scholarly journals Escherichia coli Tus protein acts to arrest the progression of DNA replication forks in vitro.

1990 ◽  
Vol 87 (7) ◽  
pp. 2481-2485 ◽  
Author(s):  
T. M. Hill ◽  
K. J. Marians
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Replication Forks

scholarly journals Termination complex in Escherichia coli inhibits SV40 DNA replication in vitro by impeding the action of T antigen helicase.

1992 ◽  
Vol 267 (8) ◽  
pp. 5361-5365
Author(s):  
M Hidaka ◽  
T Kobayashi ◽  
Y Ishimi ◽  
M Seki ◽  
T Enomoto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
T Antigen ◽  
Sv40 Dna

scholarly journals Differential Suppression of priA2::kan Phenotypes in Escherichia coli K-12 by Mutations in priA, lexA, and dnaC

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 5-13 ◽  
Author(s):  
Steven J Sandler ◽  
Hardeep S Samra ◽  
Alvin J Clark
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Mutant Allele ◽  
Wild Type ◽  
Suppressor Mutations ◽  
Dnab Helicase ◽  
K 12 ◽  
Extragenic Suppressors ◽  
Assembly Activity

Abstract First identified as an essential component of the ϕX174 in vitro DNA replication system, PriA has ATPase, helicase, translocase, and primosome-assembly activities. priA1::kan strains of Escherichia coli are sensitive to UV irradiation, deficient in homologous recombination following transduction, and filamentous. priA2::kan strains have eightfold higher levels of uninduced SOS expression than wild type. We show that (1) priA1::kan strains have eightfold higher levels of uninduced SOS expression, (2) priA2::kan strains are UVS and Rec−, (3) lexA3 suppresses the high basal levels of SOS expression of a priA2::kan strain, and (4) plasmid-encoded priA300 (K230R), a mutant allele retaining only the primosome-assembly activity of priA+, restores both UVR and Rec+ phenotypes to a priA2::kan strain. Finally, we have isolated 17 independent UVR Rec+ revertants of priA2::kan strains that carry extragenic suppressors. All 17 map in the C-terminal half of the dnaC gene. DnaC loads the DnaB helicase onto DNA as a prelude for primosome assembly and DNA replication. We conclude that priA's primosome-assembly activity is essential for DNA repair and recombination and that the dnaC suppressor mutations allow these processes to occur in the absence of priA.

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.

The Escherichia coli chaperones involved in DNA replicadon

1993 ◽  
Vol 339 (1289) ◽  
pp. 271-278 ◽  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Heat Shock ◽  
Heat Shock Proteins ◽  
Conformational Changes ◽  
Heat Shock Genes ◽  
Dnab Helicase ◽  
Initiation Of Dna Replication ◽  
Shock Proteins

Mutadons in the Escherichia coli heat shock genes, dnaK , dnaJ or grpE , alter host DNA and RNA synthesis, degradation of other proteins, cell division and expression of other heat shock genes. They also block the initiation of DNA replication of bacteriophages λ and P1, and the mini-F plasmid. An in vitro λDNA replication system, composed entirely of purified components, enabled us to describe the molecular mechanism of the dnaK , dnaJ and grpE gene products. DnaK , the bacterial hsp 70 homologue, releases λP protein from the preprimosomal complex in an ATP- and DnaJ-dependent reaction (GrpEindependent initiation of λDNA replication). In this paper, I show that, when GrpE is present, λP protein is not released from the preprimosomal complex, rather it is translocated within the complex in such a way that it does not inhibit DnaB helicase activity. Translocation of λP triggers the initiation event allowing DnaB helicase to unwind DNA near the ori λ sequence, leading to efficient λDNA replication. Chaperone activity of the DnaK -DnaJ-GrpE system is first manifested in the selective binding of these heat shock proteins to the preprimosomal complex, followed by its ATP-dependent rearrangement. I show that DnaJ not only tags the preprimosomal complex for recognition by DnaK, but also stabilizes the multi-protein structure. GrpE also participates in the binding of DnaK to the preprimosomal complex by increasing DnaK ’s affinity to those λP proteins which are already associated with DnaJ. After attracting DnaK to the preprimosomal complex, DnaJ and GrpE stimulate the ATPase activity of DnaK , triggering conformational changes in DnaK which are responsible for the rearrangement of proteins in the preprimosomal complex and recycling of these heat shock proteins. The role of DnaK , DnaJ and GrpE in λDNA replication is in sharp contrast to our understanding of their role in the oriC , P1, and probably mini-F DNA replication systems. In the cases of oriC and P1 DNA replication, these heat shock proteins activate initiation factors before they are in contact with DNA, and are not required during the subsequent steps leading to the initiation of DNA replication. The common feature of DnaK , DnaJ and GrpE action in these systems is their ATP-dependent disaggregation or rearrangement of protein complexes formed before or during initiation of DNA replication.

scholarly journals Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation

2021 ◽  
Author(s):  
Allison W. McClure ◽  
John F.X. Diffley
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genotoxic Stress ◽  
Replication Initiation ◽  
Multiple Roles ◽  
Replication Forks ◽  
Dna Replication Stress ◽  
Necessary And Sufficient

SummaryThe Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

scholarly journals Anatomy of a twin DNA replication factory

2020 ◽  
Vol 48 (6) ◽  
pp. 2769-2778
Author(s):  
Huilin Li ◽  
Nina Y. Yao ◽  
Michael E. O'Donnell
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Bacillus Subtilis ◽  
Dna Replication ◽  
In Vivo Studies ◽  
Replication Forks ◽  
Structural Studies ◽  
Replication Factory ◽  
Higher Eukaryotes

The replication of DNA in chromosomes is initiated at sequences called origins at which two replisome machines are assembled at replication forks that move in opposite directions. Interestingly, in vivo studies observe that the two replication forks remain fastened together, often referred to as a replication factory. Replication factories containing two replisomes are well documented in cellular studies of bacteria (Escherichia coli and Bacillus subtilis) and the eukaryote, Saccharomyces cerevisiae. This basic twin replisome factory architecture may also be preserved in higher eukaryotes. Despite many years of documenting the existence of replication factories, the molecular details of how the two replisome machines are tethered together has been completely unknown in any organism. Recent structural studies shed new light on the architecture of a eukaryote replisome factory, which brings with it a new twist on how a replication factory may function.

scholarly journals Nuclease dead Cas9 is a programmable roadblock for DNA replication

2018 ◽  
Author(s):  
Kelsey Whinn ◽  
Gurleen Kaur ◽  
Jacob S. Lewis ◽  
Grant Schauer ◽  
Stefan Müller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Molecular Machines ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Single Molecule Level ◽  
Replication Fork Arrest

DNA replication occurs on chromosomal DNA while processes such as DNA repair, recombination and transcription continue. However, we have limited experimental tools to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct fused to the photo-stable dL5 protein fluoromodule as a novel, targetable protein-DNA roadblock for studying replication fork arrest at the single-molecule level in vitro as well as in vivo. We find that the specifically bound dCas9–guideRNA complex arrests viral, bacterial and eukaryotic replication forks in vitro.

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