Somatic hypermutation at A/T-rich oligonucleotide substrates shows different strand polarities in Ung-deficient or -proficient backgrounds

Abstract

A/T mutations at immunoglobulin loci are introduced by DNA polymerase η (Polη) during an Msh2/6-dependent repair process which results in A's being mutated 2-fold more often than T's. This patch synthesis is initiated by a DNA incision event whose origin is still obscure. We report here the analysis of A/T oligonucleotide mutation substrates inserted at the heavy chain locus, including or not including internal C's or G's. Surprisingly, the template composed of only A's and T's was highly mutated over its entire 90-bp length, with a 2-fold decrease in mutation from the 5' to the 3' end and a constant A/T ratio of 4. These results imply that Polη synthesis was initiated from a break in the 5'-flanking region of the substrate and proceeded over its entire length. The A/T bias was strikingly altered in an Ung(-/-) background, which provides the first experimental evidence supporting a concerted action of Ung and Msh2/6 pathways to generate mutations at A/T bases. New analysis of Pms2(-/-) animals provided a complementary picture, revealing an A/T mutation ratio of 4. We therefore propose that Ung and Pms2 may exert a mutual backup function for the DNA incision that promotes synthesis by Polη, each with a distinct strand bias.

FIG 1

Knock-in strategy for the generation of A/T-rich oligonucleotide mutation substrates. Oligonucleotide mutation substrates were inserted by knock-in at the heavy chain locus, 43 bp downstream of the J_H4 segment. A schematized view of the mutation distribution within 490 bp of the J_H4 intron highlights the mutation density around the oligonucleotide insertion site. The oligonucleotides consisted of 10 repeats of a 9-mer sequence, TATTATTAA, with 4 repeats flanking 3 G's separated by the same 9-mer sequence (oligo-G). Oligo-C represents the same sequence inserted in reverse orientation. Oligo-noG and -noC consisted of the same A/T backbone, with G's and C's, respectively, omitted. The 3 segments of the oligonucleotide transgene used in subsequent mutation analysis are represented as the 5′ segment, core segment, and 3′ segment.

FIG 2

Pattern of Polη synthesis in vitro on an A/T-rich oligonucleotide substrate. (A) Scheme of the strategy used for amplification of in vitro Polη DNA synthesis products. Oligo-G was inserted into a single-stranded M13 template and subjected to Polη-mediated DNA synthesis. The primer sequence included an SP6 sequence that allows selective amplification of complete in vitro-synthesized products. (B) Total mutations generated during in vitro Polη synthesis of the oligo-G template. (C) Pattern of mutations at A/T bases generated by Polη in vitro, after correction for base composition and reported for the synthesized strand. (D) Graphical representation of the mutation profile, with an indication of each nucleotide misincorporated opposite the template base.

FIG 3

Distributions of mutations and A/T ratios along the oligonucleotide sequence. Mutation frequencies (corrected for relative nucleotide length and normalized to 100 for the 5′ segment) and A/T ratios (corrected for relative A/T composition) are presented for sequences including 1 to 3 mutations and analyzed along the 3 segments of the oligonucleotide transgenes defined in Fig. 1: 5′ part, core segment, and 3′ part. Significant trends in 5′-to-3′ mutation distributions and A/T ratios are indicated (according to Cochran-Armitage and Jonckheere-Terpstra trend tests, respectively [see Materials and Methods]). **, P < 0.01; ***, P < 0.001. KO, knockout.

FIG 4

Mutation profiles at A/T and G/C bases for the different oligonucleotide substrates. (A) Distributions of mutations are presented for each transgenic substrate, analyzed in 2 categories: total sequences and sequences with 1 to 3 mutations over the 90- or 93-bp oligonucleotide template, corrected for base composition. (B) Average A/T mutation ratios for the different transgenes. (C) Relative mutation frequencies at C's and G's versus the A/T backbone in wild-type (wt) and Ung-deficient (KO) contexts (corrected for their 30-fold lower representation level).

FIG 5

Symmetrical targeting of WA and TW motifs. (A) Mutations were analyzed for each 3-nt context, with reference to the central mutated position. (B) The distribution of mutation outcomes at the central nucleotide position is represented for each 3-nt context. Analysis of mutations was restricted to Tg-noG, for which a larger database was assembled.

FIG 6

Putative scheme of Ung/Pms2 strand targeting at the A/T oligonucleotide substrate. (A) Schematic of the impact on A/T mutagenesis of the two possible Polη synthesis profiles accounting for the observed A/T ratio of 4. Scenario 1, an 80%-20% A/T mutation profile for top-strand synthesis; scenario 2, a 100% A mutation profile, with 80% of synthesis on the top strand and 20% on the bottom strand. Only the first proposition corresponds to the pattern observed in Tg-noG, with the profile of mutations for Tg-noC (not represented) being compatible with both (Fig. 3). (B) Ung-triggered DNA incision at uracils (represented by orange scissors) is proposed to cooperate with U-G mismatch recognition by Msh2/Msh6 to promote initiation of Polη-mediated error-prone DNA synthesis at the generated nicks (18). Nearby deamination events are more likely to occur on the nontranscribed, exposed strand. (C) Initiation of DNA synthesis at internal C's of the Tg-C substrate triggered by Ung, accounting for the reduced C mutation frequency in the wt versus the Ung-deficient background (Fig. 4C). (D) Reduced A/T ratio in the Ung^−/− context due to a more moderate strand bias of the noncanonical mismatch repair involving the Pms2-Mlh1 endonuclease activity (blue scissors). The recruitment of the complete mismatch repair complex (MutSα and MutLα) is proposed to be the default pathway at the Ig locus, but Ung would take over and provide abasic sites triggering DNA incision in the specific location of the oligonucleotide substrate or in a Pms2-deficient context (see Discussion). MMR, mismatch repair.

FIG 7

A/T bias linked with Pms2 deficiency. (A) Analysis of mutations in J_H4 intronic sequences from wt, Ung^−/−, and Pms2^−/− mice. (B) Mutation profiles of wt, Ung^−/−, and Pms2^−/− mice. (C) A-over-T mutation ratios. *, P = 0.012 (two-tailed Mann-Whitney test).