DNA polymerase θ contributes to the generation of C/G mutations during somatic hypermutation of Ig genes

Abstract

Somatic hypermutation of Ig variable region genes is initiated by activation-induced cytidine deaminase; however, the activity of multiple DNA polymerases is required to ultimately introduce mutations. DNA polymerase (Pol ) has been implicated in mutations at A͞T, but polymerases involved in C͞G mutations have not been identified. We have generated mutant mice expressing DNA polymerase (Pol ) specifically devoid of polymerase activity. Compared with WT mice, Polq-inactive (Polq, the gene encoding Pol ) mice exhibited a reduced level of serum IgM and IgG1. The mutant mice mounted relatively normal primary and secondary immune responses to a T-dependent antigen, but the production of high-affinity specific antibodies was partially impaired. Analysis of the J H4 intronic sequences revealed a slight reduction in the overall mutation frequency in Polq-inactive mice. Remarkably, although mutations at A͞T were unaffected, mutations at C͞G were significantly decreased, indicating an important, albeit not exclusive, role for Pol activity. The reduction of C͞G mutations was particularly focused on the intrinsic somatic hypermutation hotspots and both transitions and transversions were similarly reduced. These findings, together with the recent observation that Pol efficiently catalyzes the bypass of abasic sites, lead us to propose that Pol introduces mutations at C͞G by replicating over abasic sites generated via uracil-DNA glycosylase.abasic site ͉ low-fidelity DNA polymerase ͉ activation-induced cytidine deaminase ͉ uracil-DNA glycosylase

DNA polymerases in adaptive immunity

“…Deletion of the sole polymerase domain of Polθ also resulted in a mild phenotype, with a small increase of A/T mutagenesis but no alteration of the G/C mutation profile 92 , a result that, again, does not fit well with the robust translesional activity of Polθ at abasic sites in vitro and its preference for insertion of As 51 . Additional experiments are therefore needed to clarify whether there is a direct role of Polθ during SHM.…”

DNA Replication to Aid Somatic Hypermutation

“…The virtual absence of dC → dG transversions in the nontranscribed strand of the Vλ1 segment DNA in Rev1 deficient mice is consistent with the role of Rev1 in inserting dC across abasic sites during SHM (Jansen et al, 2006). Pol ζ and pol θ are the only TLS polymerases known to efficiently extend the nascent DNA strand past an abasic site by copying undamaged portion of DNA template, consistent with their major role in SHM Zan et al, 2001;Masuda et al, 2005;Zan et al, 2005). Pol ζ is relatively error-free when copying undamaged DNA template.…”

“…This has been shown by the more than 70% reduction of the overall mutation frequency in a knockout mouse strain in which pol θ expression is ablated . In another knockout mouse strain, in which 99 of the 532 amino acids of the polymerase domain of pol θ were deleted and the polymerase activity was inactivated, the mutation frequency at dC/dG decreased by 40% and most of the decrease was focused on the AID deamination motif WRC, strongly suggesting that at least in SHM, bypass of abasic sites depends on pol θ (Masuda et al, 2005). An abasic site is non-instructional and all four nucleotides can be incorporated across it.…”

Mouse models of DNA polymerases

“…It can bypass thymine glycols and extend mismatched primer termini [Seki et al, 2004;Hogg et al, 2012]. Pol y has a putative role in maintenance of chromosomal stability, somatic hypermutation, and class-switch recombination of immunoglobulin genes Masuda et al, 2005Masuda et al, , 2006. In mice, Pol y is encoded by the Polq gene, which maps to chromosome 16.…”

“…These mice also displayed a decrease in transition and transversion mutations, particularly, C to G mutations at somatic hypermutation hotspots. These data suggest that Pol y introduces C to G mutations by replicating over abasic sites generated by Uracil-DNA glycosylase (UDG) [Masuda et al, 2005].…”