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PLoS One January 1, 2013; 8 (12): e82097.

Simultaneous in vitro characterisation of DNA deaminase function and associated DNA repair pathways.

Franchini DM , Incorvaia E , Rangam G , Coker HA , Petersen-Mahrt SK .

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.

PubMed ID: 24349193
PMC ID: PMC3857227
Article link: PLoS One
Grant support: [+]

Species referenced: Xenopus
Genes referenced: aicda ccno lgals4.2 pcna ube2k ung

Article Images: [+] show captions
References [+] :
Besmer, The transcription elongation complex directs activation-induced cytidine deaminase-mediated DNA deamination. 2006, Pubmed