scholarly journals DNA Helicase-SSB Interactions Critical to the Regression and Restart of Stalled DNA Replication Forks in Escherichia coli

Genes ◽  
2020 ◽  
Vol 11 (5) ◽  
pp. 471 ◽  
Author(s):  
Piero R. Bianco
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Dna Helicase ◽  
Dependent Manner ◽  
Replication Forks ◽  
Replication Restart ◽  
Helicase Dnab ◽  
Fork Regression ◽  
Ob Fold ◽  
Dna Replication Restart

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.

scholarly journals Examination of the roles of a conserved motif in the PriA helicase in structure-specific DNA unwinding and processivity

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0255409
Author(s):  
Alexander T. Duckworth ◽  
Tricia A. Windgassen ◽  
James L. Keck
Keyword(s):  
Dna Replication ◽  
Dna Helicase ◽  
Sequence Motif ◽  
Dna Unwinding ◽  
Helicase Domain ◽  
Replication Forks ◽  
Dna Helicases ◽  
Conserved Sequence ◽  
Replication Restart ◽  
Dna Replication Restart

DNA replication complexes (replisomes) frequently encounter barriers that can eject them prematurely from the genome. To avoid the lethality of incomplete DNA replication that arises from these events, bacteria have evolved “DNA replication restart” mechanisms to reload replisomes onto abandoned replication forks. The Escherichia coli PriA DNA helicase orchestrates this process by recognizing and remodeling replication forks and recruiting additional proteins that help to drive replisome reloading. We have identified a conserved sequence motif within a linker region of PriA that docks into a groove on the exterior of the PriA helicase domain. Alterations to the motif reduce the apparent processivity and attenuate structure-specific helicase activity in PriA, implicating the motif as a potential autoregulatory element in replication fork processing. The study also suggests that multiple PriA molecules may function in tandem to enhance DNA unwinding processivity, highlighting an unexpected similarity between PriA and other DNA helicases.

scholarly journals p53 orchestrates DNA replication restart homeostasis by suppressing mutagenic RAD52 and POLθ pathways

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sunetra Roy ◽  
Karl-Heinz Tomaszowski ◽  
Jessica W Luzwick ◽  
Soyoung Park ◽  
Jun Li ◽  
...  
Keyword(s):  
Dna Replication ◽  
Genomic Instability ◽  
Genome Instability ◽  
Single Cells ◽  
Replication Forks ◽  
Mutational Signatures ◽  
Development Of Resistance ◽  
Cycle Arrest ◽  
Replication Restart ◽  
Dna Replication Restart

Classically, p53 tumor suppressor acts in transcription, apoptosis, and cell cycle arrest. Yet, replication-mediated genomic instability is integral to oncogenesis, and p53 mutations promote tumor progression and drug-resistance. By delineating human and murine separation-of-function p53 alleles, we find that p53 null and gain-of-function (GOF) mutations exhibit defects in restart of stalled or damaged DNA replication forks that drive genomic instability, which isgenetically separable from transcription activation. By assaying protein-DNA fork interactions in single cells, we unveil a p53-MLL3-enabled recruitment of MRE11 DNA replication restart nuclease. Importantly, p53 defects or depletion unexpectedly allow mutagenic RAD52 and POLθ pathways to hijack stalled forks, which we find reflected in p53 defective breast-cancer patient COSMIC mutational signatures. These data uncover p53 as a keystone regulator of replication homeostasis within a DNA restart network. Mechanistically, this has important implications for development of resistance in cancer therapy. Combined, these results define an unexpected role for p53-mediated suppression of replication genome instability.

scholarly journals Multiple Genetic Pathways for Restarting DNA Replication Forks in Escherichia coli K-12

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 487-497
Author(s):  
Steven J Sandler
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Double Mutant ◽  
Synthetic Lethality ◽  
Replication Forks ◽  
Viability Assay ◽  
Replication Restart ◽  
K 12 ◽  
Redundant Functions

Abstract In Escherichia coli, the primosome assembly proteins, PriA, PriB, PriC, DnaT, DnaC, DnaB, and DnaG, are thought to help to restart DNA replication forks at recombinational intermediates. Redundant functions between priB and priC and synthetic lethality between priA2::kan and rep3 mutations raise the possibility that there may be multiple pathways for restarting replication forks in vivo. Herein, it is shown that priA2::kan causes synthetic lethality when placed in combination with either Δrep::kan or priC303:kan. These determinations were made using a nonselective P1 transduction-based viability assay. Two different priA2::kan suppressors (both dnaC alleles) were tested for their ability to rescue the priA-priC and priA-rep double mutant lethality. Only dnaC809,820 (and not dnaC809) could rescue the lethality in each case. Additionally, it was shown that the absence of the 3′-5′ helicase activity of both PriA and Rep is not the critical missing function that causes the synthetic lethality in the rep-priA double mutant. One model proposes that replication restart at recombinational intermediates occurs by both PriA-dependent and PriA-independent pathways. The PriA-dependent pathways require at least priA and priB or priC, and the PriA-independent pathway requires at least priC and rep. It is further hypothesized that the dnaC809 suppression of priA2::kan requires priC and rep, whereas dnaC809,820 suppression of priA2::kan does not.

scholarly journals Atomic force microscopy–based characterization of the interaction of PriA helicase with stalled DNA replication forks

2020 ◽  
Vol 295 (18) ◽  
pp. 6043-6052 ◽  
Author(s):  
Yaqing Wang ◽  
Zhiqiang Sun ◽  
Piero R. Bianco ◽  
Yuri L. Lyubchenko
Keyword(s):  
Atomic Force Microscopy ◽  
Dna Replication ◽  
Dna Helicase ◽  
Replication Forks ◽  
Force Microscopy ◽  
Atomic Force ◽  
Helicase Dnab ◽  
Dna Substrates ◽  
Selection Of

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3′-to-5′ direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. Single-stranded DNA–binding protein (SSB) is typically present at the abandoned forks, but it is unclear how SSB and PriA interact, although it has been shown that the two proteins interact both physically and functionally. Here, we used atomic force microscopy to visualize the interaction of PriA with DNA substrates with or without SSB. These experiments were done in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. These analyses revealed that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Such a preference has not been observed for 5′- and 3′-tailed duplexes, suggesting that it is the fork structure that plays an essential role in PriA's selection of DNA substrates. Furthermore, we found that in the absence of SSB, PriA binds exclusively to the fork regions of the DNA substrates. In contrast, fork-bound SSB loads PriA onto the duplex DNA arms of forks, suggesting a remodeling of PriA by SSB. We also demonstrate that the remodeling of PriA requires a functional C-terminal domain of SSB. In summary, our atomic force microscopy analyses reveal key details in the interactions between PriA and stalled DNA replication forks with or without SSB.

scholarly journals DpiA Binding to the Replication Origin of Escherichia coli Plasmids and Chromosomes Destabilizes Plasmid Inheritance and Induces the Bacterial SOS Response

2003 ◽  
Vol 185 (20) ◽  
pp. 6025-6031 ◽  
Author(s):  
Christine Miller ◽  
Hanne Ingmer ◽  
Line Elnif Thomsen ◽  
Kirsten Skarstad ◽  
Stanley N. Cohen
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Replication Origin ◽  
Sos Response ◽  
Dna Helicase ◽  
Replication Initiation ◽  
Chromosomal Dna ◽  
E Coli ◽  
Dna Replication Initiation ◽  
Helicase Dnab

ABSTRACT The dpiA and dpiB genes of Escherichia coli, which are orthologs of genes that regulate citrate uptake and utilization in Klebsiella pneumoniae, comprise a two-component signal transduction system that can modulate the replication of and destabilize the inheritance of pSC101 and certain other plasmids. Here we show that perturbed replication and inheritance result from binding of the effector protein DpiA to A+T-rich replication origin sequences that resemble those in the K. pneumoniae promoter region targeted by the DpiA ortholog, CitB. Consistent with its ability to bind to A+T-rich origin sequences, overproduction of DpiA induced the SOS response in E. coli, suggesting that chromosomal DNA replication is affected. Bacteria that overexpressed DpiA showed an increased amount of DNA per cell and increased cell size—both also characteristic of the SOS response. Concurrent overexpression of the DNA replication initiation protein, DnaA, or the DNA helicase, DnaB—both of which act at A+T-rich replication origin sequences in the E. coli chromosome and DpiA-targeted plasmids—reversed SOS induction as well as plasmid destabilization by DpiA. Our finding that physical and functional interactions between DpiA and sites of replication initiation modulate DNA replication and plasmid inheritance suggests a mechanism by which environmental stimuli transmitted by these gene products can regulate chromosomal and plasmid dynamics.

scholarly journals p53 suppresses mutagenic RAD52 and POLθ pathways by orchestrating DNA replication restart homeostasis

2017 ◽  
Author(s):  
Sunetra Roy ◽  
Karl-Heinz Tomaszowski ◽  
Jessica Luzwick ◽  
Soyoung Park ◽  
Jun Li ◽  
...  
Keyword(s):  
Dna Replication ◽  
Genomic Instability ◽  
Genome Instability ◽  
Single Cells ◽  
Replication Forks ◽  
Mutational Signatures ◽  
Development Of Resistance ◽  
Cycle Arrest ◽  
Replication Restart ◽  
Dna Replication Restart

ABSTRACTClassically, p53 tumor-suppressor acts in transcription, apoptosis, and cell-cycle arrest. Yet, replication-mediated genomic instability is integral to oncogenesis, and p53 mutations promote tumor progression and drug-resistance. By delineating human and murine separation-of-function p53 alleles, we find that p53 null and gain-of-function (GOF) mutations exhibit defects in restart of stalled or damaged DNA replication forks driving genomic instability independent of transcription activation. By assaying protein-DNA fork interactions in single cells, we unveil a p53-MLL3-enabled recruitment of MRE11 DNA replication restart nuclease. Importantly, p53 defects or depletion unexpectedly allow mutagenic RAD52 and POLθ pathways to hijack stalled forks, which we find reflected in p53 defective breast-cancer patient COSMIC mutational signatures. These data uncover p53 as a keystone regulator of replication homeostasis within a DNA restart network. Mechanistically, this has important implications for development of resistance in cancer therapy. Combined, these results define an unexpected role for p53 suppression of replication genome instability.

Single-molecule binding characterization of primosomal protein PriA involved in replication restart

2021 ◽  
Author(s):  
Tzu-Yu Lee ◽  
Yi-Ching Li ◽  
Min-Guan Lin ◽  
Chwan-Deng Hsiao ◽  
Hung-Wen Li
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Single Molecule ◽  
Replication Forks ◽  
Bacterial Survival ◽  
Dna Damages ◽  
Replication Restart

DNA damages lead to stalled or collapsed replication forks. Replication restart primosomes re-initiate DNA synthesis at these stalled or collapsed DNA replication forks, which is important for bacterial survival. Primosomal...

scholarly journals Delineation of the Ancestral Tus-Dependent Replication Fork Trap

2021 ◽  
Author(s):  
Casey Toft ◽  
Morgane Moreau ◽  
Jiri Perutka ◽  
Savitri Mandapati ◽  
Peter Enyeart ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Replication Fork ◽  
Wide Distribution ◽  
Replication Forks ◽  
Genome Wide ◽  
Replication Fork Arrest

In Escherichia coli, DNA replication termination is orchestrated by two clusters of Ter sites forming a DNA replication fork trap when bound by Tus proteins. The formation of a ‘locked’ Tus-Ter complex is essential for halting incoming DNA replication forks. However, the absence of replication fork arrest at some Ter sites raised questions about their significance. In this study, we examined the genome-wide distribution of Tus and found that only the six innermost Ter sites (TerA-E and G) were significantly bound by Tus. We also found that a single ectopic insertion of TerB in its non-permissive orientation could not be achieved, advocating against a need for ‘back-up’ Ter sites. Finally, examination of the genomes of a variety of Enterobacterales revealed a new replication fork trap architecture mostly found outside the Enterobacteriaceae family. Taken together, our data enabled the delineation of a narrow ancestral Tus-dependent DNA replication fork trap consisting of only two Ter sites.

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