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: To analyze the impact of selection on the saturation mutant libraries at each position we used barcoded PCR primers, specific to the generation number, to amplify the region of the AID gene centered around the loop. The PCR reaction products (4 generations × 12 positional libraries) were pooled and analyzed in a single run using 454 pyrosequencing. The reads were analyzed for the presence of two barcodes—one in the primers corresponding to the generation number and the second within the loop region encoding the identity of original diversified position (Supplementary Figure S1). Within each bin, the codons at the diversified position were cataloged and the overall frequency of each member was tracked across the generations (Figure 3 and Supplementary Figure S4). Mutations outside of the expected variable positions occurred at a low rate and did not change across generations (0.07% per nucleobase read). Notably, the two saturation mutant libraries at Phe115 that independently underwent selection show a high degree of concordance based upon two different metrics for multivariate similarity, the Euclidean distance and cosine similarity measures (Supplementary Figure S5), demonstrating the assay's reproducibility.
Affiliation: Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.