Single-molecule studies reveal reciprocating of WRN helicase core along ssDNA during DNA unwinding

Xu-Guang Xi,Xi-Miao Hou,Brigitte René,Shuo-Xing Dou,Ming Li,Bo Zhang,Wen-Qiang Wu,Olivier Mauffret,Philippe Fossé

doi:10.1038/srep43954

Abstract

Werner syndrome is caused by mutations in the WRN gene encoding WRN helicase. A knowledge of WRN helicase’s DNA unwinding mechanism in vitro is helpful for predicting its behaviors in vivo, and then understanding their biological functions. In the present study, for deeply understanding the DNA unwinding mechanism of WRN, we comprehensively characterized the DNA unwinding properties of chicken WRN helicase core in details, by taking advantages of single-molecule fluorescence resonance energy transfer (smFRET) method. We showed that WRN exhibits repetitive DNA unwinding and translocation behaviors on different DNA structures, including forked, overhanging and G-quadruplex-containing DNAs with an apparently limited unwinding processivity. It was further revealed that the repetitive behaviors were caused by reciprocating of WRN along the same ssDNA, rather than by complete dissociation from and rebinding to substrates or by strand switching. The present study sheds new light on the mechanism for WRN functioning.

Highlights

Helicases are ubiquitous enzymes moving directionally along nucleic acids by coupling the chemical energy from NTP to the separation of two annealed nucleic acid strands[1]
Using single-molecule fluorescence resonance energy transfer (smFRET), we found a novel feature of WRN in DNA unwinding: repetitive movements
To characterize the general unwinding properties of WRN, we designed a substrate to mimic a replication fork or an open telomeric end, named as Fork-17bp (Fig. 1a, right panel), which was labelled with Cy3 at the ss/dsDNA junction, and Cy5 on the biotinylated strand at the 5th nucleotide from the junction

Summary

Introduction

Helicases are ubiquitous enzymes moving directionally along nucleic acids by coupling the chemical energy from NTP to the separation of two annealed nucleic acid strands[1] They are found in all known organisms and involved in practically all aspects of nucleic acid metabolism, including replication, recombination, transcription, translation, repair, chromosome segregation and telomere maintenance[1,2,3]. Using smFRET, we found a novel feature of WRN in DNA unwinding: repetitive movements. The repetitive movements came neither from its dissociation and rebinding, nor from strand switching, rather, it came from reciprocating of the helicase along the same ssDNA This novel feature of WRN might keep dsDNA or other DNA secondary structures such as G4 and hairpin being constantly in a disrupted or unfolded state, and make contributions to the functioning of other enzymes during transcription, replication, DNA repair and telomere metabolism in vivo

Methods

Results

Conclusion