Sequence-dependent base pair stepping dynamics in XPD helicase unwinding

Zhi Qi,Yann R Chemla,Robert A Pugh,Maria Spies

doi:10.7554/elife.00334

Abstract

Helicases couple the chemical energy of ATP hydrolysis to directional translocation along nucleic acids and transient duplex separation. Understanding helicase mechanism requires that the basic physicochemical process of base pair separation be understood. This necessitates monitoring helicase activity directly, at high spatio-temporal resolution. Using optical tweezers with single base pair (bp) resolution, we analyzed DNA unwinding by XPD helicase, a Superfamily 2 (SF2) DNA helicase involved in DNA repair and transcription initiation. We show that monomeric XPD unwinds duplex DNA in 1-bp steps, yet exhibits frequent backsteps and undergoes conformational transitions manifested in 5-bp backward and forward steps. Quantifying the sequence dependence of XPD stepping dynamics with near base pair resolution, we provide the strongest and most direct evidence thus far that forward, single-base pair stepping of a helicase utilizes the spontaneous opening of the duplex. The proposed unwinding mechanism may be a universal feature of DNA helicases that move along DNA phosphodiester backbones. DOI:http://dx.doi.org/10.7554/eLife.00334.001.

Highlights

Helicases are vectorial enzymes that utilize ATP hydrolysis to translocate along single-stranded nucleic acids (NA) and separate the base pairs of the duplex
Ensemble kinetic and structural studies of SF1 PcrA (Subramanya et al, 1996; Soultanas et al, 1999; Dillingham et al, 2000, 2002), UvrD (Ail et al, 1999; Fischer et al, 2004; Lee and Yang, 2006) and Superfamily 2 (SF2) nonstructural protein 3 (NS3) (Tai et al, 1996; Kim et al, 1998; Pang et al, 2002; Levin et al, 2004; Tackett et al, 2005; Mackintosh et al, 2006; Gu and Rice, 2010) suggest these domains move as a ratchet-like inchworm, whereby the helicase translocates along single-stranded NA by 1 nucleotide during each ATP binding and hydrolysis cycle
Our analyses provide little evidence for a statistically significant 0.5-bp step size, as recently reported for NS3 helicase and interpreted as a manifestation of transient DNA looping in that system (Cheng et al, 2011)

Summary

Introduction

Helicases are vectorial enzymes that utilize ATP hydrolysis to translocate along single-stranded nucleic acids (NA) and separate the base pairs (bp) of the duplex. Ensemble kinetic and structural studies of SF1 PcrA (Subramanya et al, 1996; Soultanas et al, 1999; Dillingham et al, 2000, 2002), UvrD (Ail et al, 1999; Fischer et al, 2004; Lee and Yang, 2006) and SF2 nonstructural protein 3 (NS3) (Tai et al, 1996; Kim et al, 1998; Pang et al, 2002; Levin et al, 2004; Tackett et al, 2005; Mackintosh et al, 2006; Gu and Rice, 2010) suggest these domains move as a ratchet-like inchworm, whereby the helicase translocates along single-stranded NA by 1 nucleotide (nt) during each ATP binding and hydrolysis cycle Despite these findings, the mechanism of base pair separation has remained elusive. The mechanism of base pair separation—whether achieved by

Methods

Results

Conclusion