Biochemical and kinetic characterization of the DNA helicase and exonuclease activities of Werner syndrome protein

The WRN gene, defective in the premature aging and genome instability disorder Werner syndrome, encodes a protein with DNA helicase and exonuclease activities. In this report, cofactor requirements for WRN catalytic activities were examined. WRN helicase performed optimally at an equimolar concentration ( 1 mM) of Mg2+ and ATP with a K-m of 140 muM for the ATP-Mg2+ complex. The initial rate of WRN helicase activity displayed a hyperbolic dependence on ATP-Mg2+ concentration. Mn2+ and Ni2+ substituted for Mg2+ as a cofactor for WRN helicase, whereas Fe2+ or Cu2+ ( 10 muM) profoundly inhibited WRN unwinding in the presence of Mg2+. Zn2+ ( 100 muM) was preferred over Mg2+ as a metal cofactor for WRN exonuclease activity and acts as a molecular switch, converting WRN from a helicase to an exonuclease. Zn2+ strongly stimulated the exonuclease activity of a WRN exonuclease domain fragment, suggesting a Zn2+ binding site in the WRN exonuclease domain. A fluorometric assay was used to study WRN helicase kinetics. The initial rate of unwinding increased with WRN concentration, indicating that excess enzyme over DNA substrate improved the ability of WRN to unwind the DNA substrate. Under presteady state conditions, the burst amplitude revealed a 1: 1 ratio between WRN and DNA substrate, suggesting an active monomeric form of the helicase. These are the first reported kinetic parameters of a human RecQ unwinding reaction based on real time measurements, and they provide mechanistic insights into WRN-catalyzed DNA unwinding.