Contribution of DNA polymerase eta to immunoglobulin gene hypermutation in the mouse

Abstract

The mutation pattern of immunoglobulin genes was studied in mice deficient for DNA polymerase eta, a translesional polymerase whose inactivation is responsible for the xeroderma pigmentosum variant (XP-V) syndrome in humans. Mutations show an 85% G/C biased pattern, similar to that reported for XP-V patients. Breeding these mice with animals harboring the stop codon mutation of the 129/Olain background in their DNA polymerase iota gene did not alter this pattern further. Although this G/C biased mutation profile resembles that of mice deficient in the MSH2 or MSH6 components of the mismatch repair complex, the residual A/T mutagenesis of pol eta-deficient mice differs markedly. This suggests that, in the absence of pol eta, the MSH2-MSH6 complex is able to recruit another DNA polymerase that is more accurate at copying A/T bases, possibly pol kappa, to assume its function in hypermutation.

Figure 1.

Disruption of the mouse Polh gene. (A) Scheme of the mouse Polh gene encoding DNA polymerase η. (Closed boxes) coding exons; (open boxes) noncoding regions. (B) Scheme of the gene targeting strategy. Homologous recombination of the gene-targeting construct results in the introduction of loxP sites on both sides of exon 4. (C) Screening of mouse genotypes: deletion of exon 4 is monitored by a PCR around this exon, using primers shown in B. (D) Polη gene expression in control and gene-targeted mice: amplification of polη transcripts is performed using primers in exons 2 and 6, on twofold consecutive dilutions of RT products from total spleen RNA. The amplified product in polη^−/− animals corresponds to the splicing of exons 3–5, which generates an out-of-frame sequence.

Figure 2.

Distribution of mutations in the V_HDJ_H4 flanking sequence (“J_H4 intronic region”) of Peyer's patches PNA^high B cells from polη- and polη-polι-deficient mice. (A) Pattern of nucleotide substitution in control mice (two animals), polη-deficient mice (three animals), polη- and polι-deficient mice (two animals), and from all mutant animals together. Data are given as percent substitution after correction for base composition (490 bp starting from the exon/intron border: A, 26.3%; C, 14.9%; G, 27.4%; T, 31.4%). (B) Accumulation of mutations in individual J_H4 intronic sequences. The distribution of sequences harboring a given number of mutations relative to the total number of mutated sequences is represented for the three mouse genotypes.

Figure 3.

Distribution of mutations in the Sμ core upstream region (pre-switch) of Peyer's patches PNA^high B cells from polη- and polη-polι-deficient mice. (A) Pattern of nucleotide substitution from the same control and mutant animals described in Fig. 2. Base composition: 560 bp; A, 33.2%; C, 16.4%; G, 27.5%; T, 22.9%. (B) Accumulation of mutations in individual pre-switch sequences. Same representation as in Fig. 2 B. (C) Distribution of mutations along the pre-switch sequence. Mutations from polη-deficient mice are listed above the sequence, and mutations from double-deficient mice are listed below the sequence. “G” within RGYW motifs and “C” within WRCY motifs are underlined.