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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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Related in: MedlinePlus

Sat-Sel-Seq methodology. Oligonucleotide cassettes (light blue) containing degenerate codons (NNS) at each position (dark blue) and neighboring silent mutations (red) were cloned into AID expression plasmid to construct saturation mutant libraries. Shown is the representative sequence of a Generation 0 (G0) library for Position 115. The G0 libraries were transformed into E. coli, expressed as a cohort and subjected to rounds of selection for acquired resistance to rifampin to generate subsequent generations of the library. The generational libraries were PCR amplified with primers barcoded for the generation number and the pooled PCR samples were sequenced by 454 high-throughput sequencing. The resulting data were decoded using generational and positional bar codes to catalog the evolution of codons over the generations at each position as shown in Supplementary Figure S1.
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Figure 2: Sat-Sel-Seq methodology. Oligonucleotide cassettes (light blue) containing degenerate codons (NNS) at each position (dark blue) and neighboring silent mutations (red) were cloned into AID expression plasmid to construct saturation mutant libraries. Shown is the representative sequence of a Generation 0 (G0) library for Position 115. The G0 libraries were transformed into E. coli, expressed as a cohort and subjected to rounds of selection for acquired resistance to rifampin to generate subsequent generations of the library. The generational libraries were PCR amplified with primers barcoded for the generation number and the pooled PCR samples were sequenced by 454 high-throughput sequencing. The resulting data were decoded using generational and positional bar codes to catalog the evolution of codons over the generations at each position as shown in Supplementary Figure S1.

Mentions: We designed our method to specifically reveal the determinants of function within a small region of a larger protein (Figure 2). Our strategy for generation of the initial saturation mutagenesis libraries employed a cassette mutagenesis approach (45), which allows for high-efficiency library generation. The mutagenic cassette oligonucleotides contained two key features: a degenerate NNS codon at a single position within the targeting loop of AID and a second silent mutation at the codon immediately 3′ to the randomized codon. This silent mutation serves as an internal barcode that remains unchanged and marks the position of the original NNS codon. Twelve total saturation mutant libraries were generated, one for each position within the hotspot loop and a duplicate library for Phe115 using a different silent mutation barcode in order to assess assay reproducibility. The NNS codon renders each library inclusive of all 20 amino acids and a single stop codon, with all amino acids represented by at least one non-rare codon in E. coli. While the library is not equally represented for these variants, the use of an NNS codon allows for economical mutagenesis, and the change in codon frequency can be tracked across generations of selection.


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)

Sat-Sel-Seq methodology. Oligonucleotide cassettes (light blue) containing degenerate codons (NNS) at each position (dark blue) and neighboring silent mutations (red) were cloned into AID expression plasmid to construct saturation mutant libraries. Shown is the representative sequence of a Generation 0 (G0) library for Position 115. The G0 libraries were transformed into E. coli, expressed as a cohort and subjected to rounds of selection for acquired resistance to rifampin to generate subsequent generations of the library. The generational libraries were PCR amplified with primers barcoded for the generation number and the pooled PCR samples were sequenced by 454 high-throughput sequencing. The resulting data were decoded using generational and positional bar codes to catalog the evolution of codons over the generations at each position as shown in Supplementary Figure S1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150791&req=5

Figure 2: Sat-Sel-Seq methodology. Oligonucleotide cassettes (light blue) containing degenerate codons (NNS) at each position (dark blue) and neighboring silent mutations (red) were cloned into AID expression plasmid to construct saturation mutant libraries. Shown is the representative sequence of a Generation 0 (G0) library for Position 115. The G0 libraries were transformed into E. coli, expressed as a cohort and subjected to rounds of selection for acquired resistance to rifampin to generate subsequent generations of the library. The generational libraries were PCR amplified with primers barcoded for the generation number and the pooled PCR samples were sequenced by 454 high-throughput sequencing. The resulting data were decoded using generational and positional bar codes to catalog the evolution of codons over the generations at each position as shown in Supplementary Figure S1.
Mentions: We designed our method to specifically reveal the determinants of function within a small region of a larger protein (Figure 2). Our strategy for generation of the initial saturation mutagenesis libraries employed a cassette mutagenesis approach (45), which allows for high-efficiency library generation. The mutagenic cassette oligonucleotides contained two key features: a degenerate NNS codon at a single position within the targeting loop of AID and a second silent mutation at the codon immediately 3′ to the randomized codon. This silent mutation serves as an internal barcode that remains unchanged and marks the position of the original NNS codon. Twelve total saturation mutant libraries were generated, one for each position within the hotspot loop and a duplicate library for Phe115 using a different silent mutation barcode in order to assess assay reproducibility. The NNS codon renders each library inclusive of all 20 amino acids and a single stop codon, with all amino acids represented by at least one non-rare codon in E. coli. While the library is not equally represented for these variants, the use of an NNS codon allows for economical mutagenesis, and the change in codon frequency can be tracked across generations of selection.

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
Related in: MedlinePlus