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.
Highlights
DNA damage encompasses a large variety of chemical and physical alterations of DNA and chromatin
Principle of the In Vitro Resolution (IVR) assay In order to delineate the molecular mechanisms of activation-induced deaminase (AID)-induced lesion resolution, we designed an in vitro resolution (IVR) system, where AID was targeted to a DNA substrate and a cell extract used for the DNA repair/resolution of the AID-induced lesions
The supercoiled topology of the substrate provides both double-stranded DNA for GAL4 binding and single strand DNA (ssDNA) required for AID activity; a method which has previously been utilised for the generation of random stretches of ssDNA [14]
Summary
DNA damage encompasses a large variety of chemical and physical alterations of DNA and chromatin. DNA lesions activate DNA damage sensing proteins, which in turn activate and recruit mediators and repair factors, utilising phosphorylation, ubiquitination, sumoylation, and/or poly(ADP)ribosylation as signalling intermediates. This in turn creates an intricate network of proteinprotein interactions around the lesion; with each type of lesion inducing the formation of different networks. At the Ig locus of activated B cells, BER or MMR processing of AID-lesions does not lead to repair, but can be processed to give rise to DNA mutation and recombination. This complex relationship between the DNA repair processing and AID-lesions is emphasised by the observation that in activated B cells those AID deaminations that occur outside the Ig loci are repaired efficiently [6]
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