Mechanisms of opening and closing of the bacterial replicative helicase.

Jillian Chase,Amedee Des Georges,Edward T Eng,Martin Samuels,Brian Chait,Paul Db Olinares,David Jeruzalmi,Alex J Noble,Andrew Catalano,Kelly Molloy,Danaya Pakotiprapha

doi:10.7554/elife.41140

Abstract

Assembly of bacterial ring-shaped hexameric replicative helicases on single-stranded (ss) DNA requires specialized loading factors. However, mechanisms implemented by these factors during opening and closing of the helicase, which enable and restrict access to an internal chamber, are not known. Here, we investigate these mechanisms in the Escherichia coli DnaB helicase•bacteriophage λ helicase loader (λP) complex. We show that five copies of λP bind at DnaB subunit interfaces and reconfigure the helicase into an open spiral conformation that is intermediate to previously observed closed ring and closed spiral forms; reconfiguration also produces openings large enough to admit ssDNA into the inner chamber. The helicase is also observed in a restrained inactive configuration that poises it to close on activating signal, and transition to the translocation state. Our findings provide insights into helicase opening, delivery to the origin and ssDNA entry, and closing in preparation for translocation.

Highlights

Chromosomal replicative helicases are hexameric protein ensembles that travel ahead of the advancing replisome, dissolving duplex DNA into templates for DNA synthesis
Except that the map has been rotated by 90 ̊. (C) The E. coli DnaB helicase.bacteriophage lP complex is shown depicting the ruptured interface between DnaB subunits A and B and the deep canyon that runs through the complex
The complex has been sub-divided into three tiers: lP loader, the DnaB CTD, and the DnaB NTD tiers

Summary

Introduction

Chromosomal replicative helicases are hexameric protein ensembles that travel ahead of the advancing replisome, dissolving duplex DNA into templates for DNA synthesis. Four molecular elements cooperate to begin the process of assembling the replisome These elements are: a) a DNA sequence of length in the hundreds of basepairs called a replication origin, b) the replication initiator protein (E. coli: DnaA (Mott and Berger, 2007; Wolanski et al, 2014; Leonard and Grimwade, 2015) V. cholera: DnaA, RctB (Egan and Waldor, 2003; Val et al, 2014; Gerding et al, 2015; Fournes et al, 2018), plasmids: RepE, Pi, TrfA (Konieczny et al, 2014), phage l: O (Weigel and Seitz, 2006)), c) the replicative helicase, and d) the helicase loader. Our findings reveal insights into mechanisms of opening and closing of the helicase, and provide a coherent structural view of helicase loading at the origin of DNA replication

Results

Discussion

Materials and methods

Funding Funder National Institutes of Health