Variable deletion and duplication at recombination junction ends: Implication for staggered double-strand cleavage in class-switch recombination

Abstract

Immunoglobulin class-switch recombination (CSR) gives rise to looped-out circular DNA of a cleaved S segment, which is lost eventually after cell divisions. To understand the molecular mechanism of S region cleavage during CSR, we constructed artificial CSR substrates in which inversion-type CSR takes place to retain the cleaved S segment. Sequencing analyses of recombinant clones of these substrates revealed that varying degrees of deletions and duplications exist at CSR breakpoints, suggesting the involvement of staggered cleavage of the S region in CSR. In addition, mutations frequently found near junctions showed a similar profile of base replacement to Ig somatic hypermutation. These findings suggest that single-strand tails of staggered cleavage may be repaired by error-prone DNA synthesis. N ewly generated B cells expressing IgM and IgD migrate to peripheral lymphoid organs where they are activated by encounters with antigens, resulting in the clonal expansion of antigenspecific B cells. Activated B cell clones secrete not only IgM but also other isotypes of Ig molecules without altering the antigen specificity. This phenomenon, termed class switching, is mediated by DNA recombination in the Ig heavy (H) chain constant (C) region locus, which results in expression of a new transcription unit containing the same productively rearranged variable region (V H ) gene with a downstream C H gene other than C .Class-switch recombination (CSR) from IgM to another isotype takes place anywhere in several-kilobase G-rich regions, termed switch (S) regions, which are located 5Ј to each set of C H exons and consist of tandemly repetitive short sequences with many palindromes (1-9). CSR is accompanied by a looping-out deletion of intervening DNA sequences (10-12). Only limited sequence homology is found near the site of recombination between S and other S sequences (13-15). Because the junctions of CSR are scattered over the S regions and in some instances in proximity to but outside the S regions, CSR is a region-specific recombination event.It has been well established that the ability of a given cytokine to determine the target specificity of CSR is correlated with transcription induction (germline transcription) from a specific intron promoter located immediately upstream of each S region (16). The germline transcription is believed to regulate chromatin opening (accessibility) of the target S region to determine the isotype specificity of CSR (17, 18). Efficiency of CSR seems to be correlated quantitatively with the activity of germline transcription (19). In addition, subsequent splicing of the CSR germline transcripts seems important for CSR (20,21), but the exact role of germline transcripts per se in CSR remains to be elucidated.Previously, we described an artificial CSR substrate in which each of two S regions was directed by a different constitutive promoter, and transcripts were spliced (22). This study demonstrated that the isotype specificity of CSR was not determined by the nucleotide sequences of S regio...

Single-Stranded DNA Breaks Adjacent to Cytosines Occur during Ig Gene Class Switch Recombination

“…A similar strategy has been used to detect breaks in the human S region (22,23), yielding ladders of multiple amplified products. Other investigators have proposed CSR models that would predict not blunt, but staggered, cleavages targeted near stem-loop structures formed in ssDNA (24); certainly staggered ends would be produced if all cleavages occur adjacent to cytosines deaminated by AID.…”

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination

“…Frequent and long deletions and duplications are observed at the CSR junctions in artificial switch substrates designed to select inversion-type products (11). The results suggest that the double strand breaks (DSBs) in the S regions leading to CSR, are generated by the staggered nick-type cleavages because deletions and duplications are most likely to be generated during the repair of staggered nicked ends with single-strand overhangs by exonucleases or DNA polymerases.…”

Internal IgH class switch region deletions are position-independent and enhanced by AID expression

“…2 and 11). Notably, both doublestrand breaks and staggered single-strand breaks have been implicated as early intermediates in the CSR process (12,13). Once initiated, CSR has been shown to require an intact nonhomologous DNA end-joining system for completion (reviewed in ref.…”