A Role for the MutL Mismatch Repair Mlh3 Protein in Immunoglobulin Class Switch DNA Recombination and Somatic Hypermutation

Abstract

Class switch DNA recombination (CSR) and somatic hypermutation (SHM) are central to the maturation of the Ab response. Both processes involve DNA mismatch repair (MMR). MMR proteins are recruited to dU:dG mispairs generated by activation-induced cytidine deaminase-mediated deamination of dC residues, thereby promoting S-S region synapses and introduction of mismatches (mutations). The MutL homolog Mlh3 is the last complement of the mammalian set of MMR proteins. It is highly conserved in evolution and is essential to meiosis and microsatellite stability. We used the recently generated knockout mlh3−/− mice to address the role of Mlh3 in CSR and SHM. We found that Mlh3 deficiency alters both CSR and SHM. mlh3−/− B cells switched in vitro to IgG and IgA but displayed preferential targeting of the RGYW/WRCY (R = A or G, Y = C or T, W = A or T) motif by Sγ1 and Sγ3 breakpoints and introduced more insertions and fewer donor/acceptor microhomologies in Sμ-Sγ1 and Sμ-Sγ3 DNA junctions, as compared with mlh3+/+ B cells. mlh3−/− mice showed only a slight decrease in the frequency of mutations in the intronic DNA downstream of the rearranged JH4 gene. However, the residual mutations were altered in spectrum. They comprised a decreased proportion of mutations at dA/dT and showed preferential RGYW/WRCY targeting by mutations at dC/dG. Thus, the MMR Mlh3 protein plays a role in both CSR and SHM.

Ubiquitylated PCNA plays a role in somatic hypermutation and class-switch recombination and is required for meiotic progression

“…Genomic DNA was extracted from splenic B cells stimulated in vitro for 4 days with LPS. Junctional S -S␥3 regions were amplified using high-fidelity PfuTurbo DNA polymerase (Stratagene) in two sequential rounds with specific primers as previously described (48,49). Detailed description of cloning and sequence analysis of switch junctions is shown in SI Text.…”

The Role of MutS Homologues MSH4 and MSH5 in DNA Metabolism and Damage Response

“…MutS and MutL homologues are not only required to assist MMEJ, but they also play overlapping and distinct roles in the process of switch recombination (Ehrenstein et al 2001;Li et al 2004a;Schrader et al 1999;Schrader et al 2002). This assessment is mainly obtained through analyzing microhomology at the S-S junctions as well as the distribution of breakpoints in the absence of each protein (Ehrenstein et al 2001;Li et al 2004a;Schrader et al 1999;Wu et al 2006). Collectively, deficiency of MutS and MutL homologues has been associated with three different phenotypes at the S-S junctions-Msh2 or Mlh3 deficiency leads to a decrease in the length of microhomology (Schrader et al 2002;Wu et al 2006), Msh6-null B cells show no change of microhomology (Li et al 2004a), and an increase in the length of microhomology is evident in Mlh1-or Pms2-null B cells (Ehrenstein et al 2001;Schrader et al 2002).…”

“…This assessment is mainly obtained through analyzing microhomology at the S-S junctions as well as the distribution of breakpoints in the absence of each protein (Ehrenstein et al 2001;Li et al 2004a;Schrader et al 1999;Wu et al 2006). Collectively, deficiency of MutS and MutL homologues has been associated with three different phenotypes at the S-S junctions-Msh2 or Mlh3 deficiency leads to a decrease in the length of microhomology (Schrader et al 2002;Wu et al 2006), Msh6-null B cells show no change of microhomology (Li et al 2004a), and an increase in the length of microhomology is evident in Mlh1-or Pms2-null B cells (Ehrenstein et al 2001;Schrader et al 2002). MLH1-PMS2 acts downstream of MSH2-MSH6 during MMR, similar effect would be anticipated if the process of MMR played a predominant role.…”

DNA Replication to Aid Somatic Hypermutation

“…dU:dG that is not repaired will be replicated over to generate dC → dT and dG → dA transition mutations. MMR proteins and the impact of their deficiencies on SHM 1 Data are from the references quoted in the Table 1 of Wu et al (2006). 2 Mutations in the J H 4 intronic DNA were analyzed unless noted otherwise.…”

“…PCNA also interacts with Mlh1, which heterodimerizes with one of its three partners, Pms2, Pms1 or Mlh3. Mlh1, Pms2 and Mlh3 are involved in SHM, albeit in different ways, as mice deficient in Mlh1, Pms2 or Mlh3 display different spectra of mutations (Li et al, 2006;Stavnezer and Schrader, 2006;Wu et al, 2006). The Mlh dimer, as recruited by the Msh2-Msh6, would coordinate the mismatch recognition stage and the subsequent strand excision stage, although a MutL-independent pathway possibly exists in mlh1 −/− mice, as suggested by the moderate increase in dC/dG mutations as compared to msh2 −/− and msh6 −/− mice (Stavnezer and Schrader, 2006).…”