Alternative end-joining catalyzes robust IgH locus deletions and translocations in the combined absence of ligase 4 and Ku70

Abstract

Class switch recombination (CSR) in B lymphocytes is initiated by introduction of multiple DNA double-strand breaks (DSBs) into switch (S) regions that flank immunoglobulin heavy chain ( IgH ) constant region exons. CSR is completed by joining a DSB in the donor Sμ to a DSB in a downstream acceptor S region (e.g., Sγ1) by end-joining. In normal cells, many CSR junctions are mediated by classical nonhomologous end-joining (C-NHEJ), which employs the Ku70/80 complex for DSB recognition and XRCC4/DNA ligase 4 for ligation. Alternative end-joining (A-EJ) mediates CSR, at reduced levels, in the absence of C-NHEJ, even in combined absence of Ku70 and ligase 4, demonstrating an A-EJ pathway totally distinct from C-NHEJ. Multiple DSBs are introduced into Sμ during CSR, with some being rejoined or joined to each other to generate internal switch deletions (ISDs). In addition, S-region DSBs can be joined to other chromosomes to generate translocations, the level of which is increased by absence of a single C-NHEJ component (e.g., XRCC4). We asked whether ISD and S-region translocations occur in the complete absence of C-NHEJ (e.g., in Ku70/ligase 4 double-deficient B cells). We found, unexpectedly, that B-cell activation for CSR generates substantial ISD in both Sμ and Sγ1 and that ISD in both is greatly increased by the absence of C-NHEJ. IgH chromosomal translocations to the c-myc oncogene also are augmented in the combined absence of Ku70 and ligase 4. We discuss the implications of these findings for A-EJ in normal and abnormal DSB repair.

Elucidation of IgH 3′ region regulatory role during class switch recombination via germline deletion

“…It could be argued that the few S m -S g1 joints found in 3 0 RR-deficient B-cells mask a defect in end-joining that would result in aborted CSR (and, therefore, no joint to analyse). To test this hypothesis we analysed intra S m deletions that frequently happen in mature B-cells during CSR 20 . By using a PCR amplifying the whole S m region followed by Southern blotting, we checked that B-cell stimulation yielded fragments of different lengths corresponding to partial deletion of S m during CSR.…”

“…Altogether, this might inhibit A-EJ, by downregulating gene implicated in microhomology based DSB repair (Atm, Atmin, Ssb1 and Hdac9), and to favour NHEJ by upregulating Parp-3 41 , and Esco1, which influence the interaction between cohesion complex and DNA required for efficient NHEJ 42,43 . A-EJ has been shown to facilitate oncogenic translocations to IgH locus and to be less restricted to intrachromosomal joining than NHEJ 20 . This might be a protective mechanism limiting oncogenic translation to S m , in Figure 6 | Epigenetic marks, AID and pol II recruitments in S c3 , S c2b and S e during CSR in 3 0 RR-deficient mice.…”

Comparison of Constitutional and Replication Stress-Induced Genome Structural Variation by SNP Array and Mate-Pair Sequencing

“…Called SVs have been deposited in NCBI dbVar. chromosomal translocations (McVey and Lee 2008;Boboila et al 2010;Simsek and Jasin 2010), and entirely distinct pathways in which stalled replication structures are processed by mechanisms variably known as template switching or microhomology-mediated breakinduced replication (MMBIR) (Lee et al 2007;Hastings et al 2009a).To date, these mechanisms have largely been inferred by examination of human CNV breakpoint sequences Vissers et al 2009;Conrad et al 2010a). To begin to explore CNV mechanisms experimentally, we recently reported a system in which normal human fibroblasts were treated with the replication inhibitor aphidicolin (Arlt et al 2009).…”

“…Called SVs have been deposited in NCBI dbVar. chromosomal translocations (McVey and Lee 2008;Boboila et al 2010;Simsek and Jasin 2010), and entirely distinct pathways in which stalled replication structures are processed by mechanisms variably known as template switching or microhomology-mediated breakinduced replication (MMBIR) (Lee et al 2007;Hastings et al 2009a).…”

X-ray repair cross-complementing protein 1 (XRCC1) deficiency enhances class switch recombination and is permissive for alternative end joining

“…DSB repair in the absence of an intact NHEJ system has been collectively termed alternative end joining (A-EJ) (3,4). A-EJ could be a component-substitution form of NHEJ or a distinct pathway (or pathways) (7)(8)(9). So far, components of A-EJ have not been conclusively defined.…”