Transcription and DNA repair in immunoglobulin gene somatic hypermutations

In B lymphocytes residing in germinal centers of secondary lymphoid organs such as spleen and Peyer's patches, expressed Ig genes are targeted by the somatic hypermutation process. Resulting amino acid changes in the variable region of the antibody molecules coupled with antigen driven selection leads to affinity maturation and the selective proliferation and differentiation of high affinity B cell clones. The molecular mechanism underlying Ig somatic hypermutation remains largely obscure. However, DNA repair and transcription emerged as potential candidates in search for the factors required in Ig mutation process.;To determine whether nucleotide excision repair (NER) pathway is involved in Ig somatic hypermutation, I analyzed B lymphocytes from NER deficient human donors and found normal mutation frequency. DNA mismatch repair (MMR) deficient mice were also analyzed to examine the requirement of MMR mechanism in Ig mutation process. We found relatively normal mutation frequency but altered mutation spectra in these mice indicating that MMR is not required for the mutations to occur in the Ig genes but that its pathway indeed overlaps that of Ig mutation.;The relevance of RNA transcription in Ig mutation was approached in two ways. First, I tried to expand our knowledge of transactivating factors contributing to the specificity of somatic mutation by identifying Ig enhancer binding factors unique to the mutating B cell population. Also, the analysis of Ig mutations in mice deficient in transcription factor Spi-B showed that the germinal center defect does not correlate with defect in mutation activation.;We sought to learn more about the rules dictating the mutation targeting by designing substrates with characteristics suspected to lead to high mutation frequency. The effect of previously observed mutation hot spots and the effect of potential RNA hairpin formation were considered in designing the RS transgene. Our analysis of the RS transgene as well as other artificially designed mutation substrates suggested that hot spot effect is more relevant than RNA hairpin energy in mutation targeting. Finally, the unusually high mutation frequency observed in RS transgene presents a new tool in studying the molecular events involved in Ig somatic hypermutation.