Absence of DNA polymerase eta reveals targeting of C mutations on the nontranscribed strand in immunoglobulin switch regions

Activation-induced cytosine deaminase preferentially deaminates C in DNA on the nontranscribed strand in vitro, which theoretically should produce a large increase in mutations of C during hypermutation of immunoglobulin genes. However, a bias for C mutations has not been observed among the mutations in variable genes. Therefore, we examined mutations in the mu and gamma switch regions, which can form stable secondary structures, to look for C mutations. To further simplify the pattern, mutations were studied in the absence of DNA polymerase (pol) eta, which may produce substitutions of nucleotides downstream of C. DNA from lymphocytes of patients with xeroderma pigmentosum variant (XP-V) disease, whose polymerase eta is defective, had the same frequency of switching to all four gamma isotypes and hypermutation in mu-gamma switch sites (0.5% mutations per basepair) as control subjects. There were fewer mutations of A and T bases in the XP-V clones, similar to variable gene mutations from these patients, which confirms that polymerase eta produces substitutions opposite A and T. Most importantly, the absence of polymerase eta revealed an increase in C mutations on the nontranscribed strand. This data shows for the first time that C is preferentially mutated in vivo and pol eta generates hypermutation in the mu and gamma switch regions.     

DNA polymerase beta is able to repair breaks in switch regions and plays an inhibitory role during immunoglobulin class switch recombination

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), which converts cytosines to uracils in switch (S) regions. Subsequent excision of dU by uracil DNA glycosylase (UNG) of the base excision repair (BER) pathway is required to obtain double-strand break (DSB) intermediates for CSR. Since UNG normally initiates faithful repair, it is unclear how the AID-instigated S region lesions are converted into DSBs rather than correctly repaired by BER. Normally, DNA polymerase beta (Polbeta) would replace the dC deaminated by AID, leading to correct repair of the single-strand break, thereby preventing CSR. We address the question of whether Polbeta might be specifically down-regulated during CSR or inhibited from accessing the AID-instigated lesions, or whether the numerous AID-initiated S region lesions might simply overwhelm the BER capacity. We find that nuclear Polbeta levels are induced upon activation of splenic B cells to undergo CSR. When Polbeta(-/-) B cells are activated to switch in culture, they switch slightly better to IgG2a, IgG2b, and IgG3 and have more S region DSBs and mutations than wild-type controls. We conclude that Polbeta attempts to faithfully repair S region lesions but fails to repair them all.     

DNA polymerase eta is the sole contributor of A/T modifications during immunoglobulin gene hypermutation in the mouse

Mutations at A/T bases within immunoglobulin genes have been shown to be generated by a repair pathway involving the DNA-binding moiety of the mismatch repair complex constituted by the MSH2-MSH6 proteins, together with DNA polymerase eta (pol eta). However, residual A/T mutagenesis is still observed upon inactivation in the mouse of each of these factors, suggesting that the panel of activities involved might be more complex. We reported previously (Delbos, F., A. De Smet, A. Faili, S. Aoufouchi, J.-C. Weill, and C.-A. Reynaud. 2005. J. Exp. Med. 201:1191-1196) that residual A/T mutagenesis in pol eta-deficient mice was likely contributed by another enzyme not normally involved in hypermutation, DNA polymerase kappa, which is mobilized in the absence of the normal polymerase partner. We report the complete absence of A/T mutations in MSH2-pol eta double-deficient mice, thus indicating that the residual A/T mutagenesis in MSH2-deficient mice is contributed by pol eta, now recruited by uracil N-glycosylase, the second DNA repair pathway involved in hypermutation. We propose that this particular recruitment of pol eta corresponds to a profound modification of the function of uracil glycosylase in the absence of the mismatch repair complex, suggesting that MSH2-MSH6 actively prevent uracil glycosylase from error-free repair during hypermutation. pol eta thus appears to be the sole contributor of A/T mutations in the normal physiological context.     

A role for Msh6 but not Msh3 in somatic hypermutation and class switch recombination

Somatic hypermutation is initiated by activation-induced cytidine deaminase (AID), and occurs in several kilobases of DNA around rearranged immunoglobulin variable (V) genes and switch (S) sites before constant genes. AID deaminates cytosine to uracil, which can produce mutations of C:G nucleotide pairs, and the mismatch repair protein Msh2 participates in generating substitutions of downstream A:T pairs. Msh2 is always found as a heterodimer with either Msh3 or Msh6, so it is important to know which one is involved. Therefore, we sequenced V and S regions from Msh3- and Msh6-deficient mice and compared mutations to those from wild-type mice. Msh6-deficient mice had fewer substitutions of A and T bases in both regions and reduced heavy chain class switching, whereas Msh3-deficient mice had normal antibody responses. This establishes a role for the Msh2-Msh6 heterodimer in hypermutation and switch recombination. When the positions of mutation were mapped, several focused peaks were found in Msh6(-/-) clones, whereas mutations were dispersed in Msh3(-/-) and wild-type clones. The peaks occurred at either G or C in WGCW motifs (W = A or T), indicating that C was mutated on both DNA strands. This suggests that AID has limited entry points into V and S regions in vivo, and subsequent mutation requires Msh2-Msh6 and DNA polymerase.