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

ABSTRACT: We have designed a methodology for capture of DNA 3’ ends that allows mapping of resected DNA breaks, stalled replication forks and also normal replication fork progression. This Transferase-activated end ligation or TrAEL-seq method involves ligation of a functionalised linker to DNA 3’ ends followed by fragmentation, purification of adaptor ligated fragments, second adaptor ligation and library amplification. The major advantages of TrAEL-seq compared to other available methods are: i) an ability to map double strand breaks after resection, ii) excellent sensitivity and signal-to-noise in detecting replication fork stalling and iii) ability to map replication fork progression in unsynchronised, unlabelled populations of both yeast and mammalian cells. The samples provided here were selected to demonstrate different aspects of TrAEL-seq activity: the SfiI and dmc1 datasets shows capture of 3’ extended single strand DNA. The other yeast datasets show replication and replication fork stalling information. The RAF and RAF-GAL grown yeast samples show the effect transcriptional induction on replication fork progression. The hESC samples show the capacity to derive replication profiles from mammalian cells.

ORGANISM(S): Homo sapiens Saccharomyces cerevisiae

PROVIDER: GSE154811 | GEO | 2020/07/27

REPOSITORIES: GEO

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Genome-wide analysis of replication fork locations in hydroxyurea-treated Saccharomyces cerevisiae by TrAEL-seq

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TrAEL-seq for CUP1 locus

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Genome-wide analysis of DNA replication stress in the Human Neuroblastoma Cell Line IMR32 by TrAEL-seq

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Characterisation of mRNA expression and replication fork stalling in copper-treated yeast

Project description:Here we analyse steady state mRNA levels and yH2A-marked replication fork stalling sites in S. cerevisiae

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Temporal Regulation of Head-on Transcription at Replication Initiation Sites

Project description:Head-on collisions between the DNA replication machinery and RNA polymerase are potent genotoxic events leading to replication fork stalling, R-loop formation, and DNA breaks. Current models suggest that head-on collisions are avoided through replication initiation site (RIS) placement upstream of active genes, thus ensuring co-orientation of replication fork movement and genic transcription. However, this model does not account for pervasive transcription units, or intragenic replication initiation events. Through mining phased GRO-seq data, and developing a rigorous informatic strategy to identify RIS, we demonstrate that head-on transcription occurs frequently in a breast cancer cell line, and that this transcription is significantly downregulated during S-phase, particularly in regions susceptible to R-loop formation. Collectively, our analysis suggests the existence of a temporally tuned transcriptional regulation mechanism that functions to maintain genome stability.

2022-04-30 | GSE201746 | GEO

Capturing the dynamics of genome replication on individual ultra-long nanopore sequence reads

Project description:The replication of eukaryotic genomes is highly stochastic, making it difficult to determine the replication dynamics of individual molecules with existing methods. We now report a sequencing method for the measurement of replication fork movement on single molecules by Detecting Nucleotide Analogue signal currents on extremely long nanopore traces (D‑NAscent). Using this method, we detect BrdU incorporated by Saccharomyces cerevisiae to reveal, at a genomic scale and on single molecules, the DNA sequences replicated during a pulse labelling period. Under conditions of limiting BrdU concentration, D-NAscent detects the differences in BrdU incorporation frequency across individual molecules to reveal the location of active replication origins, fork direction, termination sites, and fork pausing/stalling events. We used sequencing reads of 20-160 kb, to generate the first whole genome single-molecule map of DNA replication dynamics and discover a new class of low frequency stochastic origins in budding yeast.

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Genome-wide ChIP-chip Rad50 in early S-phase in the presence of hydroxyurea

Project description:The Mre11-Rad50-Xrs2 (MRX) complex is related to SMC complexes that form rings capable of holding two distinct DNA strands together. MRX functions at stalled replication forks and double-strand breaks (DSB). A mutation in the N-terminal OB-fold of the 70-kD subunit of yeast replication protein A, rfa1-t11, abrogates MRX recruitment to both types of damage. The rfa1 mutation is functionally epistatic with loss of any of the MRX subunits for survival of replication fork stress or DSB recovery, although it does not compromise end resection. High resolution imaging shows that either the rfa1-t11 or the rad50 mutation lets stalled replication forks collapse, and allows the separation not only of opposing ends, but of sister chromatids at breaks. Given that cohesin loss does not provoke visible sister separation as long as the RPA -MRX contacts are intact, we conclude that MRX also serves as a structural lynchpin of sister chromatids at breaks. Rad50 is a member of the Mre11-Rad50-Xrs2 (MRX) complex which is known to associate to replication forks under hydroxyurea-stress condition. Using ChIP-chip, we show that MRX recruitment to replication forks partially rely on Rfa1 (RPA) at the genome-wide level.

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Systematic identification of fragile sites via genome-wide location analysis of gamma-H2AX

Project description:Phosphorylation of histone H2AX is an early response to DNA damage in eukaryotes. In Saccharomyces cerevisiae, DNA damage or replication fork stalling results in histone H2A phosphorylation to yield gamma-H2A (yeast gamma-H2AX) in a Mec1 (ATR)- and Tel1 (ATM)- dependent manner. Here, we describe the genome-wide location analysis of gamma-H2A as a strategy to identify loci prone to engage the Mec1 and Tel1 pathways. Remarkably, gamma-H2A enrichment overlaps with loci prone to replication fork stalling and is caused by the action of Mec1 and Tel1, indicating that these loci are prone to breakage. Moreover, about half the sites enriched for gamma-H2A map to repressed protein-coding genes, and histone deacetylases are necessary for formation of gamma-H2A at these loci. Finally, our work indicates that high resolution mapping of gamma-H2AX is a fruitful route to map fragile sites in eukaryotic genomes.

2009-12-01 | GSE18191 | GEO

Replication termination is mediated by Top2 and occurs at genomic loci containing pausing elements

Project description:[original title] Chromosome replication initiates at multiple replicons and terminates when forks converge. In Escherichia coli, the Tus-TER complex mediates polar fork converging at the terminator region and aberrant termination events challenge chromosome integrity and segregation. Since in eukaryotes termination is less characterized, we used budding yeast to identify the factors assisting fork fusion at replicating chromosomes. Using genomic and mechanistic studies we have identified and characterized 71 chromosomal termination regions (TERs). TERs contain fork pausing elements that influence fork progression and merging. The Rrm3 DNA helicase assists fork progression across TERs counteracting the accumulation of X-shaped structures. The Top2 DNA topoisomerase associates at TERs in S-phase and G2/M facilitates fork fusion and prevents DNA breaks and genome rearrangements at TERs. We propose that in eukaryotes replication fork barriers, Rrm3 and Top2 coordinate replication fork progression and fusion at termination regions thus counteracting abnormal genomic transitions. Signal tracks in BED format suitable for visualization on the UCSC genome browser can be found at http://bio.ifom-ieo-campus.it/supplementary/Fachinetti_et_al_MOLCELL_2010

2010-08-28 | GSE19061 | GEO

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