Somatic hypermutation at A/T-rich oligonucleotide substrates shows different strand polarities in Ung-deficient or -proficient backgrounds.
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.
Institut Necker-Enfants Malades, INSERM U1151-CNRS UMR 8253, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine-Site Broussais, Paris, France.
Marija
M
Zivojnovic
Institut Necker-Enfants Malades, INSERM U1151-CNRS UMR 8253, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine-Site Broussais, Paris, France.
Frédéric
F
Delbos
Institut Necker-Enfants Malades, INSERM U1151-CNRS UMR 8253, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine-Site Broussais, Paris, France.
Giulia
G
Girelli Zubani
Institut Necker-Enfants Malades, INSERM U1151-CNRS UMR 8253, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine-Site Broussais, Paris, France.
Amélie
A
Julé
Institut Imagine, INSERM UMR1163, AP-HP Hôpital Necker-Enfants Malades, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
Alexandre
A
Alcais
Institut Necker-Enfants Malades, INSERM U1151-CNRS UMR 8253, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine-Site Broussais, Paris, France claude-agnes.reynaud@inserm.fr jean-claude.weill@inserm.fr.
Jean-Claude
JC
Weill
Institut Necker-Enfants Malades, INSERM U1151-CNRS UMR 8253, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine-Site Broussais, Paris, France claude-agnes.reynaud@inserm.fr jean-claude.weill@inserm.fr.
Claude-Agnès
CA
Reynaud
Institut Necker-Enfants Malades, INSERM U1151-CNRS UMR 8253, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine-Site Broussais, Paris, France.
Sébastien
S
Storck
D020856:AT Rich Sequence / Q000235:genetics*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D045647:DNA Glycosylases / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D053843:DNA Mismatch Repair; D045643:DNA Repair Enzymes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D055879:Gene Knock-In Techniques; D056426:Genetic Loci / Q000235:genetics; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D000070976:Mismatch Repair Endonuclease PMS2; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D059645:Mutation Rate; D009841:Oligonucleotides / Q000235:genetics*; D027041:Somatic Hypermutation, Immunoglobulin / Q000235:genetics*; D013379:Substrate Specificity; D019076:Transgenes / Q000235:genetics; D051981:Uracil-DNA Glycosidase / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D000099270:Y-Family DNA Polymerases
D000251:Adenosine Triphosphatases; D004247:DNA; D045647:DNA Glycosylases; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D000070976:Mismatch Repair Endonuclease PMS2; D009841:Oligonucleotides; D051981:Uracil-DNA Glycosidase; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases; C499533:Pms2 protein, mouse