Figure 7

Proposed mechanism of hAID-catalyzed deamination. (a) hAID binds to ssDNA and slides bidirectionally along this molecule. (b–e) Occasionally, the enzyme pauses to deaminate C or 5 mC. There are three elements that determine the enzyme’s propensity to pause: (1) the network of interactions between the catalytic pocket and the pyrimidine ring that position the latter (red circles; the shade of the color corresponds to the strength of the interactions, with darker colors corresponding to stronger interactions); (2) the network of interactions between hAID and the ssDNA sugar-phosphate backbone (one or two green circles); (3) the stacking interactions between the aromatic residues of the protein and nucleobase(s) adjacent to C/5 mC (blue lines; the shade of the color and the number of lines correspond to the stability of the interactions, with darker colors and more lines indicating increased stability. (b) The ability of wt hAID to pause and deaminate C is determined by (1) strong stabilization of the pyrimidine ring in the catalytic pocket; (2) N51 residue interactions with the sugar-phosphate ssDNA backbone; and (3) stable stacking interactions (W84:Y114:G). (c) The ability of wt hAID to pause and deaminate 5 mC is determined by (1) sufficient stabilization of the pyrimidine ring in the catalytic pocket; (2) double anchoring of the sugar-phosphate ssDNA backbone (by interactions specific to the N51 residue and to the methyl group); and (3) unstable stacking (W84:Y114:G) or alternative stacking (A:Y114:G). (d) The N51A mutant failed to deaminate C due to (1) insufficient stabilization of the pyrimidine ring and (2) the lack of an ssDNA backbone anchoring. The stacking (W84:Y114:G) is stable in this case. (e) The N51A mutant retains its activity on 5 mC because of (1) sufficient stabilization of the pyrimidine ring and (2) anchoring of the sugar-phosphate ssDNA backbone mediated by the methyl group. In this case, the stacking (W84:Y114:G) is unstable, or alternative stacking (A:Y114:G) is formed.