Inactivation of the mouse Polh
gene was performed by a conditional knock-out strategy that introduces loxP sites flanking exon 4, an exon containing the DE polymerase motifs conserved in all Y-family polymerases and indispensable for polη catalytic function ( and references 15
). This strategy was designed with the aim of crossing the animals obtained with mice harboring other DNA repair defects, allowing the follow-up of the B cell lineage in case of lethality of their combined deficiencies. Deletion of exon 4 was performed here in the targeted embryonic stem (ES) cell clone, leaving in place a single loxP site and, therefore, minimizing disturbance around the gene. The Xpo5
gene, encoding exportin-5 involved in the nuclear export of microRNAs, indeed initiates 200 bp upstream from the first Polh
exon in opposite transcriptional orientation; affecting expression of this gene is, therefore, likely to result in an early embryonic lethal phenotype (25
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 (more ...)
The heterozygotes obtained by breeding chimeras with C57Bl/6 mice were crossed together and screened for the presence of the stop codon mutation born by the Poli
gene from the 129/Ola genetic background of the ES cells. Wild-type, homozygous polη-deficient, and double polη-polι mutant mice were selected for analysis. Deletion of exon 4 of polη resulted at the transcriptional level in the direct splicing of exons 3–5, an out-of-frame junction that introduces a stop codon 11 amino acids downstream in exon 5 and generates a truncated polη protein of 102 amino acids ( D). Apart from deletion of exon 4, such a truncated protein would lack domains involved in nuclear localization and repair foci formation (19
Wild-type, polη-deficient, and double polη-polι mutant mice were analyzed for the mutation pattern of their Ig locus. Different types of sequences were analyzed: intronic sequences flanking rearranged JH4 sequences (“JH4 intronic sequences”) and a 560-bp region upstream from the repeats of the Sμ core sequence (hereafter referred to as “pre-switch”), both from Peyer's patch PNAhigh B cells ().
Somatic mutations in JH4 intronic sequences and Sμ core upstream sequences from normal and mutant mice
No B cell anomalies were observed in either type of polymerase-deficient animals. Ig sequences from polη−/−
B cells displayed the same specific pattern as those obtained from memory B cells of XP-V patients; i.e., a marked reduction of mutations at A/T base pairs: 86.1% mutations at G/C base pairs for the JH
4 intron and 88.6% for the pre-switch sequence (vs. 89.3 and 89.8% for the corresponding Ig gene sequences in XP-V patients; and , , and reference 17
). Mutation frequencies were somewhat lower for polη-deficient animals compared with wild type when JH
4 intronic sequences are considered, but similar for the pre-switch sequences. Such a quantitative dissociation has been described for MSH6- and for UNG/MSH2-deficient animals (5
Figure 2. Distribution of mutations in the VHDJH4 flanking sequence (“JH4 intronic region”) of Peyer's patches PNAhigh B cells from polη- and polη-polι-deficient mice. (A) Pattern of nucleotide substitution in control mice (more ...)
Figure 3. Distribution of mutations in the Sμ core upstream region (pre-switch) of Peyer's patches PNAhigh B cells from polη- and polη-polι-deficient mice. (A) Pattern of nucleotide substitution from the same control and mutant animals (more ...)
Pattern of nucleotide changes in JH4 intronic sequences of normal, polη-, and MSH2-deficient mice
We next compared the mutation profile of mice harboring both polη and polι inactivation. These profiles appear extremely similar, either in their distribution along the sequence (shown for the pre-switch sequence in C), or in their base substitution characteristics ( A and 3 A). The lack of phenotype of polι-deficient mice on Ig gene hypermutation is, therefore, not due to a compensatory role of polη in this specific strain.
All further analyses were, therefore, performed on pooled data from both types of mutant mice, allowing us to compare larger databases (366 mutations for the JH
4 intron and 277 for the pre-switch sequence). The targeting was slightly more pronounced for C than for G, in particular for the pre-switch sequence. Such an increase has been described in switch junctions from XP-V patients (18
). However, among the three types of sequences that we have analyzed in similar patients, we could not observe this bias in either switch junctions or JH
4 introns, the specific increase in C mutations being only significant in pre-switch sequences from XP-V individuals (17
). The general relevance of this observation is, therefore, difficult to assess at this stage. Moreover, among G/C mutations, no difference from wild type is noticeable in the relative proportion of transitions and transversions ().
MSH2 deficiency, together with defects in MSH6 and Exo1, results in a similar bias toward G/C mutagenesis (7
). We, therefore, wanted to compare the mutation pattern in the JH
4 intronic sequence between Msh2−/−
animals. This pattern was established using data from both MSH2- and Exo1-deficient animals, as well as MSH2-ATPase mutant mice (326 mutations), taking into account only data obtained using Pfu polymerase, to exclude any contribution of less accurate enzymes whose intrinsic error spectrum might specifically impinge on the A/T pattern. At G/C base pairs, these mutant mice show a strong bias toward transitions, which has been interpreted as an increased replicative bypass of uracils, the absence of the mismatch-binding complex leading to an increased fraction of deaminated bases escaping detection and repair. Polh−/−
animals, in contrast, show a transition/transversion distribution at G/C bases similar to controls. Strikingly, the mutation pattern at A/T bases pairs shows the reverse situation. MSH2 deficiency shows a distribution similar to wild type, whereas polη deficiency results in a strong transversion bias. This transversion bias is consistent enough to emerge from both types of sequences analyzed (JH
4 and pre-switch), and in both mouse and human polη deficiencies (). Moreover, among transversions at A/T bases, most of them are A to C or T to G changes, which represent more than half of all A/T mutations. Such a mutation bias has been described for DNA polymerase κ (26
). It is thus possible that, in the absence of polη, the mismatch binding complex is able to recruit a different translesional enzyme, which, due to its specific misincorporation pattern, would be less mutagenic at A/T base pairs, leading to a lower frequency of A/T mutations with a specific transversion bias. Because polκ-deficient animals do not harbor the slightest alteration of their A/T mutation pattern (28
), it is probable that this enzyme is not a normal actor of the Ig gene mutation process, being on duty only in case of absence of the regular partner of the MSH2–MSH6 complex (i.e., polη).
Polμ is another enzyme with such a transversion bias (29
). However, contrary to polη and polκ, its preferred misincorporation is at copying A, and not T. It is therefore unlikely, in the mouse at least, that polμ could generate the A over T bias of mutations in JH
4 sequences, present in control mice and conserved in polη-deficient animals.
UNG and MSH2–MSH6 are the only two repair pathways handling uracils generated by AID during Ig gene hypermutation. The model of Neuberger et al. (5
) posits that G/C mutations are introduced by replication over the uracils or the abasic sites generated by uracil glycosylase, both events occurring on the DNA strand opposed to the lesion and without repair. Replication over the abasic site would involve translesional DNA polymerases in their “classical” role of damage bypass. Effectively, in the AID-dependent mutations observed in the chicken cell line DT40 that are almost entirely restricted to G/C bases, Rev1 has been proposed to be the major enzyme involved (30
). As the mutation pattern of DT40 is more biased toward G to C and C to G transversions (the hallmark of Rev1) than it is in the mouse, it is so far unclear how many translesional polymerases are involved in the G/C mutation pattern in mouse (and human) B cells. Mutations at A/T bases are more difficult to explain on a strict replication mode within this model.
In another scenario, the MSH2–MSH6 complex would recruit polη for an error-prone repair of the lesion (i.e., on the same DNA strand) that would remove the uracil or the abasic site and create mismatches at nearby A/T bases because this enzyme is inherently more mutagenic at copying Ts. Along this line, specific modifications of AID in hypermutating B cells might also actively recruit UNG at the site of the lesion (31
), driving the G/C mutagenesis to proceed differently from a strict saturation of the normal repair of uracils. In such a scheme, the dichotomy between translesion bypass and error-prone repair for the G/C versus A/T mutation pathways might have to be reassessed.
The absence of phenotype of the polι mutation of the 129 strains obviously questions the relevance of the polι-dependent mutagenic process that we described in the BL2 Burkitt's lymphoma cell line (20
). In fact, it has been reported that such a polι-dependent mutagenesis can be induced at the Ig locus in B cell lines infected by the hepatitis C virus (32
). These mutations would be related to the specific metabolic alterations brought upon infection by this oncogenic virus, alterations that may be shared by many B cell lymphomas (33
). Alternatively, inactivation of polι in the 129/Ola mouse strain might show leakiness, in particular in activated B cells, by either read-trough of the stop codon or alternative splicing. A classical inactivation of the Poli
gene and the analysis of mice cumulating several deficiencies in the activities involved in hypermutation might contribute to address these issues.