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.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01452-13.

ACKNOWLEDGMENTS

We thank the SEAT (Villejuif, France) for its expertise in generation of ES-derived mouse lines and mouse breeding. We thank Deborah Barnes and Michael Liskay for providing Ung^−/− and Pms2^−/− mice, respectively. We thank Jérôme Mégret (Cytometry core facility of the Structure Fédérative de Recherche Necker) for cell sorting.

The Développement du Système Immunitaire team is supported by the Ligue contre le Cancer (Équipe Labelisée) and the Fondation Princesse Grace. Marija Zivojnovic was supported by a 3-year allocation from the Ecole Normale Supérieure and by the Ligue Nationale contre le Cancer for her fourth year of Ph.D. research.