DNA polymerases β and λ do not directly affect Ig variable region somatic hypermutation although their absence reduces the frequency of mutations

During somatic hypermutation (SHM) of antibody variable (V) region genes, activation-induced cytidine deaminase (AID) converts dC to dU, and dUs can either be excised by uracil DNA glycosylase (UNG), by mismatch repair, or replicated over. If UNG excises the dU, the abasic site could be cleaved by AP-endonuclease (APE), introducing the single-strand DNA breaks (SSBs) required for generating mutations at A:T bp, which are known to depend upon mismatch repair and DNA Pol η. DNA Pol β or λ could instead repair the lesion correctly. To assess the involvement of Pols β and λ in SHM of antibody genes, we analyzed mutations in the VDJh4 3′ flanking region in Peyer's patch germinal center (GC) B cells from polβ^?/?polλ^?/?, polλ^?/?, and polβ^?/? mice. We find that deficiency of either or both polymerases results in a modest but significant decrease in V region SHM, with Pol β having a greater effect, but there is no effect on mutation specificity, suggesting they have no direct role in SHM. Instead, the effect on SHM appears to be due to a role for these enzymes in GC B cell proliferation or viability. The results suggest that the BER pathway is not important during V region SHM for generating mutations at A:T bp. Furthermore, this implies that most of the SSBs required for Pol η to enter and create A:T mutations are likely generated during replication instead. These results contrast with the inhibitory effect of Pol β on mutations at the Ig Sμ locus, Sμ DSBs and class switch recombination (CSR) reported previously. We show here that B cells deficient in Pol λ or both Pol β and λ proliferate normally in culture and undergo slightly elevated CSR, as shown previously for Pol β-deficient B cells.     

The SbcCD complex of Deinococcus radiodurans contributes to radioresistance and DNA strand break repair in vivo and exhibits Mre11–Rad50 type activity in vitro

Deinococcus radiodurans lacks a homologue of the recB and recC genes, and the sbcA/B genes, of Escherichia coli. Thus, DNA strand break repair in Deinococcus proceeds by pathways that do not utilize these proteins. Unlike E. coli, the absence of recBC and sbcA/sbcB, and presence of only sbcC and sbcD in Deinococcus, indicates an enigmatic role of SbcCD in this bacterium. Studies on sbcCD mutation in Deinococcus showed nearly a 100-fold increase in gamma radiation sensitivity as compared to wild type. The mutant showed a higher rate of in vivo DNA degradation during the post-irradiation recovery period that corresponds to the RecA-dependent DSB repair phase. These cells showed a typical NotI pattern of DNA reassembly during the early phase of DSB repair, but were defective for the subsequent RecA-dependent phase II of DSB repair. Hydrogen peroxide had no effect on cell survival of the mutant. While its tolerance to higher doses of UVC and mitomycin C was significantly decreased as compared to wild type. Purified recombinant SbcCD proteins showed single-stranded endonuclease and 3′ → 5′ double-stranded DNA exonuclease activities similar to that of the Mre11–Rad50 complex, which is required for DNA strand break repair in higher organisms. These results suggested that the Mre11–Rad50 type nuclease activity of SbcCD proteins contributes to the radiation resistance of D. radiodurans perhaps by promoting the RecA-dependent DSB repair required for polyploid genome maturation.