Differences in the enzymatic efficiency of human and bony fish AID are mediated by a single residue in the C terminus modulating single‐stranded DNA binding

Abstract

Activation-induced cytidine deaminase (AID) mediates antibody diversification by deaminating deoxycytidines to deoxyuridine within immunoglobulin genes. However, it also generates genome-wide DNA lesions, leading to transformation. Though the biochemical properties of AID have been described, its 3-dimensional structure has not been determined. Hence, to investigate the relationship between the primary structure and biochemical characteristics of AID, we compared the properties of human and bony fish AID, since these are most divergent in amino acid sequence. We show that AIDs of various species have different catalytic rates that are thermosensitive and optimal at native physiological temperatures. Zebrafish AID is severalfold more catalytically robust than human AID, while catfish AID is least active. This disparity is mediated by a single amino acid difference in the C terminus. Using functional assays supported by models of AID core and surface structure, we show that this residue modulates activity by affecting ssDNA binding. Furthermore, the cold-adapted catalytic rates of fish AID result from increased ssDNA binding affinity at lower temperatures. Our work suggests that AID may generate DNA damage with variable efficiencies in different organisms, identifies residues critical in regulating AID activity, and provides insights into the evolution of the APOBEC family of enzymes.

Zinc-binding Domain-dependent, Deaminase-independent Actions of Apolipoprotein B mRNA-editing Enzyme, Catalytic Polypeptide 2 (Apobec2), Mediate Its Effect on Zebrafish Retina Regeneration

“…1, A and B). Other work has suggested that temperature impacts the activity of zebrafish AID (27,32). To test if this is also true of zebrafish Apobec2a,2b, we performed mutagenesis assays at 25°C instead of 37°C.…”

“…Briefly, BL21(DE3) cultures harboring the indicated plasmid were grown at 37°C in 2 ml of LB containing 100 g/ml ampicillin to an A 600 of 0.5. 1 ml of LB containing 100 g/ml ampicillin, 3 mM isopropyl-␤-D-thiogalactopyranoside was then added, and the culture was grown overnight with shaking at either 37 or 25°C (27,28). The next day, 1 ml of the culture was harvested, spun down, and spread onto a plate containing LB and 100 g/ml rifampicin.…”

Activation induced deaminase C-terminal domain links DNA breaks to end protection and repair during class switch recombination

“…E. coli mutation assays were performed using the Δung BW310 strain as described (24). AID variants were subcloned as NheI-NotI fragments from pEGFP-N3 into pTrc23-24 (21) express GST-AID fusions and purified as described (51). An end-labeled bubble substrate containing a 7-nt-long single-stranded region with the motif TGC, previously described to be an optimal AID substrate, was used in activity assays and EMSAs (52).…”

Catalytic Pocket Inaccessibility of Activation-Induced Cytidine Deaminase Is a Safeguard against Excessive Mutagenic Activity

“…the canonical position of the three Zn-coordinating residues (H56, C87, and C90) above a glutamic acid residue (E58), the equivalent of which in all other cytidine/cytosine deaminases serves as the catalytic proton-donating residue for deamination ( Figure 3C) (Conticello et al, 2007b). Second, this catalytic motif forms an indented pocket on the surface with dimensions and electrostatic properties that allow docking of a dC nucleotide in deamination-feasible conformations ( Figure 3) Dancyger et al, 2012;Abdouni et al, 2013). That a viable catalytic pocket is formed by the three-dimensional convergence of the four catalytic residues from throughout the model (a2, a3, loops 2, 4, 6, and 8), provides overall confidence ( Figures 3A-3D).…”

“…In 2012 and 2013, we presented several core and surface charge/topology models of bony fish and human AID. These models were based on the resolved partial A3G structure template and had features that could explain differences in biochemical properties among bony fish and human AID Dancyger et al, 2012;Abdouni et al, 2013).…”