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High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase.

Gajula KS, Huwe PJ, Mo CY, Crawford DJ, Stivers JT, Radhakrishnan R, Kohli RM - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

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Functional determinants for the Leu113-Pro123 loop revealed by Sat-Sel-Seq. (A) Horizontal bar plots depicting the prevalence of amino acids from the four generational libraries, G0–G3. At position Leu113, stringent selection occurs in favor of the wild type (WT) residue. Alternative patterns of selection are evident at (B) position Phe115, where aromatic residues are selected by G3, (C) position Glu117 that remains highly diversified after selection or (D) position Arg119, where mutant variant R119G outcompetes the WT residue over generations. The individual data from other positions are provided in Supplementary Figure S4A. (E) Normalized logo plot for residues 113–123 summarizing the frequency of each amino acid at each position in the final selected library. An alternative representative of the G3 data scaled relative to the frequency of each amino acid in the G0 population is provided in Supplementary Figure S4B.
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Figure 3: Functional determinants for the Leu113-Pro123 loop revealed by Sat-Sel-Seq. (A) Horizontal bar plots depicting the prevalence of amino acids from the four generational libraries, G0–G3. At position Leu113, stringent selection occurs in favor of the wild type (WT) residue. Alternative patterns of selection are evident at (B) position Phe115, where aromatic residues are selected by G3, (C) position Glu117 that remains highly diversified after selection or (D) position Arg119, where mutant variant R119G outcompetes the WT residue over generations. The individual data from other positions are provided in Supplementary Figure S4A. (E) Normalized logo plot for residues 113–123 summarizing the frequency of each amino acid at each position in the final selected library. An alternative representative of the G3 data scaled relative to the frequency of each amino acid in the G0 population is provided in Supplementary Figure S4B.

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.


High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase.

Gajula KS, Huwe PJ, Mo CY, Crawford DJ, Stivers JT, Radhakrishnan R, Kohli RM - Nucleic Acids Res. (2014)

Functional determinants for the Leu113-Pro123 loop revealed by Sat-Sel-Seq. (A) Horizontal bar plots depicting the prevalence of amino acids from the four generational libraries, G0–G3. At position Leu113, stringent selection occurs in favor of the wild type (WT) residue. Alternative patterns of selection are evident at (B) position Phe115, where aromatic residues are selected by G3, (C) position Glu117 that remains highly diversified after selection or (D) position Arg119, where mutant variant R119G outcompetes the WT residue over generations. The individual data from other positions are provided in Supplementary Figure S4A. (E) Normalized logo plot for residues 113–123 summarizing the frequency of each amino acid at each position in the final selected library. An alternative representative of the G3 data scaled relative to the frequency of each amino acid in the G0 population is provided in Supplementary Figure S4B.
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Related In: Results  -  Collection

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Figure 3: Functional determinants for the Leu113-Pro123 loop revealed by Sat-Sel-Seq. (A) Horizontal bar plots depicting the prevalence of amino acids from the four generational libraries, G0–G3. At position Leu113, stringent selection occurs in favor of the wild type (WT) residue. Alternative patterns of selection are evident at (B) position Phe115, where aromatic residues are selected by G3, (C) position Glu117 that remains highly diversified after selection or (D) position Arg119, where mutant variant R119G outcompetes the WT residue over generations. The individual data from other positions are provided in Supplementary Figure S4A. (E) Normalized logo plot for residues 113–123 summarizing the frequency of each amino acid at each position in the final selected library. An alternative representative of the G3 data scaled relative to the frequency of each amino acid in the G0 population is provided in Supplementary Figure S4B.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Show MeSH