Fig. 3
From: GEN1 as a risk factor for human congenital anomalies of the kidney and urinary tract
Nuclease activity of GEN1 variant proteins at 5’ flap DNA. As a HJ enzymatic lyase, GEN1 protein’s dissociation ability was partly manifested by the degradation of the equal 5’ flap (substrate) by different concentrations of purified proteins. In this experiment, seven gradient concentrations were applied (0, 0.5, 1, 2, 4, 8 and 16nM). GEN1 (p.R401X,508) showed the most significant reduction in enzymatic hydrolytic capacity. The other five mutant proteins [GEN1 (p.T105R), GEN1 (p.D149N), GEN1 (p.H369L), GEN1 (p.I577T), and GEN1 (p. T105R + p.I577T)] impaired the enzymatic hydrolysis of 5’ flap. The impaired enzymatic hydrolytic capacity in the control group variants was less significant than that in CAKUT group. (A) EMSA of GEN1-5’ flap complexes. 5’ flap DNA was hydrolyzed by WT and mutant GEN1 proteases. The upper band of each plot represents undigested 5’ flap DNA, and the lower band represents enzymatically hydrolyzed 5’ flap DNA. The band concentration represents 5’ flap concentration. (B) Grayscale statistical chart. The results are reported as mean ± SD for three individual experiments. * sites in the non-CAKUT group. Abbreviations: EMSA, electrophoretic mobility shift assay; HJ, Holliday junction; SD, standard deviation; WT, wild type