The Balance between Recombination Enzymes and Accessory Replicative Helicases in Facilitating Genome Duplication.

Aisha Syeda,John Atkinson,Peter Mcglynn,Robert Lloyd

doi:10.3390/genes7080042

Aisha Syeda, John Atkinson + Show 2 more

Open Access

https://doi.org/10.3390/genes7080042

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Abstract

Accessory replicative helicases aid the primary replicative helicase in duplicating protein-bound DNA, especially transcribed DNA. Recombination enzymes also aid genome duplication by facilitating the repair of DNA lesions via strand exchange and also processing of blocked fork DNA to generate structures onto which the replisome can be reloaded. There is significant interplay between accessory helicases and recombination enzymes in both bacteria and lower eukaryotes but how these replication repair systems interact to ensure efficient genome duplication remains unclear. Here, we demonstrate that the DNA content defects of Escherichia coli cells lacking the strand exchange protein RecA are driven primarily by conflicts between replication and transcription, as is the case in cells lacking the accessory helicase Rep. However, in contrast to Rep, neither RecA nor RecBCD, the helicase/exonuclease that loads RecA onto dsDNA ends, is important for maintaining rapid chromosome duplication. Furthermore, RecA and RecBCD together can sustain viability in the absence of accessory replicative helicases but only when transcriptional barriers to replication are suppressed by an RNA polymerase mutation. Our data indicate that the minimisation of replisome pausing by accessory helicases has a more significant impact on successful completion of chromosome duplication than recombination-directed fork repair.

Highlights

The replication machineries of all organisms encounter many potential barriers whilst duplicating their genomes, presenting a major challenge to the maintenance of genetic stability [1,2]
Individual nucleoprotein complexes may have a low probability of halting a replication fork but the large number of barriers encountered creates a substantial risk of failure to complete high fidelity genome duplication [6,7]
One key feature of E. coli cells lacking the strand exchange protein RecA in otherwise unperturbed cells is elevated levels of RecBCD-dependent chromosome degradation [55]. This degradation is manifest as formation of cells with a range of different numbers of chromosome equivalents as detected by flow cytometry [55] (see Figure 1A (i,iii))

Summary

Introduction

The replication machineries of all organisms encounter many potential barriers whilst duplicating their genomes, presenting a major challenge to the maintenance of genetic stability [1,2] These barriers include damage to the template, non-B form DNA structures, topological strain and proteins bound to the DNA. Individual nucleoprotein complexes may have a low probability of halting a replication fork but the large number of barriers encountered creates a substantial risk of failure to complete high fidelity genome duplication [6,7] Replisomes paused at these barriers retain activity but this activity is lost as a function of time [8,9,10,11]. If clearance or bypass of the barrier does not occur prior to loss of paused replisome function the replication machinery must be reloaded back onto the chromosome to facilitate

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