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

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.

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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.

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DNA end resection and its role in DNA replication and DSB repair choice in mammalian cells

Experimental & Molecular Medicine ◽

10.1038/s12276-020-00519-1 ◽

2020 ◽

Vol 52 (10) ◽

pp. 1705-1714 ◽

Cited By ~ 1

Author(s):

Fei Zhao ◽

Wootae Kim ◽

Jake A. Kloeber ◽

Zhenkun Lou

Keyword(s):

Dna Repair ◽

Dna Replication ◽

Mammalian Cells ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

General Process ◽

Break Repair ◽

Dna End Resection ◽

End Resection

Abstract DNA end resection has a key role in double-strand break repair and DNA replication. Defective DNA end resection can cause malfunctions in DNA repair and replication, leading to greater genomic instability. DNA end resection is initiated by MRN-CtIP generating short, 3′-single-stranded DNA (ssDNA). This newly generated ssDNA is further elongated by multiple nucleases and DNA helicases, such as EXO1, DNA2, and BLM. Effective DNA end resection is essential for error-free homologous recombination DNA repair, the degradation of incorrectly replicated DNA and double-strand break repair choice. Because of its importance in DNA repair, DNA end resection is strictly regulated. Numerous mechanisms have been reported to regulate the initiation, extension, and termination of DNA end resection. Here, we review the general process of DNA end resection and its role in DNA replication and repair pathway choice.

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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.

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

10.1101/398438 ◽

2018 ◽

Cited By ~ 1

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.

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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.

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Chromatin remodeler Fft3 plays a dual role at blocked DNA replication forks

Life Science Alliance ◽

10.26508/lsa.201900433 ◽

2019 ◽

Vol 2 (5) ◽

pp. e201900433 ◽

Cited By ~ 2

Author(s):

Anissia Ait-Saada ◽

Olga Khorosjutina ◽

Jiang Chen ◽

Karol Kramarz ◽

Vladimir Maksimov ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Fission Yeast ◽

Chromatin Remodeling ◽

Replication Fork ◽

Strand Break ◽

Replication Forks ◽

Damage Repair ◽

Single Strand Annealing ◽

Strand Annealing

Here, we investigate the function of fission yeast Fun30/Smarcad1 family of SNF2 ATPase-dependent chromatin remodeling enzymes in DNA damage repair. There are three Fun30 homologues in fission yeast, Fft1, Fft2, and Fft3. We find that only Fft3 has a function in DNA repair and it is needed for single-strand annealing of an induced double-strand break. Furthermore, we use an inducible replication fork barrier system to show that Fft3 has two distinct roles at blocked DNA replication forks. First, Fft3 is needed for the resection of nascent strands, and second, it is required to restart the blocked forks. The latter function is independent of its ATPase activity.

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Replication Fork Slowing and Stalling are Distinct, Checkpoint-Independent Consequences of Replicating Damaged DNA

10.1101/122895 ◽

2017 ◽

Author(s):

Divya Ramalingam Iyer ◽

Nicholas Rhind

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Fission Yeast ◽

Single Molecule ◽

Replication Fork ◽

Replication Forks ◽

Origin Firing ◽

Dna Combing ◽

Fork Stalling

AbstractIn response to DNA damage during S phase, cells slow DNA replication. This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. Slowing of replication allows for tolerance of DNA damage and suppresses genomic instability. Although the mechanisms of origin inhibition by the intra-S checkpoint are understood, major questions remain about how the checkpoint regulates replication forks: Does the checkpoint regulate the rate of fork progression? Does the checkpoint affect all forks, or only those encountering damage? Does the checkpoint facilitate the replication of polymerase-blocking lesions? To address these questions, we have analyzed the checkpoint in the fission yeast Schizosaccharomyces pombe using a single-molecule DNA combing assay, which allows us to unambiguously separate the contribution of origin and fork regulation towards replication slowing, and allows us to investigate the behavior of individual forks. Moreover, we have interrogated the role of forks interacting with individual sites of damage by using three damaging agents—MMS, 4NQO and bleomycin—that cause similar levels of replication slowing with very different frequency of DNA lesions. We find that the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. Finally, using a novel analytic approach, we rigorously identify fork stalling events in our combing data and show that they play a previously unappreciated role in shaping replication kinetics in response to DNA damage.Author SummaryFaithful duplication of the genome is essential for genetic stability of organisms and species. To ensure faithful duplication, cells must be able to replicate damaged DNA. To do so, they employ checkpoints that regulate replication in response to DNA damage. However, the mechanisms by which checkpoints regulate DNA replication forks, the macromolecular machines that contain the helicases and polymerases required to unwind and copy the parental DNA, is unknown. We have used DNA combing, a single-molecule technique that allows us to monitor the progression of individual replication forks, to characterize the response of fission yeast replication forks to DNA damage that blocks the replicative polymerases. We find that forks pass most lesions with only a brief pause and that this lesion bypass is checkpoint independent. However, at a low frequency, forks stall at lesions, and that the checkpoint is required to prevent these stalls from accumulating single-stranded DNA. Our results suggest that the major role of the checkpoint is not to regulate the interaction of replication forks with DNA damage, per se, but to mitigate the consequences of fork stalling when forks are unable to successfully navigate DNA damage on their own.

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Double-Strand Break Generation under Deoxyribonucleotide Starvation in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00411-07 ◽

2007 ◽

Vol 189 (15) ◽

pp. 5782-5786 ◽

Cited By ~ 30

Author(s):

Estrella Guarino ◽

Israel Salguero ◽

Alfonso Jiménez-Sánchez ◽

Elena C. Guzmán

Keyword(s):

Escherichia Coli ◽

Replication Fork ◽

Thermal Inactivation ◽

Strand Break ◽

Double Strand Break ◽

Replication Forks ◽

Thymine Starvation ◽

Hydroxyurea Treatment ◽

Deoxynucleoside Triphosphate ◽

Stalled Replication Forks

ABSTRACT Stalled replication forks produced by three different ways of depleting deoxynucleoside triphosphate showed different capacities to undergo “replication fork reversal.” This reaction occurred at the stalled forks generated by hydroxyurea treatment, was impaired under thermal inactivation of ribonucleoside reductase, and did not take place under thymine starvation.

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Evidence for Biased Holliday Junction Cleavage and Mismatch Repair Directed by Junction Cuts during Double-Strand-Break Repair in Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.21.10.3425-3435.2001 ◽

2001 ◽

Vol 21 (10) ◽

pp. 3425-3435 ◽

Cited By ~ 13

Author(s):

Mark D. Baker ◽

Erin C. Birmingham

Keyword(s):

Homologous Recombination ◽

Mismatch Repair ◽

Mammalian Cells ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Holliday Junction ◽

Chromosomal Dna ◽

Dna Breaks ◽

Break Repair

ABSTRACT In mammalian cells, several features of the way homologous recombination occurs between transferred and chromosomal DNA are consistent with the double-strand-break repair (DSBR) model of recombination. In this study, we examined the segregation patterns of small palindrome markers, which frequently escape mismatch repair when encompassed within heteroduplex DNA formed in vivo during mammalian homologous recombination, to test predictions of the DSBR model, in particular as they relate to the mechanism of crossover resolution. According to the canonical DSBR model, crossover between the vector and chromosome results from cleavage of the joint molecule in two alternate sense modes. The two crossover modes lead to different predicted marker configurations in the recombinants, and assuming no bias in the mode of Holliday junction cleavage, the two types of recombinants are expected in equal frequency. However, we propose a revision to the canonical model, as our results suggest that the mode of crossover resolution is biased in favor of cutting the DNA strands upon which DNA synthesis is occurring during formation of the joint molecule. The bias in junction resolution permitted us to examine the potential consequences of mismatch repair acting on the DNA breaks generated by junction cutting. The combination of biased junction resolution with both early and late rounds of mismatch repair can explain the marker patterns in the recombinants.

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