Biochemical and Kinetic Characterization of the DNA Helicase and Exonuclease Activities of Werner Syndrome Protein

Saba Choudhary,Joshua A Sommers,Robert M Brosh

doi:10.1074/jbc.m401901200

Saba Choudhary, Joshua A Sommers + Show 1 more

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https://doi.org/10.1074/jbc.m401901200

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Abstract

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 Mg(2+) and ATP with a K(m) of 140 microm for the ATP-Mg(2+) complex. The initial rate of WRN helicase activity displayed a hyperbolic dependence on ATP-Mg(2+) concentration. Mn(2+) and Ni(2+) substituted for Mg(2+) as a cofactor for WRN helicase, whereas Fe(2+) or Cu(2+) (10 microm) profoundly inhibited WRN unwinding in the presence of Mg(2+).Zn(2+) (100 microm) was preferred over Mg(2+) as a metal cofactor for WRN exonuclease activity and acts as a molecular switch, converting WRN from a helicase to an exonuclease. Zn(2+) strongly stimulated the exonuclease activity of a WRN exonuclease domain fragment, suggesting a Zn(2+) 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.

Highlights

It is generally believed that RecQ helicases play an important role in the maintenance of genome stability [21,22,23]; the precise molecular and cellular functions of RecQ helicases are not well understood
To better understand the catalytic functions of the Werner syndrome protein and how they may be important in DNA metabolism, we investigated the cofactor requirements for WRN helicase and exonuclease activities
Fluorescence Resonance Energy Transfer for Measuring WRN Helicase Unwinding of Duplex DNA—To begin to understand the mechanism of duplex DNA unwinding catalyzed by the WRN helicase, we performed kinetic analyses of the unwinding reaction using a fluorometric helicase assay and a more traditional gel-based assay that uses a radiolabeled DNA substrate

Summary

Introduction

It is generally believed that RecQ helicases play an important role in the maintenance of genome stability [21,22,23]; the precise molecular and cellular functions of RecQ helicases are not well understood. To better understand the WRN helicase mechanism, we utilized a fluorometric assay to monitor helicase-catalyzed unwinding of duplex DNA [38] This assay uses the principle of fluorescence resonance energy transfer to observe the unwinding of duplex DNA in real time. Upon helicase-catalyzed unwinding of the duplex and separation of the complementary strands, F and HF are no longer in close proximity, and the fluorescence emission from F excitation can be detected by a photosensor (Fig. 1B) This method of measuring helicase-catalyzed unwinding of duplex DNA using fluorescence stopped-flow instrumentation is valuable for kinetic analyses where data are collected continuously throughout the reaction in real time. We applied fluorescence resonance energy transfer technology to study the kinetics of WRN helicase activity under presteady state conditions in which the enzyme is saturated with an excess of DNA substrate [39]. Mechanistic information on the WRN helicase reaction was determined from these analyses and provides the first kinetic parameters of a human RecQ unwinding reaction based on real time measurements

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