INSIGHT FROM AID-INDUCED DNA DAMAGE RESOLUTION: CELLULAR CONTEXT MATTERS

When genome integrity is perturbed, surveillance and repair mechanisms are activated to restore genome integrity through high fidelity DNA repair. However, in some physiological situations, those mechanisms are channeled away from integrity towards mutations and recombinations. During the diversification of the immunoglobulin locus in B cells, Activation Induced Deaminase (AID) triggers the physiological introduction of mutations. The current work was based on the observation that upon simple lesion generation, AID-induced deamination of a cytosine to uracil, the resolution by the molecular mechanism of DNA repair can lead to different outcomes. This homeostatic outcome, error-free or error-prone, is governed by specific cellular context and processes associated with DNA. To uncover the regulation of the pathway choice an in vitro system, named in vitro resolution (IVR), was developed. In the 1st phase of the IVR, AID was targeted to a DNA plasmid for uracil lesion generation. In the 2nd phase, a cellular extract resolved the lesions via Base Excision Repair [BER, divided in short patch (SP)-BER or long patch (LP)-BER] or Mismatch Repair (MMR). The quantitative nature of the IVR provided a novel means to precisely quantitate the contribution of each single DNA repair pathway. This set-up allowed us to evaluate how different cellular environments influenced the choice. Cell origins presented quantitative differences in DNA repair kinetics: a) overall sensitivity, b) non-B cells activating non-canonical MMR first, c) B cells activating SP-BER first, and d) LP-BER is significantly activated only in B cells. To understand the possible molecular mechanisms, we analysed single components known to influence DNA repair, such as transcription, protein availability, and chromatin. Changing the DNA substrate to either prefer or avoid forming nucleosomes, we uncovered significant changes in AID deamination preference and in DNA repair pathway choice. DNA with nucleosome favourable base-stacking preferred LP-BER, while non-nucleosome stacked DNA preferred SP-BER and MMR. Overall our findings provide novel insight into the cellular context that can influence DNA repair. The use of B cells and cancer cell lines can recapitulate in vivo Ig locus diversification, and our findings have a direct bearing in understanding mechanisms of tumorigenesis.