| Abstract: | Fanconi anemia is a rare genetic disorder that can lead to bone marrow failure, congenital abnormalities, and increased risk for leukemia and cancer. Cells with loss-of-function mutations in the FANC pathway are characterized by chromosome fragility, altered mutability, and abnormal regulation of the nonhomologous end-joining (NHEJ) pathway. Somatic hypermutation (SHM) and immunoglobulin (Ig) class switch recombination (CSR) enable B cells to produce high-affinity antibodies of various isotypes. Both processes are initiated after the generation of dG:dU mismatches by activation-induced cytidine deaminase. Whereas SHM involves an error-prone repair process that introduces novel point mutations into the Ig gene, the mismatches generated during CSR are processed to create double-stranded breaks (DSBs) in DNA, which are then repaired by the NHEJ pathway. As several lines of evidence suggest a possible role for the FANC pathway in SHM and CSR, we analyzed both processes in B cells derived from Fanca−/− mice. Here we show that Fanca is required for the induction of transition mutations at A/T residues during SHM and that despite globally normal CSR function in splenic B cells, Fanca is required during CSR to stabilize duplexes between pairs of short microhomology regions, thereby impeding short-range recombination downstream of DSB formation.To respond to the enormous variety of pathogens they might encounter, B lymphocytes have evolved processes to alter their genetic material to assemble novel functional Ig genes through site-specific V(D)J recombination. Later, contact with antigens can induce two different activation-induced cytidine deaminase (AID)–dependent processes, which are known as somatic hypermutation (SHM) and class switch recombination (CSR). SHM is responsible for the targeted introduction of point mutations into the variable (V) region of the Ig gene, creating Ig variants with enhanced affinity for a particular antigen. CSR allows for exchange of the initial IgM constant region (Cμ) with a downstream constant region (Cδ, Cγ, Cε, or Cα) through deletional recombination to generate different classes of effector antibodies (Alt et al., 2013).AID, which converts cytosines into uracils, initiates both SHM and CSR by creating dU:dG mismatches (Alt et al., 2013). During SHM, mutations arise from these mismatches through several different mechanisms: (a) DNA replication across the uracil leads to transition mutations; (b) abasic sites generated by the base excision repair glycosylase UNG may be replicated in an error-prone manner by the translesional synthesis (TLS) polymerase REV1 to yield either transition or transversion mutations at the site of the C/G base pair; and (c) mismatch repair (MMR) proteins (MSH2/MSH6 and EXO1) can trigger excision and error-prone resynthesis of short stretches of DNA by the TLS polymerase Polη, thus spreading mutations to surrounding U/G base pairs (Liu and Schatz, 2009). In contrast, during CSR, both base excision repair and MMR proteins induce double-stranded breaks (DSBs) within the switch (S) regions situated upstream of each Ig CH gene. These breaks are then repaired by either the classical nonhomologous end-joining (NHEJ) pathway, which directly rejoins DNA ends with minimal modification of the broken ends, or by the alternative NHEJ pathway, which makes use of sequence microhomologies (MHs) to associate and rejoin distal DSBs (Alt et al., 2013).Fanconi anemia (FA) is a rare, inherited chromosomal breakage disorder presenting bone marrow failure and cancer predisposition. FA is genetically heterogeneous, with 16 FANC genes (named A through Q) identified to date. After DNA damage or replicative stress, eight FANC proteins (FANCA, -B, -C, -E, -F, -G, -L, and -M) assemble into the FANCcore complex, which is necessary for the monoubiquitination and nuclear foci formation of both FANCD2 and FANCI. Monoubiquitinated FANCD2/FANCI heterodimer functionally or biochemically interacts with FANCD1/BRCA2, FANCN/PALB2, FANCJ/BRIP1, FANCO/RAD51C, FANCP/SXL4, and FANCQ/XPF to eliminate DNA lesions and rescue replication (Kottemann and Smogorzewska, 2013). FANC proteins promote homologous recombination and suppress NHEJ repair (Adamo et al., 2010; Pace et al., 2010). Moreover, FANC pathway disruption has been associated with abnormalities of several proteins involved in SHM or CSR, including the TLS polymerases REV1 and Polη (Kim et al., 2012; Fu et al., 2013; Renaud and Rosselli, 2013), the MMR protein MSH2 (Williams et al., 2011), the helicase BLM, and the DSB repair protein MRE11 (Pichierri et al., 2002, 2004; Naim and Rosselli, 2009). High levels of FANCD2 have been detected in germinal center (GC) cells in the spleen, tonsil, and reactive lymph nodes (Hölzel et al., 2003). Finally, expression of the Fanca mRNA, but not the Fancg mRNA, is specifically increased in GC B cells, which show high levels of SHM and CSR (Heng and Painter, 2008). In light of the above, we decided to analyze SHM and CSR in B cells derived from Fanca−/− mice to determine a possible involvement of FANCA in the process of secondary Ig diversification. |
