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Nucleic Acids Res. 2014 Sep;42(15):9964-75. doi: 10.1093/nar/gku689. Epub 2014 Jul 26.

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

Author information

1
Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
2
Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
3
Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
4
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
5
Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA rkohli@upenn.edu.

Abstract

Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9-11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement. To this end, we developed a methodology (Sat-Sel-Seq) that couples saturation mutagenesis at each position across the targeting loop, with iterative functional selection and next-generation sequencing. This high-throughput mutational analysis revealed dominant characteristics for residues within the loop and additionally yielded enzymatic variants that enhance deaminase activity. 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.

PMID:
25064858
PMCID:
PMC4150791
DOI:
10.1093/nar/gku689
[Indexed for MEDLINE]
Free PMC Article

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