination is further suggested by a mutation that affects DSB repair and CJ formation, similarly to mutations in DNA-PKcs, but which maps to human chromosome 10 and must therefore be different from DNA-PKcs [75••].

Conclusions In recent years, a clearer picture has emerged about the way in which V(D)J recombination occurs and about the roles that RAG proteins and the different NHEJ components play in the joining of coding- and signal-ends. There is strong evidence that the RAG proteins are involved in the processing of coding ends, for example in the opening of covalently sealed hairpins and for the removal of 3′ flap structures. However, the possibility remains that ubiquitously expressed DNA-repair factors, for example a complex of Nbs1−Mre11−Rad50, also participate in the processing of hairpinned coding ends and further experiments are needed to determine which processing activities are involved in vivo. Recent data suggest that all components of the DNA-PK holoenzyme play a role for both SJ and CJ formation although alternative DNA-repair pathways may be able to mediate SJ formation in the absence of DNA-PKcs. The DNA-PK components are crucial elements of the RAG postcleavage/end-joining complex. These are possibly required for the remodeling of the postcleavage-complex architecture allowing, for example, the addition of N-region sequences by TdT. DNA-PK components also appear to interact with XRCC4−DNA-ligase-IV on DNA ends, possibly guiding ligation events to the formation of correct SJs and CJs. Despite the recent increase in knowledge about the functions of DNA end-joining activities in V(D)J recombination,

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their mechanistic and biochemical relationship requires further investigation. In addition, there are indications that still-unknown factors are essential for both NHEJ and V(D)J recombination, and their identification may help to fill in some missing links in knowledge about the mechanism of DNA end-joining in V(D)J recombination.

Acknowledgements We would like to thank Fraser McBlane and Heinz Jacobs for helpful suggestions on the manuscript. Most of this review was written at The Basel Institute for Immunology, Basel, Switzerland, which was founded and supported by F. Hoffmann-LaRoche, and is now transformed into a Roche Center for Medical Genomics (RCMG).

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest •• of outstanding interest 1.

Tonegawa S: Somatic generation of antibody diversity. Nature 1983, 302:575-581.

2.

Schlissel MS, Stanhope-Baker P: Accessibility and the developmental regulation of V(D)J recombination. Semin Immunol 1997, 9:161-170.

3. ••

McMurry MT, Krangel MS: A role for histone acetylation in the developmental regulation of V(D)J recombination. Science 2000, 287:495-498. This study analyses histone H3 acetylation, using chromosome immunoprecipitation (ChIP) for various human TCRδ minilocus transgenic constructs with different wild-type and mutant Eα and Eδ enhancer elements, as well as for the endogenous murine TCRα/δ locus. The authors find a tight correlation between activation of gene loci for V(D)J recombination and histone H3 hyperacetylation in these regions.

4. McBlane F, Boyes J: Stimulation of V(D)J recombination by histone • acetylation. Curr Biol 2000, 10:483-486. The authors of this study demonstrate that V(D)J recombination on the κ light-chain locus in an Abelson murine leukemia virus (A-MuLV)-transformed cell line can be activated by treatment with trichostatin A, a drug that inhibits histone deacetylation and thereby results in global nucleosome hyperacetylation. The activation of V(D)J recombination was shown to be specific for the Κ light-chain locus and was not caused by any changes in recombination-activating gene (RAG) expression or activity. The authors therefore conclude that the accessibility of the κ light-chain locus in vivo is increased by histone hyperacetylation. However, inhibition of RAG cleavage by nucleosomes in vitro could not be overcome by histone hyperacetylation, suggesting a regulation at the level of higher-order chromatin structures in vivo. 5. ••

Bassing CH, Alt FW, Hughes MM, D’Auteuil M, Wehrly TD, Woodman BB, Gartner F, White JM, Davidson L, Sleckman BP: Recombination signal sequences restrict chromosomal V(D)J recombination beyond the 12/23 rule. Nature 2000, 405:583-586. This study describes the impact of targeted mutations within endogenous TCR Dβ1 and Jβ1.2 elements in mice on the ordered rearrangements of the TCRβ chain gene locus, where Dβ→Jβ rearrangements usually precede Vβ→DJβ rearrangements. Surprisingly, this study demonstrates that the quality of the RSS appended to the Dβ and Jβ coding regions is crucial for restricting Dβ→Jβ and Vβ→DJβ rearrangements. For instance, deletion of the Dβ1 does not lead to direct Vβ1−Jβ1.2 rearrangements, unless the 12-RSS 5′ of Jβ1.2 is replaced by the 12-RSS 5′ of Dβ1. Conversely, replacement of the 12-RSS 5′ of Dβ1 by the 12-RSS 5′ of Jβ1.2 no longer allows Vβ→DJβ rearrangements. 6.

McBlane JF, van Gent DC, Ramsden DA, Romeo C, Cuomo CA, Gellert M, Oettinger MA: Cleavage at the V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell 1995, 83:387-395.

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Oettinger MA, Schatz DG, Gorka C, Baltimore D: RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science 1990, 248:1517-1523.

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Mombaerts P, Iacomini J, Johnson RS, Herrup K, Tonegawa S, Papaioannou VE: RAG-1-deficient mice have no mature B and T lymphocytes. Cell 1992, 68:869-877.

10. Shinkai Y, Rathbun G, Lam KP, Oltz EM, Stewart V, Mendelsohn M, Charron J, Datta M, Young F, Stall AM, Alt FW: RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 1992, 68:855-867. 11. Gilfillan S, Dierich A, Lemeur M, Benoist C, Mathis D: Mice lacking TdT: mature animals with an immature lymphocyte repertoire. Nature 1993, 261:1175-1178. 12. Featherstone C, Jackson SP: Ku, a DNA repair protein with multiple cellular functions? Mutat Res 1999, 434:3-15. 13. Gottlieb TM, Jackson SP: The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell 1993, 72:131-142. 14. West RB, Yaneva M, Lieber MR: Productive and nonproductive complexes of Ku and DNA-dependent protein kinase at DNA termini. Mol Cell Biol 1998, 18:5908-5920. 15. Hammarsten O, Chu G: DNA-dependent protein kinase: DNA binding and activation in the absence of Ku. Proc Natl Acad Sci USA 1998, 95:525-530. 16. Blunt T, Finnie NJ, Taccioli GE, Smith GCM, Demengeot J, Gottlieb TM, Mizuta R, Varghese AJ, Alt FW, Jeggo PA, Jackson SP: Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell 1995, 80:813-823. 17.

Blunt T, Gell D, Fox M, Taccioli GE, Lehmann AR, Jackson SP, Jeggo PA: Identification of a nonsense mutation in the carboxylterminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc Natl Acad Sci USA 1996, 93:10285-10290.

18. Danska JS, Holland DP, Mariathasan S, Williams KM, Guidos CJ: Biochemical and genetic defects in the DNA-dependent protein kinase in murine scid lymphocytes. Mol Cell Biol 1996, 16:5507-5517. 19. Gao Y, Chaudhuri J, Zhu C, Davidson L, Weaver DT, Alt FW: A targeted DNA-PKcs-null mutation reveals DNA-PK-independent functions for KU in V(D)J recombination. Immunity 1998, 9:367-376. 20. Taccioli GE, Amatucci AG, Beamish HJ, Gell D, Xiang XH, Torres Arzayus MI, Priestley A, Jackson SP, Marshak Rothstein A, Jeggo PA, Herrera VL: Targeted disruption of the catalytic subunit of the DNA-PK gene in mice confers severe combined immunodeficiency and radiosensitivity. Immunity 1998, 9:355-366. 21. Nussenzweig A, Chen C, da Costa Soares V, Sanchez M, Sokol K, Nussenzweig MC, Li GC: Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature 1996, 382:551-555. 22. Zhu C, Bogue MA, Lim DS, Hasty P, Roth DB: Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell 1996, 86:379-389. 23. Gu Y, Seidl KJ, Rathbun GA, Zhu C, Manis JP, van der Stoep N, Davidson L, Cheng HL, Sekiguchi JM, Frank K et al.: Growth retardation and leaky SCID phenotype of Ku70-deficient mice. Immunity 1997, 7:653-665. 24. Ouyang H, Nussenzweig A, Kurimasa A, da Costa Soares V, Li X, Cordon-Cardo C, Li WH, Cheong N, Nussenzweig M, Iliakis G et al.: Ku70 is required for DNA repair but nor for T cell antigen receptor recombination in vivo. J Exp Med 1997, 186:921-929. 25. Li Z, Otevrel T, Gao Y, Cheng HL, Seed B, Stamato TD, Taccioli GE, Alt FW: The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination. Cell 1995, 83:1079-1089. 26. Grawunder U, Wilm M, Wu X, Kulesza P, Wilson TE, Mann M, Lieber MR: Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature 1997, 388:492-495. 27.

Critchlow SE, Bowater RP, Jackson SP: Mammalian DNA doublestrand break repair protein XRCC4 interacts with DNA ligase IV. Curr Biol 1997, 7:588-598.

28. Frank KM, Sekiguchi JM, Seidl KJ, Swat W, Rathbun GA, Cheng HL, Davidson L, Kangaloo L, Alt FW: Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV. Nature 1998, 396:173-177.

How to make ends meet in V(D)J recombination Grawunder and Harfst 193

29. Gao Y, Sun Y, Frank KM, Dikkes P, Fujiwara Y, Seidl KJ, Sekiguchi JM, Rathbun GA, Swat W, Wang J et al.: A critical role for DNA endjoining proteins in both lymphogenesis and neurogenesis. Cell 1998, 95:891-902. 30. Critchlow SE, Jackson SP: DNA end-joining: from yeast to man. Trends Biochem Sci 1998, 23:394-398. 31. Fugmann SD, Lee AI, Shockett PE, Villey IJ, Schatz DG: The RAG proteins and V(D)J recombination: complexes, ends, and transposition. Annu Rev Immunol 2000, 18:495-527. 32. Ramsden DA, Paull TT, Gellert M: Cell-free V(D)J recombination. Nature 1997, 388:488-491. 33. van Gent DC, McBlane JF, Ramsden DA, Sadofsky MJ, Hesse JE, Gellert M: Initiation of V(D)J recombination in a cell-free system. Cell 1995, 81:925-934. 34. van Gent DC, Ramsden DA, Gellert M: The RAG1 and RAG2 proteins establish the 12/23 rule in V(D)J recombination. Cell 1996, 85:107-113. 35. Eastman QM, Leu TMJ, Schatz DG: Initiation of V(D)J recombination in vitro obeying the 12/23 rule. Nature 1996, 380:85-88. 36. Sawchuk DJ, Weis-Garcia F, Malik S, Besmer E, Bustin M, Nussenzweig MC, Cortes P: V(D)J recombination: modulation of RAG1 and RAG2 cleavage activity on 12/23 substrates by whole cell extract and DNA-bending proteins. J Exp Med 1997, 185:2025-2032. 37.

Hiom K, Gellert M: A stable RAG1-RAG2-DNA complex that is active in V(D)J cleavage. Cell 1997, 88:65-72.

38. Melek M, Gellert M, van Gent DC: Rejoining of DNA by the RAG1 and RAG2 proteins. Science 1998, 280:301-303. 39. Agrawal A, Eastman QM, Schatz DG: Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system. Nature 1998, 394:744-751. 40. Hiom K, Melek M, Gellert M: DNA transposition by the RAG1 and RAG2 proteins: a possible source of oncogenic translocations. Cell 1998, 94:463-470. 41. van Gent DC, Mizuuchi K, Gellert M: Similarities between initiation of V(D)J recombination and retroviral integration. Science 1996, 271:1592-1594. 42. Besmer E, Mansilla-Soto J, Cassard S, Sawchuk DJ, Brown G, Sadofsky M, Lewis SM, Nussenzweig MC, Cortes P: Hairpin coding end opening is mediated by RAG1 and RAG2 proteins. Mol Cell 1998, 2:817-828. 43. Shockett PE, Schatz DG: DNA hairpin opening mediated by the RAG1 and RAG2 proteins. Mol Cell Biol 1999, 19:4159-4166. 44. Roth DB, Menetski JP, Nakajima PB, Bosma MJ, Gellert M: V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding end in scid mouse thymocytes. Cell 1992, 70:983-991. 45. Han JO, Erskine LA, Purugganan MM, Stamato TD, Roth DB: V(D)J recombination intermediates and non-standard products in XRCC4-deficient cells. Nucleic Acids Res 1998, 26:3769-3775. 46. Agrawal A, Schatz DG: RAG1 and RAG2 form a stable postcleavage synaptic complex with DNA containing signal ends in V(D)J recombination. Cell 1997, 89:43-53. 47. ••

Paull TT, Gellert M: Nbs1 potentiates ATP-driven DNA unwinding and endonuclease cleavage by the Mre11/Rad50 complex. Genes Dev 1999, 13:1276-1288. This study describes the in vitro activity of a ternary complex of the purified human DNA-repair proteins, Nbs1, Mre11 and Rad50. The authors find that the Nbs1 protein is required to allow Mre11 nuclease to endonucleolytically open fully paired hairpin structures, which are V(D)J reaction intermediates for coding-joint formation. This activity appears to be mediated by a partial unwinding of the DNA duplex close to the hairpin tip, mediated by the Nbs1 protein. The activity of Nbs1−Mre11−Rad50 would therefore be compatible with a proposed coding-end hairpin-opening activity. 48. Carney JP, Maser RS, Olivares H, Davis EM, Le Beau M, Yates JR III, Hays L, Morgan WF, Petrini JH: The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of doublestrand break repair to the cellular DNA damage response. Cell 1998, 93:477-486. 49. Varon R, Vissinga C, Platzer M, Cerosaletti KM, Chrzanowska KH, Saar K, Beckmann G, Seemanova E, Cooper PR, Nowak NJ et al.:

Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome. Cell 1998, 93:467-476. 50. Liang F, Jasin M: Ku80-deficient cells exhibit excess degradation of extrachromosomal DNA. J Biol Chem 1996, 271:14405-14411. 51. Bogue MA, Wang C, Zhu C, Roth DB: V(D)J recombination in Ku86deficient mice: distinct effects on coding, signal, and hybrid joint formation. Immunity 1997, 7:37-47. 52. Mahajan KN, Gangi-Peterson L, Sorscher DH, Wang J, Gathy KN, • Mahajan NP, Reeves WH, Mitchell BS: Association of terminal deoxynucleotidyl transferase with Ku. Proc Natl Acad Sci USA 1999, 96:13926-13931. The authors report the capability of TdT to physically interact with the Ku protein complex, as well as with the individual Ku70 and Ku86 components, suggesting that Ku recruits TdT into the recombination-activating gene (RAG) postcleavage complex. The Ku−TdT interaction appears to be DNAindependent and is mediated by amino-terminal 131 amino acids of TdT, which contain a BRCT (BRCA-1 carboxyl terminus) domain. BRCT domains have previously been shown to mediate protein−protein interaction between DNA-repair proteins. This result provides a biochemical explanation for the lack of nontemplated nucleotide (N)-region sequences in rare coding joints from Ku-deficient animals. 53. Gerstein RM, Lieber MR: Extent to which homology can constrain coding exon junctional diversity in V(D)J recombination. Nature 1993, 363:625-627. 54. Santagata S, Besmer E, Villa A, Bozzi F, Allingham JS, Sobacchi C, •• Haniford DB, Vezzoni P, Nussenzweig MC, Pan ZQ, Cortes P: The RAG1/RAG2 complex constitutes a 3′′ flap endonuclease: implications for junctional diversity in V(D)J and transpositional recombination. Mol Cell 1999, 4:935-947. This study shows that the recombination-activating gene (RAG)-1−RAG-2 complex displays preferential binding to double-strand−single-strand DNA junctions in vitro and that it is capable of endonucleolytically removing 3′ single-stranded DNA extensions of annealed oligonucleotide substrates. Both activities are independent of recombination signal sequence (RSS) binding of RAG1−RAG2. These findings make it conceivable that a complex of RAG1−RAG2 itself participates in the trimming of proposed nonhomologous-DNA end-joining intermediates in V(D)J recombination. 55. Paull TT, Gellert M: The 3′′ to 5′′ exonuclease activity of Mre 11 facilitates repair of DNA double-strand breaks. Mol Cell 1998, 1:969-979. 56. Paull TT, Gellert M: A mechanistic basis for Mre11-directed DNA •• joining at microhomologies. Proc Natl Acad Sci USA 2000, 97:6409-6414. The authors of this study show that the in vitro 3′→5′ exonuclease activity of purified human Mre11 protein on DNA-ends is stimulated by the addition of noncompatible DNA ends. However, when regions of internal microhomology between two DNA-ends are reached, Mre11 nuclease activity is repressed. The authors further show that Mre11 allows the rejoining of linearized plasmids with incompatible DNA-ends by X-ray cross-complementation group (XRCC)4−DNA-ligase-IV. These findings suggest that Mre11 may be involved in trimmimg of 3′ overhanging ends during microhomologydirected DNA end-joining and possibly V(D)J recombination. 57.

Harrington JJ, Lieber MR: The characterization of a mammalian DNA structure-specific endonuclease. EMBO J 1994, 13:1235-1246.

58. Wu X, Wilson TE, Lieber MR: A role for FEN-1 in nonhomologous • DNA end joining: the order of strand annealing and nucleolytic processing events. Proc Natl Acad Sci USA 1999, 96:1303-1308. The authors find that deletion of RAD27, encoding the Saccharomyces cerevisiae homologue of the mammalian 5′-flap endonuclease FEN-1, leads to a shift in the spectrum of NHEJ products of intracellularly recircularized plasmid substrates. Specifically, those joining-products that would require 5′-flap processing are 4.4-fold reduced in comparison with joining-products recovered from wild-type or exonuclease 1 (EXO1)-deficient yeast cells. The effect on substrate-joining in RAD27-deficient yeast could be reconstituted with a wild-type RAD27 expression vector. The authors propose a role for RAD27 (FEN-1) in processing microhomology-annealed DNA ends. 59. Grawunder U, Zimmer D, Fugmann S, Schwarz K, Lieber MR: DNA ligase IV is essential for V(D)J recombination and DNA doublestrand break repair in human precursor lymphocytes. Mol Cell 1998, 2:477-484. 60. McElhinny SA, Snowden CM, McCarville J, Ramsden DA: Ku recruits • the XRCC4-ligase IV complex to DNA ends. Mol Cell Biol 2000, 20:2996-3003. This paper analyzes the effect of Ku proteins on the activity of purified X-ray cross-complementation group (XRCC)4−DNA-ligase-IV for DNA end ligation in vitro. The authors show that XRCC4−DNA-ligase-IV has undetectable DNA-binding activity on its own but that the complex is recruited to DNA ends

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by direct protein−protein interaction with Ku. The study further shows that the catalytic activity of XRCC4−DNA-ligase-IV for end joining is stimulated by addition of Ku proteins and that Ku specifically interacts with XRCC4−DNAligase-IV but not with DNA ligases I or II (see also annotation to [61••]).

69. Wiler R, Leber R, Moore BB, van Dyk LF, Perryman LE, Meek K: Equine severe combined immunodeficiency: a defect in V(D)J recombination and DNA-dependent protein kinase activity. Proc Natl Acad Sci USA 1995, 92:11485-11489.

61. Chen L, Trujillo K, Sung P, Tomkinson AE: Interactions of the DNA •• ligase IV-XRCC4 complex with DNA ends and the DNA-dependent protein kinase. J Biol Chem 2000, 275:26196-26205. The authors of this study characterize the activities of a purified X-ray cross-complementation group (XRCC)4−DNA-ligase-IV complex in DNA end-joining in vitro in the absence and presence of DNA-PK components. In contrast to the study by McElhinny et al. [60•], this paper states that XRCC4−DNA-ligase-IV binds to DNAends on its own, as shown both by electrophoretic mobility shift assay (EMSA) and by atomic force microscopy (AFM) analyses. In agreement with [60•], this study finds that Ku proteins interact with XRCC4−DNA-ligase-IV at DNA ends. In the assays presented in this study, addition of Ku proteins inhibits the ligation of DNA substrates by XRCC4−DNA-ligase-IV. However, interestingly, the authors find that addition of DNA-PK catalytic subunit or DNA-PK holoenzyme to plasmid-religation assays by XRCC4−DNA-ligase-IV favors intramolecular ligation events.

70. Errami A, He DM, Friedl AA, Overkamp WJ, Morolli B, Hendrickson EA, Eckardt-Schupp F, Oshimura M, Lohman PH, Jackson SP, Zdzienicka MZ: XR-C1, a new CHO cell mutant which is defective in DNA-PKcs, is impaired in both V(D)J coding and signal joint formation. Nucleic Acids Res 1998, 26:3146-3153.

62. Modesti M, Hesse JE, Gellert M: DNA binding of XRCC4 protein is •• associated with V(D)J recombination but not with stimulation of DNA ligase IV activity. EMBO J 1999, 18:2008-2018. This detailed analysis of in vitro functions of purified X-ray cross-complementation group (XRCC)4−DNA-ligase-IV demonstrates that XRCC4 displays DNA-binding activity that can be competed for most efficiently by nicked and linear, but not supercoiled, DNA. The authors further show that the stimulation of DNA ligase IV activity by XRCC4 occurs at the level of adenylation of DNA ligase IV. Mutational analysis further indicates that the DNA-binding activity of XRCC4 is essential for its in vivo function whereas the activation of ligase activity is not. Collectively, the data indicate that XRCC4 may be required to efficiently target DNA ligase IV to sites of DNA breaks. 63. Teo SH, Jackson SP: Lif1p targets the DNA ligase Lig4p to sites of • DNA double-strand breaks. Curr Biol 2000, 10:165-168. In this study, the authors report that the Saccharomyces cerevisiae homologue of mammalian purified X-ray cross-complementation group (XRCC)4, Lif1p (ligase 4 interacting factor), is able to bind DNA and to stimulate Lig4p (yeast DNA ligase IV) activity in vitro. The authors further show that Lif1p is required to target Lig4p to HO-endonuclease-induced DSBs, as shown by chromatin immunoprecipitation experiments. This recruitment is dependent on the presence of Ku. 64. Gu Y, Jin S, Gao Y, Weaver DT, Alt FW: Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination. Proc Natl Acad Sci USA 1997, 94:8076-8081. 65. Lieber MR, Hesse JE, Lewis S, Bosma GC, Rosenberg N, Mizuuchi K, Bosma MJ, Gellert M: The defect in murine severe combined immune deficiency: joining of signal sequences but not coding segments in V(D)J recombination. Cell 1988, 55:7-16. 66. Bogue MA, Jhappan C, Roth DB: Analysis of variable (diversity) joining recombination in DNA dependent protein kinase (DNAPK)-deficient mice reveals DNA-PK-independent pathways for both signal and coding joint formation. Proc Natl Acad Sci USA 1998, 95:15559-15564. 67.

Zdzienicka MZ: Mammalian X-ray-sensitive mutants which are defective in non-homologous (illegitimate) DNA double-strand break repair. Biochimie 1999, 81:107-116.

68. Kulesza P, Lieber MR: DNA-PK is essential only for coding joint formation in V(D)J recombination. Nucleic Acids Res 1998, 26:3944-3948.

71. Fukumura R, Araki R, Fujimori A, Mori M, Saito T, Watanabe F, Sarashi M, Itsukaichi H, Eguchi-Kasai K, Sato K et al.: Murine cell line SX9 bearing a mutation in the dna-pkcs gene exhibits aberrant V(D)J recombination not only in the coding joint but also in the signal joint. J Biol Chem 1998, 273:13058-13064. β 72. Shin EK, Rijkers T, Pastink A, Meek K: Analyses of TCRβ • rearrangements substantiate a profound deficit in recombination signal sequence joining in SCID foals: implications for the role of DNA-dependent protein kinase in V(D)J recombination. J Immunol 2000, 164:1416-1424. This paper describes a quantitative analysis of signal joint (SJ) and coding joint (CJ) formation on the endogenous TCRβ locus in severe combined immunodeficiency (scid) foals, that carry a mutation in DNA-PKcs (catalytic subunit). In contrast to the mouse scid mutation, the equine scid mutation had previously been reported to cause a deficiency in both SJ and CJ formation [69]. The level of SJ formation in scid foals was found to be four-logs reduced compared with wild-type controls. Both SJ and CJ formation could be restored by expression of a wild-type DNA-PKcs cDNA. This result excludes the possibility that the equine scid mutation leads to the expression of a dominant-negative form of DNA-PKcs. It suggests that DNA-PKcs is involved in SJ formation and that an alternative SJ pathway exists in scid cells from other species, conferring relatively normal signal-joining. 73. Fukumura R, Araki R, Fujimori A, Tsutsumi Y, Kurimasa A, Li GC, • Chen DJ, Tatsumi K, Abe M: Signal joint formation is also impaired in DNA-dependent protein kinase catalytic subunit knockout cells. J Immunol 2000, 165:3883-3889. This study desribes a detailed analysis of SJ formation with V(D)J recombination substrates in cell lines from DNA-PKcs-deficient mice. The study shows that different cell lines carrying the same DNA-PKcs mutation display a 2–4.5-fold reduction in SJ formation, with only a 14–19% SJ fidelity. This phenotype can be reconstituted by expression of wild-type DNA-PKcs cDNA. Like other studies [69–71], these results indicate that DNA-PKcs is also involved in SJ formation and that an alternative repair pathway may allow SJ formation to occur, albeit at lower levels and with less precision. 74. Baumann P, West SC: DNA end-joining catalyzed by human cellfree extracts. Proc Natl Acad Sci USA 1998, 95:14066-14070. 75. Moshous D, Li L, Chasseval R, Philippe N, Jabado N, Cowan MJ, •• Fischer A, de Villartay JP: A new gene involved in DNA doublestrand break repair and V(D)J recombination is located on human chromosome 10p. Hum Mol Genet 2000, 9:583-588. The authors of this study present a more precise linkage analysis for an unidentified mutation mapping to human chromosome 10p that leads to a high incidence of T−B− SCID (severe combined immunodeficiency) in Athabascan-speaking Navajo and Apache Native-Americans. The mutation is shown to selectively affect CJ formation in extracellular plasmid V(D)J recombination assays and indicates the presence of a novel gene required for V(D)J recombination in vivo.