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

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.

Download Full-text

Delineation of the Ancestral Tus-Dependent Replication Fork Trap

10.20944/preprints202111.0539.v1 ◽

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.

Download Full-text

Defining the prototypical DNA replication fork trap in bacteria

10.1101/2021.07.20.453168 ◽

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.

Download Full-text

Delineation of the Ancestral Tus-Dependent Replication Fork Trap

International Journal of Molecular Sciences ◽

10.3390/ijms222413533 ◽

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.

Download Full-text

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

PLoS Biology ◽

10.1371/journal.pbio.3000886 ◽

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.

Download Full-text

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

10.1101/2020.08.10.243931 ◽

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.

Download Full-text

Faculty Opinions recommendation of Genome-wide Nucleotide-Resolution Mapping of DNA Replication Patterns, Single-Strand Breaks, and Lesions by GLOE-Seq.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.737786716.793576729 ◽

2020 ◽

Author(s):

Susan Gerbi ◽

Miiko Sokka

Keyword(s):

Dna Replication ◽

Single Strand ◽

Strand Breaks ◽

Single Strand Breaks ◽

Genome Wide ◽

Resolution Mapping ◽

Nucleotide Resolution

Download Full-text

Implications of DNA replication for eukaryotic gene expression

Journal of Cell Science ◽

10.1242/jcs.99.2.201 ◽

1991 ◽

Vol 99 (2) ◽

pp. 201-206 ◽

Cited By ~ 8

Author(s):

A.P. Wolffe

Keyword(s):

Gene Expression ◽

Dna Replication ◽

Replication Fork ◽

Recent Progress ◽

Gene Activity ◽

Nucleosome Assembly ◽

Developmental Processes ◽

Eukaryotic Gene Expression ◽

Eukaryotic Gene

DNA replication has a key role in many developmental processes. Recent progress in understanding events at the replication fork suggests mechanisms for both establishing and maintaining programs of eukaryotic gene activity. In this review, I discuss the consequences of replication fork passage for preexisting chromatin structures and describe how the mechanism of nucleosome assembly at the replication fork may facilitate the formation of either transcriptionally active or repressed chromatin.

Download Full-text

Set1-dependent H3K4 methylation becomes critical for DNA replication fork progression in response to changes in S phase dynamics in Saccharomyces cerevisiae

10.1101/2020.10.26.355008 ◽

2020 ◽

Author(s):

Christophe de La Roche Saint-André ◽

Vincent Géli

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Dna Replication ◽

Replication Fork ◽

Genetic Material ◽

S Phase ◽

Replication Forks ◽

H3k4 Methylation ◽

Origin Firing ◽

Phase Dynamics

AbstractDNA replication is a highly regulated process that occurs in the context of chromatin structure and is sensitive to several histone post-translational modifications. In Saccharomyces cerevisiae, the histone methylase Set1 is responsible for the transcription-dependent deposition of H3K4 methylation (H3K4me) throughout the genome. Here we show that a combination of a hypomorphic replication mutation (orc5-1) with the absence of Set1 (set1Δ) compromises the progression through S phase, and this is associated with a large increase in DNA damage. The ensuing DNA damage checkpoint activation, in addition to that of the spindle assembly checkpoint, restricts the growth of orc5-1 set1Δ. Interestingly, orc5-1 set1Δ is sensitive to the lack of RNase H activity while a reduction of histone levels is able to counterbalance the loss of Set1. We propose that the recently described Set1-dependent mitigation of transcription-replication conflicts becomes critical for growth when the replication forks accelerate due to decreased origin firing in the orc5-1 background. Furthermore, we show that an increase of reactive oxygen species (ROS) levels, likely a consequence of the elevated DNA damage, is partly responsible for the lethality in orc5-1 set1Δ.Author summaryDNA replication, that ensures the duplication of the genetic material, starts at discrete sites, termed origins, before proceeding at replication forks whose progression is carefully controlled in order to avoid conflicts with the transcription of genes. In eukaryotes, DNA replication occurs in the context of chromatin, a structure in which DNA is wrapped around proteins, called histones, that are subjected to various chemical modifications. Among them, the methylation of the lysine 4 of histone H3 (H3K4) is carried out by Set1 in Saccharomyces cerevisiae, specifically at transcribed genes. We report that, when the replication fork accelerates in response to a reduction of active origins, the absence of Set1 leads to accumulation of DNA damage. Because H3K4 methylation was recently shown to slow down replication at transcribed genes, we propose that the Set1-dependent becomes crucial to limit the occurrence of conflicts between replication and transcription caused by replication fork acceleration. In agreement with this model, stabilization of transcription-dependent structures or reduction histone levels, to limit replication fork velocity, respectively exacerbates or moderates the effect of Set1 loss. Last, but not least, we show that the oxidative stress associated to DNA damage is partly responsible for cell lethality.

Download Full-text

Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication

Science Advances ◽

10.1126/sciadv.abc0330 ◽

2020 ◽

Vol 6 (38) ◽

pp. eabc0330 ◽

Cited By ~ 1

Author(s):

D. T. Gruszka ◽

S. Xie ◽

H. Kimura ◽

H. Yardimci

Keyword(s):

Dna Replication ◽

Real Time ◽

Single Molecule ◽

Replication Fork ◽

Epigenetic Inheritance ◽

Replication Forks ◽

Replication Fork Progression ◽

Egg Extracts ◽

Fork Stalling ◽

The Moment

During replication, nucleosomes are disrupted ahead of the replication fork, followed by their reassembly on daughter strands from the pool of recycled parental and new histones. However, because no previous studies have managed to capture the moment that replication forks encounter nucleosomes, the mechanism of recycling has remained unclear. Here, through real-time single-molecule visualization of replication fork progression in Xenopus egg extracts, we determine explicitly the outcome of fork collisions with nucleosomes. Most of the parental histones are evicted from the DNA, with histone recycling, nucleosome sliding, and replication fork stalling also occurring but at lower frequencies. Critically, we find that local histone recycling becomes dominant upon depletion of endogenous histones from extracts, revealing that free histone concentration is a key modulator of parental histone dynamics at the replication fork. The mechanistic details revealed by these studies have major implications for our understanding of epigenetic inheritance.

Download Full-text