Abstract
In Escherichia coli, DNA replication forks stall on average once per cell cycle. When this occurs, replisome components disengage from the DNA, exposing an intact, or nearly intact fork. Consequently, the fork structure must be regressed away from the initial impediment so that repair can occur. Regression is catalyzed by the powerful, monomeric DNA helicase, RecG. During this reaction, the enzyme couples unwinding of fork arms to rewinding of duplex DNA resulting in the formation of a Holliday junction. RecG works against large opposing forces enabling it to clear the fork of bound proteins. Following subsequent processing of the extruded junction, the PriA helicase mediates reloading of the replicative helicase DnaB leading to the resumption of DNA replication. The single-strand binding protein (SSB) plays a key role in mediating PriA and RecG functions at forks. It binds to each enzyme via linker/OB-fold interactions and controls helicase-fork loading sites in a substrate-dependent manner that involves helicase remodeling. Finally, it is displaced by RecG during fork regression. The intimate and dynamic SSB-helicase interactions play key roles in ensuring fork regression and DNA replication restart.
Highlights
The precise duplication of the genome relies on the DNA replication, DNA repair and genetic recombination machinery working closely together [1,2,3,4,5]
protein A (PriA) binds to an single-stranded DNA (ssDNA) hairpin structure in φX174 called the n'primosome assembly site (PAS), leading to the assembly of the primosome, a complex responsible for primer RNA synthesis and duplex DNA unwinding at a replication fork [104,105]
Stalled DNA replication fork rescue is essential. It requires physical and functional interactions between the single-strand binding protein (SSB) protein and the DNA helicases, PriA and RecG.SSB-helicase binding is critical to the loading of these enzymes onto stalled forks
Summary
The precise duplication of the genome relies on the DNA replication, DNA repair and genetic recombination machinery working closely together [1,2,3,4,5]. When the replisome encounters a nick in the leading strand, the structure of the fork will collapse and require the processing of the nascent double-strand break by RecBCD followed by strand invasion catalyzed by RecA This results in the formation of a displacement loop that is used to reload the replicative helicase DnaB leading to the restart of DNA replication [15]. For many years a conundrum existed in the fork regression field as it was thought that the branch-specific DNA helicases RecG or RuvAB could each catalyze this reaction This conundrum was resolved using a combination of bulk-phase biochemical and single-molecule approaches. RecG [46,47,48]
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