Are the SSB-Interacting Proteins RecO, RecG, PriA and the DnaB-Interacting Protein Rep Bound to Progressing Replication Forks in Escherichia coli?

Bentchikou, Esma; Chagneau, Carine; Long, Emilie; Matelot, Mélody; Allemand, Jean-François; Michel, Bertrand

doi:10.1371/journal.pone.0134892

Cited by 15 publications

(22 citation statements)

References 41 publications

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“…Our finding of multiple Rep molecules colocalized with the replisome compares to a previous recent live cell imaging study of fluorescently-labeled Rep, and other repair and replisome proteins (28). Here, although the authors did not have an independent fork marker for visualizing simultaneous Rep and fork colocalization, they observed Rep foci in locations consistent with fork localization.…”

Section: Discussioncontrasting

confidence: 47%

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Single-molecule live cell imaging of Rep reveals the dynamic interplay between an accessory replicative helicase and the replisome

Syeda

Wollman

Harvgreaves

et al. 2018

Preprint

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DNA replication requires strategies to cope with nucleoprotein barriers that impair the efficient translocation of the replisome. Biochemical and genetic studies indicate accessory helicases play essential roles in continuity of replication in the presence of nucleoprotein barriers, but how they operate in the native cellular environment is unclear. With high-speed single-molecule microscopy we determine the dynamic patterns of localization of genomically-encoded fluorescent protein constructs of the bacterial accessory helicase Rep and core replisome protein DnaQ in live E. coli cells. We demonstrate that Rep colocalizes with 70% of replication forks. Colocalisation is dependent upon interaction with replicative helicase DnaB, with an underlying hexameric stoichiometry of Rep indicating maximal occupancy of the single DnaB hexamer within the replisome. We find that Rep associates dynamically with the replisome with an average dwell time of 6.5 ms dependent on ATP hydrolysis, indicating rapid binding then translocation away from the fork. We also imaged the PriC replication restart factor given the known Rep-PriC functional interaction and observe Rep-replisome association is also dependent on the presence of PriC. Our findings suggest two Rep-replisome populations in vivo: one involving Rep continually associating with DnaB then translocating away to aid nucleoprotein barrier removal ahead of the fork, another assisting PriC-dependent reloading of DnaB if replisome progression fails. These new findings reveal how a single type of helicase is recruited to the replisome to provide two independent ways of underpinning replication of proteinbound DNA, a problem that all organisms face as they replicate their genomes. Significance statementAll organisms face the challenge of proteins bound to DNA acting as barriers to prevent DNA replication. We have performed fluorescence imaging experiments on living bacteria to track the positions of the replication machinery, a protein called Rep which is involved in removing these barriers, and a protein called PriC believed to be involved with reloading the replication machinery if the original replication machinery breaks down. We find that Rep is very dynamic with continual binding and movement away from the replication machinery. Association with the replication machinery depends on both binding to the replication machinery directly and on PriC. Thus Rep can circumvent barriers in two independent ways: a strategy which may be relevant to all organisms.

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Section: Discussioncontrasting

confidence: 47%

“…Indeed, a recent live cell single-molecule imaging study failed to detect any Rep molecules present at the replisome (28). Furthermore, accessory helicases at the fork may have more than one function and more than one interaction partner.…”

mentioning

confidence: 99%

Single-molecule live cell imaging of Rep reveals the dynamic interplay between an accessory replicative helicase and the replisome

Syeda

Wollman

Harvgreaves

et al. 2018

Preprint

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“…RecG and RecO bind to the unstructured C-terminus of SSB in vitro [33,46]. However, visualization of RecG and RecO molecules in a DNA replication fork in vivo has been unsuccessful so far [64,69]. We hypothesized that a competition between RecG and RecO binding to SSB-ssDNA via the C-terminus of SSB could play a critical role in regulating pathways of DNA metabolisms at the interface of DNA replication, repair and homologous recombination when DNA replication turned out to be difficulty due to some situations aforementioned.…”

Section: Experimental Rationalementioning

confidence: 99%

“…It catalyzes single stranded DNA annealing of complementary oligonucleotides complexed with SSB in an ATP-independent manner [36][37][38][39]. RecR can inhibit the annealing activity of RecO by binding to it, forming RecOR complex [22,34,[40][41][42] [55][56][57][58][59][60][61][62][63][64]. Some of them interact with the unstructured C-terminus of SSB when working at the interface of DNA replication and recombination [34, 42, 62, and 65].…”

Section: Introductionmentioning

confidence: 99%

RecO impedes RecG-SSB binding to impair the strand annealing recombination pathway inE.coli

Pan

Yang

Jiang

et al. 2019

Preprint

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Faithful duplication of genomic DNA relies not only on the fidelity of DNA replication itself, but also on fully functional DNA repair and homologous recombination machinery. We report a molecular mechanism responsible for deciding homologous recombinational repair pathways during replication dictated by binding of RecO and RecG to SSB in E.coli. Using a RecG-yfp fusion protein, we found that RecG-yfp foci appeared only in the Δ recG, Δ recO and Δ recA, Δ recO double mutants. Surprisingly, foci were not observed in wild-type Δ recG, or double mutants where recG and either recF or, separately recR were deleted. In addition, formation of RecG-yfp foci in the Δ recO::kan R required wildtype ssb, as ssb-113 could not substitute. This suggests that RecGand RecO binding to SSB is competitive. We also found that the UV resistance of recO alone mutant increased to certain extent by supplementing RecG. In an ssb-113 mutant, RecO and RecG worked following a different pattern. Both RecO and RecG were able to participate in repairing UV damages when grown at permissive temperature, while they could also be involved in making DNA double strand breaks when grown at nonpermissive temperature. So, our results suggested that differential binding of RecG and RecO to SSB in a DNA replication fork in Escherichia coli.may be involved in determining whether the SDSA or DSBR pathway of homologous recombinational repair is used. Author summarySingle strand DNA binding proteins (SSB) stabilize DNA holoenzyme and prevent single strand DNA from folding into non-B DNA structures in a DNA replication fork. It has also been revealed that SSB can also act as a platform for some proteins working in DNA repair and recombination to access DNA molecules when DNA replication fork needs to be reestablished. In Escherichia coli, several proteins working primarily in DNA repair and recombination were found to participate in DNA replication fork resumption by physically interacting with SSB, including RecO and RecG etc. However the hierarchy of these proteins interacting with SSB in Escherichia coli has not been well defined. In this study, we demonstrated a differential binding of RecO and RecG to SSB in DNA replication was used to establish a RecO-dependent pathway of replication fork repair by abolishing a RecG-dependent replication fork repair. We also show that, RecG and RecO could randomly participate in DNA replication repair in the absence of a functional SSB, which may be responsible for the generation of DNA double strand breaks in an ssb-113 mutant in Escherichia coli.

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“…In bacteria, SSB proteins assemble onto DNA as homodimers or tetramers, each unit composed of an N-terminal DNA binding domain and a C-terminal intrinsically disordered tail that mediates interaction with numerous proteins (Kozlov et al, 2015 ). The bacterial SSB proteins are mainly associated with the replication machinery at the replicating forks and serve as central hubs to coordinate DNA replication and repair (Lecointe et al, 2007 ; Costes et al, 2010 ; Antony et al, 2013 ; Bentchikou et al, 2015 ). As in bacteria, the eukaryotic single strand binding protein RPA has multiple roles in protecting ssDNA, sensing and promoting repair of DNA damage via PPIs (Oakley and Patrick, 2010 ; Maréchal and Zou, 2015 ).…”

Section: Identification Of Phospho-proteins Involved In Dna-related Pmentioning

confidence: 99%

Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria

et al. 2016

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In all living organisms, the phosphorylation of proteins modulates various aspects of their functionalities. In eukaryotes, protein phosphorylation plays a key role in cell signaling, gene expression, and differentiation. Protein phosphorylation is also involved in the global control of DNA replication during the cell cycle, as well as in the mechanisms that cope with stress-induced replication blocks. Similar to eukaryotes, bacteria use Hanks-type kinases and phosphatases for signal transduction, and protein phosphorylation is involved in numerous cellular processes. However, it remains unclear whether protein phosphorylation in bacteria can also regulate the activity of proteins involved in DNA-mediated processes such as DNA replication or repair. Accumulating evidence supported by functional and biochemical studies suggests that phospho-regulatory mechanisms also take place during the bacterial cell cycle. Recent phosphoproteomics and interactomics studies identified numerous phosphoproteins involved in various aspect of DNA metabolism strongly supporting the existence of such level of regulation in bacteria. Similar to eukaryotes, bacterial scaffolding-like proteins emerged as platforms for kinase activation and signaling. This review reports the current knowledge on the phosphorylation of proteins involved in the maintenance of genome integrity and the regulation of cell cycle in bacteria that reveals surprising similarities to eukaryotes.

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Are the SSB-Interacting Proteins RecO, RecG, PriA and the DnaB-Interacting Protein Rep Bound to Progressing Replication Forks in Escherichia coli?

Cited by 15 publications

References 41 publications

Single-molecule live cell imaging of Rep reveals the dynamic interplay between an accessory replicative helicase and the replisome

Single-molecule live cell imaging of Rep reveals the dynamic interplay between an accessory replicative helicase and the replisome

RecO impedes RecG-SSB binding to impair the strand annealing recombination pathway inE.coli

Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria

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