Hexameric helicase G40P unwinds DNA in single base pair steps.

Michael Schlierf,Xiaojiang S Chen,Ganggang Wang,Taekjip Ha

doi:10.7554/elife.42001

Abstract

Most replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Binding of a single ATPγS could stall unwinding, demonstrating highly coordinated ATP hydrolysis between six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis.

Highlights

Helicases are essential enzymes for all life forms and catalyze the separation of double-stranded nucleic acids into single-stranded nucleic acids, and many of these enzymes involved in DNA repair, DNA recombination and transcription termination are linked to human diseases (Crampton et al, 2006; Enemark and Joshua-Tor, 2008; Enemark and Joshua-Tor, 2006; Gai et al, 2004; Johnson et al, 2007; Lionnet et al, 2007; Lohman et al, 2008; Manosas et al, 2009; Pandey et al, 2009; Patel and Picha, 2000; Rasnik et al, 2006b; Ribeck et al, 2010; Rothenberg et al, 2007; Singleton et al, 2000; Thomsen and Berger, 2009; Wang et al, 2008; Yodh et al, 2010)
For T7 gp4 helicase-primase, structural and ensemble kinetic data (Crampton et al, 2006; Liao et al, 2005; Singleton et al, 2000) suggested a sequential hydrolysis mechanism during DNA translocation, and with the one-to-one coupling between nucleotide unwinding and base pair unwinding (Pandey and Patel, 2014), but the estimated unwinding step size is either larger than 1 bp (Johnson et al, 2007) or is variable depending on the GC content of the duplex DNA (Donmez and Patel, 2008; Syed et al, 2014)
FRET between the donor (Cy3) and the acceptor (Cy5) fluorophores conjugated to the fork was used to follow individual DNA unwinding in real time (Figure 1—figure supplement 1)

Summary

Introduction

Helicases are essential enzymes for all life forms and catalyze the separation of double-stranded nucleic acids (dsNA) into single-stranded nucleic acids (ssNA), and many of these enzymes involved in DNA repair, DNA recombination and transcription termination are linked to human diseases (Crampton et al, 2006; Enemark and Joshua-Tor, 2008; Enemark and Joshua-Tor, 2006; Gai et al, 2004; Johnson et al, 2007; Lionnet et al, 2007; Lohman et al, 2008; Manosas et al, 2009; Pandey et al, 2009; Patel and Picha, 2000; Rasnik et al, 2006b; Ribeck et al, 2010; Rothenberg et al, 2007; Singleton et al, 2000; Thomsen and Berger, 2009; Wang et al, 2008; Yodh et al, 2010). Schlierf et al found that the primase DnaG could prevent G40P from moving backwards on the DNA, a new and unexpected function of DnaG These findings contribute to an ongoing debate among researchers with partially contradictory models for how DNA helicases unwind the DNA double helix. For T7 gp helicase-primase, structural and ensemble kinetic data (Crampton et al, 2006; Liao et al, 2005; Singleton et al, 2000) suggested a sequential hydrolysis mechanism during DNA translocation, and with the one-to-one coupling between nucleotide unwinding and base pair unwinding (Pandey and Patel, 2014), but the estimated unwinding step size is either larger than 1 bp (Johnson et al, 2007) or is variable depending on the GC content of the duplex DNA (Donmez and Patel, 2008; Syed et al, 2014). In the most comprehensive analysis of stepping by a ring-shaped motor on DNA, the DNA packaging motor from f29 was shown to package dsDNA in a hierarchy of non-integer, 2.5 bp steps, pausing after packaging 10 bp (Moffitt et al, 2009)

Results and discussion

A T G40P

D P i slippage low ATP concentration d

Materials and methods

Design of the unwinding substrate

Funding Funder National Institutes of Health