Targeting of the somatic hypermutation mechanism

During humoral immunity to T cell-dependent antigens, responding B lymphocytes selectively mutate their antibody variable region genes at a high rate. This, together with the process of clonal selection, ultimately enhances the affinity and specificity of the antibody molecule and memory B cells that express it as a receptor. Immunoglobulin somatic hypermutation is the only known instance where DNA mutations are physiologically induced; they are not a haphazard result of environmental insult, biological accident, or metabolic stress. Despite several decades of investigation, the mutation mechanism has remained unresolved, largely due to the convoluted nature of experimental systems used to study it.; Due to the limitations of available experimental systems, I extensively analyzed the microsequence context of somatic mutations in order to investigate the molecular basis of the mutation mechanism. Somatic mutations preferentially occur within specific oligonucleotide motifs, and this targeting is consistent in all heavy and light chain antibody genes of humans and mice, thus suggesting the existence of a common mechanism. The mutation targeting analyses also implicate evolution of germline variable gene sequences to direct somatic mutations to specific codon positions in a manner that regulates the frequency of amino acid replacements to the benefit of the antibody Product. Finally, I have obtained results suggesting that somatic hypermutation occurs preferentially, perhaps exclusively, at two bases on both strands of DNA. These analyses have addressed fundamental characteristics of somatic hypermutation and have helped to shape the working hypotheses about its mechanism.; In addition to the above strategy, I devised a novel method that entails introducing recombinant retroviruses into B cells destined to undergo an immune response. However, despite the presence of all the necessary elements, the retroviruses failed to undergo somatic hypermutation. Experiments suggested that the inability to recruit the mutation mechanism was probably not due to the transfer system or a lack of transcription. One possible explanation is that methylation of the retroviral long terminal repeats prevents somatic hypermutation from occurring. Therefore, in contrast to all other promoters studied, these results suggest that retroviral promoters fail to support the mutation mechanism.