Indirect and direct evidence for DNA double–strand breaks in hypermutating immunoglobulin genes

Abstract

The generation of a diverse antigen receptor repertoire is fundamental for the functionality of the adaptive immune system. While the V(D)J recombination process that generates the primary antigen receptor repertoire is understood in great detail, it is still unclear by which mechanism immunoglobulin (Ig) genes are further diversi¢ed by somatic hypermutation. Using mouse strains that carry a non-functional, prede¢ned V H D H J H gene segment in their IgH locus we demonstrate DNA double-strand breaks (DSBs) in and around V H D H J H in B cells undergoing somatic hypermutation. The generation of these DSBs depends on transcriptional activity, and their distribution along the V H D H J H segment parallels that of point mutations in the hypermutation domain. Furthermore, similar to hot spots of somatic hypermutation, 50^60% of all DSBs occur preferentially at RGYW motifs. DSBs may transiently dissociate the Ig promoter from the intronic enhancer to block further transcription and to initiate an error-prone nonhomologous DSB repair pathway. In accord with this model large deletions are frequently produced, along with point mutations, in a V H D H J H segment inserted together with its promoter into the IgH locus in inverted orientation. Our data suggest that DSBs are reaction intermediates of the mechanism underlying somatic hypermutation.

AID-GFP chimeric protein increases hypermutation of Ig genes with no evidence of nuclear localization

“…Deletions have been shown to be an integral part of hypermutation (22), and, therefore, we believe that far from being artifactual, there is a mechanistic basis for the increase. It is possible that the increase reflects an alteration in the balance of the different steps of the establishment and fixation of mismatches.…”

The Block in Immunoglobulin Class Switch Recombination Caused by Activation-Induced Cytidine Deaminase Deficiency Occurs Prior to the Generation of DNA Double Strand Breaks in Switch μ Region

“…2, the CSR-induced DNA breaks that we observed do not preferentially occur on C residues. This reflects the fact that DSBs are probably the consequence of ssDNA breaks occurring on two C residues in close proximity on each DNA strand, leading to secondary DSBs following base excision repair (21,36). The required repair mechanisms are known to be distinct in SHM and CSR.…”

Evolving responsively: adaptive mutation

“…114) has garnered strong support from recent reports of DNA breaks as intermediates in the process (reviewed in REF. 115). These might be associated with transcription, a postulate not tested in the lac system.…”