Search for trans-acting factors in somatic mutation of immunoglobulin genes

Abstract

Somatic hypermutation of immunoglobulin (Ig) genes plays a major role in diversifying and improving the efficiency of the antibody repertoire, yet its mechanism remains elusive. In the cultured cell system previously established in our laboratory, mutation of a transfected mu or gamma2a transfected mini-gene is quantified by reversion of an amber codon in the variable (V) region. We found that in the Ag8.653 and MPC11 cell lines, both transfected Ig mini-genes were uniformly mutated at low rates, while Sp2/0 transfectant clones varied from each other by a factor of 100,000. In order to investigate the mutability of these Ig substrates in vivo, we engineered transgenic mouse lines carrying the mu or gamma2a heavy chain mini-genes. By sequencing transgenic and endogenous Ig V regions collected from splenic germinal center (GC) B cells, we found that the mu transgene was mutated at a high frequency, only 10-fold below the endogenous frequency. In Peyer's patches, the mu transgene was mutated more frequently in GC B cells than in non-GC B cells, and all base changes recovered were single point mutations, providing further evidence that the ectopically integrated passenger transgene was targeted and regulated by the normal mechanism of somatic mutation.;Since the DNA mismatch repair (MMR) system functions primarily to identify base mismatches introduced during DNA replication, it is likely to impinge upon V region mutation. In eukaryotic MMR, MSH2 dimerizes with MSH6 to recognize single nucleotide mispairs, while a complex of MSH2 with MSH3 is required for 2--4 base pair mutations. The MLH1/PMS2 heterodimer is subsequently recruited and ultimately, the mismatched DNA is excised and resynthesized correctly. Mice deficient in MSH6 or MLH1 exhibited a decrease in mutation frequency of Peyer's patch B cell Ig sequences, as well as an impaired hapten-specific antibody response. In mice lacking MSH6 or both MSH3 and MSH6, base substitutions were preferentially targeted to G and C nucleotides and highly clustered in an intrinsic hot spot. Others have described a similar phenotype in Msh2 -/- mice, indicating that the MSH2-MSH6 heterodimer specifically modulates the pattern of somatic hypermutation.