Somatic hypermutation of immunoglobulin genes : correlation with the cell cycle and contribution of mutagenic repair pathways

Abstract : Somatic hypermutation is a localized mutagenesis, essentially targeted to the immunoglobulin V region, and occurring during the immune response. This process is triggered by AID (activation-induced cytidine deaminase) that deaminates cytosines into uracils at the Ig locus. This lesion is further processed by Ung or the Msh2-Msh6 complex, with an abnormal outcome for both pathways that results in an increased mutation load. The Msh2-Msh6 complex recruits Pol eta to generate a short patch DNA synthesis with mostly mutations at A and T bases. To get further insight into this error-prone repair process, we have generated hypermutation substrates consisting in an A/T oligonucleotide of 100 bases with or without 3 cytidines in its core region, inserted by knock-in at the heavy chain Ig locus. Our aim was to compare the mutation frequency, distribution and mutation profile of substrates with C on either the coding or the non-coding strand on WT or Ung-deficient background, taking into account that Pol eta is a preferred A to G mutator. Our results suggest that Pol eta resynthesis may proceed on the coding strand, whatever the strand localization of the uracil, thus contradicting previous reports. Unexpectedly, our results revealed a cooperation between the Ung pathway and the endonuclease activity of the mismatch repair, with both of them providing the single-strand nick that allows initiation of the error-prone process that generates mutations at A and T bases. These results resolve the apparent paradox of the non-involvement of the mismatch repair effector complex (Mlh1-Pms2) in hypermutation, by proposing that it works redundantly with UNG, in a distribution of tasks that will depend upon the sequence context and the intensity of deamination activity. We have also constructed cell cycle restricted mutants of AID, to study in which phase of the cell cycle this atypical, mismatch repair driven, error-prone synthesis is taking place. Using the Fucci restriction system (degrons based on Cdt1 or Geminin peptides), we have generated AID constructs with proper restriction in either G1 or S/G2/M phases. These retroviral constructs have been used to transduce mouse hematopoietic stem cells from either AID -deficient mice and to restore immunodeficient animals, in order to analyze their immune response. We report that restriction of AID expression in S/G2/M part of the cycle yielded only background mutation frequency, while AID operating in the G1 phase is able to generate an equal proportion of A/T and G/C mutations at the Ig loci, thus demonstrating that uracils generated in G1 are substrates for both Ung- and mismatch repair pathways.
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Marija Zivojnovic. Somatic hypermutation of immunoglobulin genes : correlation with the cell cycle and contribution of mutagenic repair pathways. Human health and pathology. Université René Descartes - Paris V, 2013. English. ⟨NNT : 2013PA05T070⟩. ⟨tel-00998385⟩

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