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

2021 ◽  
Vol 22 (24) ◽  
pp. 13533
Author(s):  
Casey J. Toft ◽  
Morgane J. J. 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.

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.

scholarly journals Defining the prototypical DNA replication fork trap in bacteria

2021 ◽  
Author(s):  
Patrick M Schaeffer ◽  
Andrew Ellington ◽  
Jiri Perutka ◽  
Peter Enyeart ◽  
Savitri Mandapati ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Replication Fork ◽  
Wide Distribution ◽  
Replication Forks ◽  
Opposite Orientation ◽  
Genome Wide ◽  
Replication Fork Arrest

In Escherichia coli, DNA replication termination is orchestrated by two opposite 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. 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 ectopic insertion of a TerB sequence in its non-permissive orientation could not be achieved, advocating against the necessity for 'back-up' Ter sites due to the inefficient formation of a 'locked' Tus-Ter complex. Finally, examination of the genomes of a variety of Enterobacterales revealed two major types of replication fork traps including a prototypical architecture consisting of two unique Ter sequences in opposite orientation.

scholarly journals Nascent chromatin occupancy profiling reveals locus and factor specific chromatin maturation dynamics behind the DNA replication fork

2018 ◽  
Author(s):  
Mónica P. Gutiérrez ◽  
Heather K. MacAlpine ◽  
David M. MacAlpine
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Histone Variants ◽  
Replication Forks ◽  
Nucleosome Assembly ◽  
Genome Wide ◽  
Dna Binding Factors ◽  
Nucleotide Resolution ◽  
Kinetics Of ◽  
Regulatory Landscape

AbstractProper regulation and maintenance of the epigenome is necessary to preserve genome function. However, in every cell division, the epigenetic state is disassembled and then re-assembled in the wake of the DNA replication fork. Chromatin restoration on nascent DNA is a complex and regulated process that includes nucleosome assembly and remodeling, deposition of histone variants, and the re-establishment of transcription factor binding. To study the genome-wide dynamics of chromatin restoration behind the DNA replication fork, we developed Nascent Chromatin Occupancy Profiles (NCOPs) to comprehensively profile nascent and mature chromatin at nucleotide resolution. While nascent chromatin is inherently less organized than mature chromatin, we identified locus specific differences in the kinetics of chromatin maturation that were predicted by the epigenetic landscape, including the histone variant H2A.Z which marked loci with rapid maturation kinetics. The chromatin maturation at origins of DNA replication was dependent on whether the origin underwent initiation or was passively replicated from distal-originating replication forks suggesting distinct chromatin assembly mechanisms between activated and disassembled pre-replicative complexes. Finally, we identified sites that were only occupied transiently by DNA-binding factors following passage of the replication fork which may provide a mechanism for perturbations of the DNA replication program to shape the regulatory landscape of the genome.

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.

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

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kelsey S. Whinn ◽  
Gurleen Kaur ◽  
Jacob S. Lewis ◽  
Grant D. Schauer ◽  
Stefan H. Mueller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Molecular Machines ◽  
Enzymatic Activities ◽  
Replication Forks ◽  
Dna Substrates ◽  
Replication Fork Arrest

Abstract Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9–guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro.

scholarly journals Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq

PLoS Biology ◽  
2021 ◽  
Vol 19 (3) ◽  
pp. e3000886
Author(s):  
Neesha Kara ◽  
Felix Krueger ◽  
Peter Rugg-Gunn ◽  
Jonathan Houseley
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Replication Fork ◽  
Strand Break ◽  
Double Strand Break ◽  
Replication Forks ◽  
Dna Breaks ◽  
Genome Wide ◽  
Fork Stalling ◽  
End Resection

Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3′ ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3′ ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

scholarly journals Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq

2020 ◽  
Author(s):  
Neesha Kara ◽  
Felix Krueger ◽  
Peter Rugg-Gunn ◽  
Jonathan Houseley
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Replication Fork ◽  
High Sensitivity ◽  
Strand Break ◽  
Double Strand Break ◽  
Replication Forks ◽  
Dna Breaks ◽  
Biochemical Purification ◽  
Genome Wide

AbstractUnderstanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods sensitive to both changes in replication fork structure and the formation of recombinogenic DNA ends. Here we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single stranded DNA 3’ ends genome-wide and with base pair resolution. TrAEL-seq labels DNA breaks, and profiles both stalled and processive replication forks in yeast and mammalian cells. Replication forks stalling at defined barriers and expressed genes are detected by TrAEL-seq with exceptional signal-to-noise, most likely through labelling of DNA 3’ ends exposed during fork reversal. TrAEL-seq also labels unperturbed processive replication forks to yield maps of replication fork direction similar to those obtained by Okazaki fragment sequencing, however TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3’ ends also allows accurate detection of double strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double strand break hotspots in a dmc1Δ mutant. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

scholarly journals Single-molecule insight into stalled replication fork rescue in Escherichia coli

2021 ◽  
Author(s):  
Piero R Bianco ◽  
Yue Lu
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Protein Complexes ◽  
Replication Forks ◽  
Step Process ◽  
Sequential Action ◽  
Single Molecule Studies ◽  
Insight Into

Abstract DNA replication forks stall at least once per cell cycle in Escherichia coli. DNA replication must be restarted if the cell is to survive. Restart is a multi-step process requiring the sequential action of several proteins whose actions are dictated by the nature of the impediment to fork progression. When fork progress is impeded, the sequential actions of SSB, RecG and the RuvABC complex are required for rescue. In contrast, when a template discontinuity results in the forked DNA breaking apart, the actions of the RecBCD pathway enzymes are required to resurrect the fork so that replication can resume. In this review, we focus primarily on the significant insight gained from single-molecule studies of individual proteins, protein complexes, and also, partially reconstituted regression and RecBCD pathways. This insight is related to the bulk-phase biochemical data to provide a comprehensive review of each protein or protein complex as it relates to stalled DNA replication fork rescue.

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