Somatic hypermutation in 5′ flanking regions of heavy chain antibody variable regions

Abstract

The aim of this study has been to determine the distribution of somatic mutations in the 5' flanking regions of rearranged immunoglobulin heavy chain variable region genes (VDJ). We sequenced the 5' flanking region in 12 secondary immune response antibodies produced in C57BL/6j mice against the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to chicken-gamma-globulin. In these and previously published sequences, almost 97% of the mutations occurred in the transcribed region of the gene, and only a minority of genes (5/29) contained mutations upstream of the transcription start (cap) site. No potential germ-line donor was found for a cluster of five base changes previously found in a single heavy chain gene, 3B62. However, the uniqueness of this mutational cluster and its distance from the normally mutated region suggests that the nucleotide changes may not be due to the normal mutator mechanism. Thus, as this was the only instance of somatic mutations that far upstream of the promoter/cap site region, the reverse transcriptase model for somatic hypermutation is still a possibility. The data are consistent with a mutational mechanism that requires transcription of the rearranged target V(D)J gene which appears to result in the generation of a positively skewed asymmetrical distribution of somatic mutations. A single mode is centered near the V(D)J and a long tail extends into the 3' non-translated region of the J-C intron. Two classes of model could explain this mutation distribution pattern: those where transcription products (RNA, cDNA) are the direct mutational substrates, or those that postulate local unfolding of the chromatin around a V(D)J rearrangement directly exposing the DNA of the transcribed region to specific mutational enzymes.

Multiple sequences from downstream of the Jκ cluster can combine to recruit somatic hypermutation to a heterologous, upstream mutation domain

“…However, the 5' border of the mutation domain in normal Ig genes is in the vicinity of the promoter [15][16][17][18], some 100-200 nucleotides downstream of the transcription start site [19,20]. This positioning of the 5' border of the mutation domain with respect to the start site remains even in the g G-C O transgene when the g -globin gene provides both the promoter and the bulk of the mutation domain (Fig.…”

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

“…This`ligation protection phenomenon' might relate to the presence of a transient DNA hairpin or, alternatively, a covalently bound peptide, which renders the 3' break site inert to the ligation of an adapter molecule. (Lebecque & Gearhart 1990;Peters & Storb 1996;Rothen£uh et al 1993;Weber et al 1991), DSBs are found in an area of 1.3 kb downstream of the promoter and peak around CDR2 and CDR3 (not shown, see Bross et al 2000). The distribution of DSBs (¢gure 3a) along the targeted V H B1-8 gene is remarkably similar to that of the somatic point mutations (Fukita et al 1998).…”

Evaluation of Molecular Models for the Affinity Maturation of Antibodies: Roles of Cytosine Deamination by AID and DNA Repair

“…This would predict that the 5′ edge of SHM could be upstream of its promoter. However, the SHM data clearly show that hypermutations rarely occur 5′ of the promoter 10,139 . Some modifications to this model may be necessary to accommodate this fact.…”