title: Simultaneous in vitro characterisation of DNA deaminase function and associated DNA repair pathways.
abstract: During immunoglobulin (Ig) diversification, activation-induced deaminase (AID) initiates somatic hypermutation and class switch recombination by catalysing the conversion of cytosine to uracil. The synergy between AID and DNA repair pathways is fundamental for the introduction of mutations, however the molecular and biochemical mechanisms underlying this process are not fully elucidated. We describe a novel method to efficiently decipher the composition and activity of DNA repair pathways that are activated by AID-induced lesions. The in vitro resolution (IVR) assay combines AID based deamination and DNA repair activities from a cellular milieu in a single assay, thus avoiding synthetically created DNA-lesions or genetic-based readouts. Recombinant GAL4-AID fusion protein is targeted to a plasmid containing GAL4 binding sites, allowing for controlled cytosine deamination within a substrate plasmid. Subsequently, the Xenopus laevis egg extract provides a source of DNA repair proteins and functional repair pathways. Our results demonstrated that DNA repair pathways which are in vitro activated by AID-induced lesions are reminiscent of those found during AID-induced in vivo Ig diversification. The comparative ease of manipulation of this in vitro systems provides a new approach to dissect the complex DNA repair pathways acting on defined physiologically lesions, can be adapted to use with other DNA damaging proteins (e.g. APOBECs), and provide a means to develop and characterise pharmacological agents to inhibit these potentially oncogenic processes.
authors.affiliation: DNA Editing in Immunity and Epigenetics, IFOM-Fondazione Instituto FIRC di Oncologia Molecolare, Milano, Italy ; DNA Editing Lab, Clare Hall Laboratories, London Research Institute, South Mimms, United Kingdom.
authors.forename: Don-Marc
authors.initials: DM
authors.lastname: Franchini
authors.affiliation: DNA Editing in Immunity and Epigenetics, IFOM-Fondazione Instituto FIRC di Oncologia Molecolare, Milano, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Incorvaia
authors.affiliation: DNA Editing in Immunity and Epigenetics, IFOM-Fondazione Instituto FIRC di Oncologia Molecolare, Milano, Italy ; DNA Editing Lab, Clare Hall Laboratories, London Research Institute, South Mimms, United Kingdom.
authors.forename: Gopinath
authors.initials: G
authors.lastname: Rangam
authors.affiliation: DNA Editing Lab, Clare Hall Laboratories, London Research Institute, South Mimms, United Kingdom.
authors.forename: Heather A
authors.initials: HA
authors.lastname: Coker
authors.affiliation: DNA Editing in Immunity and Epigenetics, IFOM-Fondazione Instituto FIRC di Oncologia Molecolare, Milano, Italy ; DNA Editing Lab, Clare Hall Laboratories, London Research Institute, South Mimms, United Kingdom.
authors.forename: Svend K
authors.initials: SK
authors.lastname: Petersen-Mahrt
mesh_terms: D000818:Animals; D019921:Biotinylation; D002457:Cell Extracts; D003564:Cytidine Deaminase / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006801:Humans; D010063:Ovum / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D014981:Xenopus; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D002457:Cell Extracts; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase