Identification of a basal system for unwinding a bacterial chromosome origin.

Tomas T Richardson,Heath Murray,Omar Harran,Simone Pelliciari,Theodor Sperlea,Daniel Stevens

doi:10.15252/embj.2019101649

Tomas T Richardson, Heath Murray + Show 4 more

Open Access

https://doi.org/10.15252/embj.2019101649

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Abstract

Genome duplication is essential for cell proliferation, and DNA synthesis is generally initiated by dedicated replication proteins at specific loci termed origins. In bacteria, the master initiator DnaA binds the chromosome origin (oriC) and unwinds the DNA duplex to permit helicase loading. However, despite decades of research it remained unclear how the information encoded within oriC guides DnaA‐dependent strand separation. To address this fundamental question, we took a systematic genetic approach in vivo and identified the core set of essential sequence elements within the Bacillus subtilis chromosome origin unwinding region. Using this information, we then show in vitro that the minimal replication origin sequence elements are necessary and sufficient to promote the mechanical functions of DNA duplex unwinding by DnaA. Because the basal DNA unwinding system characterized here appears to be conserved throughout the bacterial domain, this discovery provides a framework for understanding oriC architecture, activity, regulation and diversity.

Highlights

Accurate transmission of genetic material is a fundamental requirement for the viability of all cells
The unwinding region of the bipartite B. subtilis oriC is located between the dnaA and dnaN genes and has been termed incC
To enable identification of essential DnaA-boxes within incC without selecting for suppressor mutations, we utilized a strain in which DNA replication can conditionally initiate from a plasmid origin integrated into the chromosome (Fig 1B) (Richardson et al, 2016)

Summary

Introduction

Accurate transmission of genetic material is a fundamental requirement for the viability of all cells. DNA replication must commence once (and only once) per cell cycle to ensure rigorous coordination of genome duplication and segregation. Dysfunction of DNA replication initiation can lead to improper chromosome inheritance, disease and cell death. Throughout the domains of life, conserved proteins containing AAA+ (ATPase Associated with various cellular Activities) domains assemble into dynamic multimeric complexes on double-stranded DNA (dsDNA) and direct loading of the replicative helicase (Bleichert et al, 2017). Subsequent helicase activation promotes assembly of the replication machinery that catalyses DNA synthesis. The ring-shaped hexameric helicases that drive bidirectional replication from a chromosome origin (oriC) are loaded around single-stranded DNA (ssDNA).

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