Fig. 4: Kinetics of adenine glycosylase activity and dissociation constants, Kd.
A Full-time course adenine glycosylase assays for determination of kinetic parameters for WT, V246F, and R309C MUTYH. Left panel; time course reaction of MBP-MUTYH adenine excision under single turnover condition ([E] = 100 nM active concentration, [A:OG] = 20 nM). Right panel; kinetics of adenine excision under multiple turnover conditions ([E] = 2.5–5 nM active concentration, [A:OG] = 20 nM). Data fitting was used to obtained the glycosylase (k2) and turnover (k3) rate constants (See “Methods” for more information). B DNA binding isotherms of electrophoretic mobility shift assays (EMSA) for WT, R241Q, V246F and R309C. Left panel; DNA binding isotherms obtained with substrate adenine analog (fA) across OG (10 pM) titrated with increasing concentrations of MBP-MUTYH (0–190 nM). Right panel; DNA binding isotherms obtained with abasic site analog (THF) across OG (10 pM) titrated with increasing concentrations of MBP-MUTYH (0-3000 nM). C DNA binding isotherms obtained for KD determination with fA:OG- and THF:OG-containing DNA duplex binding with WT, R241Q and N238S MBP-MUTYH. Data for R241Q are shown in both panel B and C for comparison. All experiments were repeated in triplicate and the error bars the standard deviation from the mean. Source data are provided as a Source Data file.
