VDJ Recombination Artemis and Its In Vivo Role in Hairpin Opening

Artemis is the newest player in VDJ recombination and double strand break repair. First identified in radiation-sensitive and immune-deficient patients, it was recently shown to interact with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and have nuclease activity, becoming the most popular candidate for the opening of hairpin coding ends. Reports presented in this issue (1) and in the December issue of Molecular Cell (2) address the role of Artemis in vivo by studying the effect of its deletion on the generation of intermediates and products of VDJ recombination in mouse ES cells, MEFs, and thymocytes. In all three systems analyzed, a defect in coding joint formation was observed, which was highly dependent on the cellular background and included inefficient and partially defective opening of hairpin coding ends. In addition, Artemis-deficient ES cells and Artemis-deficient MEFs show spontaneous chromosomal abnormalities, including telomere fusions, indicating that Artemis is also required for maintenance of genomic stability. This commentary will focus on the results regarding the role of Artemis in VDJ recombination. VDJ recombination, also known as antigen receptor gene rearrangement, is the process that assembles the variable domain of immunoglobulin and TCR genes. The hallmark of this reaction is the production of a large repertoire of antigen receptors with different specificities, a characteristic that is essential to the normal functioning of the immune system. VDJ recombination is directed to the immunoglobulin and TCR loci by highly conserved recombination signal sequences (RSSs) comprised of a heptamer and a nonamer motif with an intervening 12- or 23-bp spacer. Efficient recombination in vivo occurs almost exclusively between RSSs with different spacers, which is referred to as the 12/23 rule (3). Several proteins that mediate VDJ recombination in vivo have been identified (4). The lymphoid-specific components of the recombination machinery are RAG-1, RAG-2, and terminal deoxynucleotidyl transferase (TdT). RAG-1 and RAG-2 together constitute the recombinase. TdT, although not essential for catalysis, plays an important role increasing diversity by mediating the incorporation of nontemplate-dependent nucleotides. The nonlymphoid-restricted components identified so far are DNA-PKcs, Ku70, Ku80, XRCC4, ligase 4, and the most recently identified Artemis. All these proteins are involved in DNA double strand break repair as well as VDJ recombination. These nonlymphoid-specific components are likely to participate in the processing and joining steps of VDJ recombination, but their specific architectural or catalytic roles in the reaction remain largely unclear. Two other nonlymphoid-specific components, HMG1 and HMG2, have been implicated in VDJ recombination. In vitro experiments showed that these proteins increase the efficiency of cleavage by RAGs (5, 6). The role of HMG1 and HMG2 in VDJ recombination in vivo remains to be investigated. During the initial stages of the reaction, RAG-1/RAG-2 form a complex with the RSS, which is in part stabilized by the interactions between the nonamer binding domain of RAG-1 and the nonamer motif. Bridging of 12 and 23 RSSs in a synaptic complex is critical for DNA cleavage and it seems to be facilitated by the DNA bending proteins HMG1 and HMG2 as well as other yet unidentified cellular factors (Fig. 1) . Within the synaptic complex, RAG-1/RAG-2 efficiently introduce a nick at each RSS via a hydrolysis reaction at the heptamer/coding flank border, generating a 3′ hydroxyl end. Subsequently, a transesterification reaction that resembles the mechanism of transpositional recombination creates a double strand break as the free 3′ hydroxyl of the nicked strand is used in a nucleophilic attack on the opposing strand generating a covalently sealed hairpin intermediate, known as the hairpin coding end (5, 6). Opening and processing of this hairpin intermediate is a critical step in the generation of junctional diversity and has been the topic of intensive analysis in the last few years. Figure 1. Schematic representation of protein–DNA complexes in VDJ recombination. Blue and yellow rectangles represent the coding segments to be recombined (exemplified as Vλ and Jλ). The 12-RSS and 23-RSS are represented as white and black ... In vitro experiments demonstrated that after RSS cleavage, RAG-1/RAG-2 and HMG1 proteins remain bound in a complex with the resulting 5′ phosphorylated blunt signal end and the hairpin coding end in what is known as the postcleavage complex (7). At this stage, the generation of junctional diversity is initiated by opening of hairpin coding ends leading to the formation of open double strand ends, which may contain single stranded overhangs. Also at this stage, the open coding ends are potential substrates for nontemplate nucleotide additions by TdT, polymerase extension leading to palindromic nucleotides, nuclease attack generating small coding element deletions, and microhomology-directed nonhomologous end joining (NHEJ) via annealing of short complementary stretches of the overhangs. These coding end intermediates, arising at this late stage of VDJ recombination, have common branched structures characterized by duplex/single strand junctions lacking consensus binding motifs (8). Many of these recombination intermediates could be processed by the nuclease activity of Artemis, RAG-1/RAG-2, or the Mre11/Rad50/NBS1 (MRN) complex. Evidence supporting the participation of these protein complexes in hairpin coding end opening and processing will be further discussed. Ultimately, the VDJ recombination reaction is concluded with the joining of the processed coding ends and the unprocessed signal ends by the ubiquitously expressed double strand break repair proteins. Although it is not clearly known when the double strand break repair factors are incorporated in the recombination complex, it is plausible that they coexist with RAG-1/RAG-2 during the opening and processing of the coding ends as suggested in the scheme presented in Fig. 1. Furthermore, evidence accumulated so far suggests that coding and signal joints are formed independently (4), which argues for the existence of different protein–DNA complexes during the later stages of VDJ recombination.