Figure 4.

LIG1 syndrome mutations decrease catalytic efficiency and increase abortive ligation. (A) Representative time course from multiple turnover reactions containing 10 nM enzyme, 500 nM DNA, 2 mM MgCl₂ (1 mM Mg²⁺_free) and 1 mM ATP. Initial rates from multiple turnover reactions were determined from the slope of linear fits to the amount of substrate reacted. (B) The steady-state DNA dependence was determined with 0.1–10 nM LIG1 and increasing amounts of the DNA substrate. The resulting data were fit by the Michaelis–Menten equation to obtain k_cat and K_M,DNA values for WT and mutant LIG1 enzymes. (C) The R641L and R771W mutants have substantially reduced catalytic efficiency (k_cat/K_M values from the fits in panel b). (D) Representative denaturing gel image from a ligation assay containing 5 nM R641L, 500 nM DNA, 2 mM MgCl₂ (1 mM Mg²⁺_free) and 1 mM ATP highlighting buildup of the aborted intermediate. (E) The fraction of abortive ligation events observed in the steady-state magnesium dependences. The physiologically-relevant [Mg²⁺]_free (∼1 mM) is marked by a dashed line. R641L and R771W LIG1 exhibit enhanced rates of abortive ligation compared to WT at low [Mg²⁺]_free. All values are mean ± SD (n ≥ 3).