Involvement of Artemis in nonhomologous end-joining during immunoglobulin class switch recombination

Abstract

DNA double-strand breaks (DSBs) introduced in the switch (S) regions are intermediates during immunoglobulin class switch recombination (CSR). These breaks are subsequently recognized, processed, and joined, leading to recombination of the two S regions. Nonhomologous end-joining (NHEJ) is believed to be the principle mechanism involved in DSB repair during CSR. One important component in NHEJ, Artemis, has however been considered to be dispensable for effi cient CSR. In this study, we have characterized the S recombinational junctions from Artemis-defi cient human B cells. S -S ␣ junctions could be amplifi ed from all patients tested and were characterized by a complete lack of " direct " end-joining and a remarkable shift in the use of an alternative, microhomology-based endjoining pathway. S -S ␥ junctions could only be amplifi ed from one patient who carries " hypomorphic " mutations. Although these S -S ␥ junctions appear to be normal, a significant increase of an unusual type of sequential switching from immunoglobulin (Ig)M, through one IgG subclass, to a different IgG subclass was observed, and the S ␥ -S ␥ junctions showed long microhomologies. Thus, when the function of Artemis is impaired, varying modes of CSR junction resolution may be used for different S regions. Our fi ndings strongly link Artemis to the predominant NHEJ pathway during CSR.

Artemis splice defects cause atypical SCID and can be restored in vitro by an antisense oligonucleotide

“…30,31 Previously described Artemis-deficient patients showed a strong dependence on long microhomologies and a complete lack of 'direct end-joining'. 23 The average length of microhomology, defined as successive nucleotides that were shared by both the Sm and Sa regions at the CSR junctions, was not significantly different from control in patient 1 (3.8 ± 5.2 bp versus 1.8±3.2 bp in controls) and in patient 2 (2.0±2.7 bp versus 1.8 ± 3.2 bp in controls). Furthermore, the pattern of Sm-Sa junctions was indistinguishable from that in controls (Table 4).…”

“…In mature B cells, Artemis is involved in CSR. 23,38 Artemisdeficiency results in CSR characterized by strong dependence of long microhomologies in the S junctions and a complete lack of ''direct end-joining''. We showed that reduced levels of WT Artemis and residual Artemis activity resulted in normal S-junction formation.…”

“…29 Artemis has recently been shown to be involved in CSR. 23 Switch (S) junctions resulting from in vivo (CSR) events, were cloned and sequenced from B cells of patient 1 and 2. Unique Sm-Sa1 sequences, representing independent CSR events, were subsequently compared with Sm and Sa junctions (n ¼ 154) from healthy adult controls.…”

“…Unlike classical SCID, patients with Omenn's syndrome have severe erythroderma, increased IgE levels and eosinophilia. 22,23 Hypomorphic RAG1 and RAG2 mutations have also been reported to cause primary immunodeficiency disease with granulomatous skin lesions. 24 Here, we present two SCID patients with different types of Artemis splicing defects, both leading to an atypical Artemis-SCID phenotype, characterized by a later onset and milder disease course and, in one patient, severe localized granulomatous skin lesions.…”

Programmed DNA breaks in lymphoid cells: repair mechanisms and consequences in human disease

“…Therefore the role of the DNA–PK complex in CSR was considered to be in regulating end processing, possibly by phosphorylating other proteins 25. However, it has since been shown that Artemis is indeed involved in CSR, where its nuclease activity may function in resolving more complex lesions that are generated during CSR 26. The polymerases involved in CSR are thought to include the error prone polymerases, polymerase η and REV1 27, 28…”