The Fanconi Anemia Core Complex Is Dispensable during Somatic Hypermutation and Class Switch Recombination

To generate high affinity antibodies during an immune response, B cells undergo somatic hypermutation (SHM) of their immunoglobulin genes. Error-prone translesion synthesis (TLS) DNA polymerases have been reported to be responsible for all mutations at template A/T and at least a fraction of G/C transversions. In contrast to A/T mutations which depend on PCNA ubiquitination, it remains unclear how G/C transversions are regulated during SHM. Several lines of evidence indicate a mechanistic link between the Fanconi Anemia (FA) pathway and TLS. To investigate the contribution of the FA pathway in SHM we analyzed FancG-deficient B cells. B cells deficient for FancG, an essential member of the FA core complex, were hypersensitive to treatment with cross-linking agents. However, the frequencies and nucleotide exchange spectra of SHM remained comparable between wild-type and FancG-deficient B cells. These data indicate that the FA pathway is not involved in regulating the outcome of SHM in mammals. In addition, the FA pathway appears dispensable for class switch recombination

MSH2/MSH6 Complex Promotes Error-Free Repair of AID-Induced dU:G Mispairs as well as Error-Prone Hypermutation of A:T Sites

Mismatch repair of AID-generated dU:G mispairs is critical for class switch recombination (CSR) and somatic hypermutation (SHM) in B cells. The generation of a previously unavailable Msh2−/−Msh6−/− mouse has for the first time allowed us to examine the impact of the complete loss of MutSα on lymphomagenesis, CSR and SHM. The onset of T cell lymphomas and the survival of Msh2−/−Msh6−/− and Msh2−/−Msh6−/−Msh3−/− mice are indistinguishable from Msh2−/− mice, suggesting that MSH2 plays the critical role in protecting T cells from malignant transformation, presumably because it is essential for the formation of stable MutSα heterodimers that maintain genomic stability. The similar defects on switching in Msh2−/−, Msh2−/−Msh6−/− and Msh2−/−Msh6−/−Msh3−/− mice confirm that MutSα but not MutSβ plays an important role in CSR. Analysis of SHM in Msh2−/−Msh6−/− mice not only confirmed the error-prone role of MutSα in the generation of strand biased mutations at A:T bases, but also revealed an error-free role of MutSα when repairing some of the dU:G mispairs generated by AID on both DNA strands. We propose a model for the role of MutSα at the immunoglobulin locus where the local balance of error-free and error-prone repair has an impact in the spectrum of mutations introduced during Phase 2 of SHM

Unique DNA Repair Gene Variations and Potential Associations with the Primary Antibody Deficiency Syndromes IgAD and CVID

BACKGROUND: Despite considerable effort, the genetic factors responsible for >90% of the antibody deficiency syndromes IgAD and CVID remain elusive. To produce a functionally diverse antibody repertoire B lymphocytes undergo class switch recombination. This process is initiated by AID-catalyzed deamination of cytidine to uridine in switch region DNA. Subsequently, these residues are recognized by the uracil excision enzyme UNG2 or the mismatch repair proteins MutSalpha (MSH2/MSH6) and MutLalpha (PMS2/MLH1). Further processing by ubiquitous DNA repair factors is thought to introduce DNA breaks, ultimately leading to class switch recombination and expression of a different antibody isotype. METHODOLOGY/PRINCIPAL FINDINGS: Defects in AID and UNG2 have been shown to result in the primary immunodeficiency hyper-IgM syndrome, leading us to hypothesize that additional, potentially more subtle, DNA repair gene variations may underlie the clinically related antibody deficiencies syndromes IgAD and CVID. In a survey of twenty-seven candidate DNA metabolism genes, markers in MSH2, RAD50, and RAD52 were associated with IgAD/CVID, prompting further investigation into these pathways. Resequencing identified four rare, non-synonymous alleles associated with IgAD/CVID, two in MLH1, one in RAD50, and one in NBS1. One IgAD patient carried heterozygous non-synonymous mutations in MLH1, MSH2, and NBS1. Functional studies revealed that one of the identified mutations, a premature RAD50 stop codon (Q372X), confers increased sensitivity to ionizing radiation. CONCLUSIONS: Our results are consistent with a class switch recombination model in which AID-catalyzed uridines are processed by multiple DNA repair pathways. Genetic defects in these DNA repair pathways may contribute to IgAD and CVID

DNA polymerases in adaptive immunity.: DNA polymerases in adaptive immunity

review articleInternational audienceTo cope with an unpredictable variety of potential pathogenic insults, the immune system must generate an enormous diversity of recognition structures, and it does so by making stepwise modifications at key genetic loci in each lymphoid cell. These modifications proceed through the action of lymphoid-specific proteins acting together with the general DNA-repair machinery of the cell. Strikingly, these general mechanisms are usually diverted from their normal functions, being used in rather atypical ways in order to privilege diversity over accuracy. In this Review, we focus on the contribution of a set of DNA polymerases discovered in the past decade to these unique DNA transactions