9964–9975 Nucleic Acids Research, 2014, Vol. 42, No. 15 Published online 26 July 2014 doi: 10.1093/nar/gku689 High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase Kiran S. Gajula1, Peter J. Huwe2,†, Charlie Y. Mo3,†, Daniel J. Crawford1, James T. Stivers4, Ravi Radhakrishnan2 and Rahul M. Kohli1,3,* 1Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA, 2Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA, 3Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA and 4Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA Received June 7, 2014; Revised July 16, 2014; Accepted July 16, 2014 ABSTRACT tional scanning and may find general utility for high- throughput analysis of protein function. Antibody maturation is a critical immune process governed by the enzyme activation-induced deam- inase (AID), a member of the AID/APOBEC DNA INTRODUCTION deaminase family. AID/APOBEC deaminases prefer- Enzyme families often share a central well-structured cat- entially target cytosine within distinct preferred se- alytic core, with different specificities among family mem- quence motifs in DNA, with specificity largely con- bers encoded by variable regions surrounding the active ferred by a small 9–11 residue protein loop that dif- site core (1,2). This mechanism for fulfilling the need for fers among family members. Here, we aimed to deter- specialization while maintaining core function is evident in / mine the key functional characteristics of this protein the family of AID APOBEC cytosine deaminase enzymes, loop in AID and to thereby inform our understanding which play an important role in adaptive and innate immu- nity. of the mode of DNA engagement. To this end, we Activation-induced deaminase (AID) is the central B-cell developed a methodology (Sat-Sel-Seq) that couples enzyme that governs two diversity-generating reactions that saturation mutagenesis at each position across the are essential for antibody maturation: somatic hypermuta- targeting loop, with iterative functional selection and tion (SHM) and class switch recombination (CSR) (3). In next-generation sequencing. This high-throughput SHM, deamination of cytosine bases within the variable re- mutational analysis revealed dominant characteris- gion of the immunoglobulin (Ig) locus populates the gene tics for residues within the loop and additionally with uracil bases. Error-prone repair of these uracil lesions yielded enzymatic variants that enhance deaminase generates variations within antibody complementarity de- activity. To rationalize these functional requirements, termining regions (CDRs) which can result in enhanced we performed molecular dynamics simulations that antigen binding and increase the effectiveness of adaptive suggest that AID and its hyperactive variants can en- immune responses. In CSR, deamination can alter the na- ture of the immune response that follows antigen binding. gage DNA in multiple specific modes. These find- CSR results from the introduction of uracil lesions into op- ings align with AID’s competing requirements for posite strands of DNA in the switch regions upstream of specificity and flexibility to efficiently drive antibody constant genes. Resolution of the resulting dsDNA breaks maturation. Beyond insights into the AID-DNA inter- juxtaposes the variable region encoding antigen specificity face, our Sat-Sel-Seq approach also serves to further with different constant regions to change the antibody from expand the repertoire of techniques for deep posi- IgM to an alternative isotype. The related APOBEC3 family enzymes (APOBEC3A-H in the human genome) play a role on the other arm of the immune system, contributing to in- *To whom correspondence should be addressed. Tel: +1 215 573 7523; Fax: +1 215 349 5111; Email: [email protected] †The authors wish it to be known that, in their opinion, the second and third authors contributed equally. Present Address: Rahul M. Kohli, Division of Infectious Diseases, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. C The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2014, Vol. 42, No. 15 9965 nate immune responses to retroviruses, such as HIV (4). Tar- MATERIALS AND METHODS geted deamination of cytosines in viral genomic interme- Cloning, expression and purification of human AID diates can lead to degradation, prevent viral integration or result in highly mutated and non-functional viral genomes Synthetic oligonucleotides for cloning and assays were pur- (5–8). chased from Integrated DNA Technologies and sequences As part of their mechanism of targeted deamination, are available upon request. Alanine scanning loop variants AID/APOBEC enzymes engage cytosine in the context of were generated by overlap extension polymerase chain reac- its neighboring nucleotides within ssDNA. AID prefers to tion (PCR) as previously described (17). Inserts were cloned mutate WRC motifs (W = A/T, R = A/G), which are highly into the EcoRI-XhoI region of AID-expressing pET41b populated within both the target CDRs and switch regions vector (Novagen) also containing an N-terminal maltose- in the Ig locus (9). APOBEC3 enzymes are also directed to binding protein, with the human AID gene codon opti- various hotspot motifs for deamination, with well charac- mized for expression in Escherichia coli as previously de- terized targeting of CCC by A3G and TC for A3A as exam- scribed (17). Plasmids were co-transformed with the chap- ples (5,8,10,11). Targeting of preferred ‘hotspot’ sequences erone trigger factor for heterologous expression in E. coli by the deaminases can be essential to their physiological BL21 (DE3) pLysS (Novagen). Enzyme expression and pu- function, as altered hotspot targeting of AID compromises rification were carried out as previously described (26). SHM and CSR (12,13). Hotspot targeting has also been key to deciphering the role of APOBEC3 family members in Deamination assays and sequence preference profiles driving mutagenesis in cancerous cells, as the deaminases leave a distinctive mutational signature with clustered mu- Similar to previously described protocols (17), the sub- tations enriched for a characteristic TC sequence context strate was a 27-mer oligonucleotide containing a single C (14,15). within an AGC context. For fluorescence-based assays, 1 Notably, engagement of target sequences by ␮M substrate containing a 3-fluorescein was incubated AID/APOBEC enzymes does not show the level of fi- with 1 ␮M enzyme, 1 unit uracil DNA glycosylase (UDG; delity seen with many other DNA modifying enzymes, such NEB) and 1 ␮g RNase A (Fermentas) in 20 mM Tris-Cl (pH as restriction enzymes. For AID, the differences between 8), 1 mM dithiothreitol (DTT), 1 mM ethylenediaminete- deamination of best (hotspot) and worst (coldspot) sub- traacetic acid (EDTA) for 3 h at 30◦C followed by heating strates is ∼12- to 30-fold, raising the question of how such to 95◦C for 20 min. For kinetic assays, the substrate was 32P loose specificity can be achieved (16,17). While the lack end-labeled by standard methods, gel purified and quanti- of a DNA-bound structure for any AID/APOBEC family fied using liquid scintillation counting. An end-labeled 40- member leaves many open questions (18–23), structure- mer was also generated as an internal standard. In the as- guided experiments by several groups have helped to say, 50–300 nM radioactive substrate was incubated with localize some of the determinants for deamination target- 15 nM standard oligonucleotide under reaction conditions ing. In particular, one highly divergent 9–11 amino acid described above for 1 h at 30◦C (within linear product for- protein loop situated between the ␤4strandand␣4 helix mation range). Abasic sites formed in the substrates were in AID was suggested to be a candidate for conferring cleaved by adding NaOH (100 mM final), an equal volume sequence preferences to the enzymes (8). In early studies, of formamide and heating to 95◦C for 20 min. Samples were selective point mutations in this loop altered the spectrum then separated on 20% Tris/Borate/EDTA (TBE), 7 M of deaminase activity (24). Even more strikingly, when the Urea polyacrylamide gels (45–50◦C). For assays with fluo- loop from one family member was replaced by the loop rescent substrate, gels were imaged using a Typhoon imager from a second family member, the sequence targeting of (GE healthcare) and the products quantified using Quan- the acceptor enzyme was noted to shift to that of the donor tityOne (Biorad). For kinetic assays, gels were imaged via (12,13,17,25). storage phosphor screen on the Typhoon imager, quantified Given the significance of the hotspot recognition loop in using custom MATLAB software, and the total amount of AID and the larger APOBEC family, we aimed to elucidate deamination was calculated using the known concentration the specific functional requirements of the residues