High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase.
Bottom Line: To rationalize these functional requirements, we performed molecular dynamics simulations that suggest that AID and its hyperactive variants can engage DNA in multiple specific modes.These findings align with AID's competing requirements for specificity and flexibility to efficiently drive antibody maturation.Beyond insights into the AID-DNA interface, our Sat-Sel-Seq approach also serves to further expand the repertoire of techniques for deep positional scanning and may find general utility for high-throughput analysis of protein function.
Affiliation: Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.Show MeSH
Mentions: We next aimed to understand why certain mutations were preferred in the Sat-Sel-Seq procedure. Across all positions, we selected mutants that represented >20% of the total count in G3 and evaluated these mutants in the context of our two complementary deaminase assays. In the rifampin assay, the majority of selected variants had activity equivalent to or greater than AID-WT, within the limits of statistical significance (Figure 4A). When the individual point mutants were purified and evaluated using the in vitro deamination assay, all of the variants that were favored over AID-WT in Sat-Sel-Seq showed increased deaminase activity. Among the variants, one notable mutation, R119G, demonstrated the largest enhancement in activity in both the rifampin assay (∼4-fold) and the in vitro enzymatic assay (at least 3-fold).
Affiliation: Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.