Genome-wide analysis of replication fork locations in hydroxyurea-treated Saccharomyces cerevisiae by TrAEL-seq
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ABSTRACT:
PROVIDER: PRJNA987973 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR25033721.fastq.gz | Fastqsanger.gz | |||
| SRR25033722.fastq.gz | Fastqsanger.gz | |||
| SRR25033723.fastq.gz | Fastqsanger.gz | |||
| SRR25033724.fastq.gz | Fastqsanger.gz | |||
| SRR25033725.fastq.gz | Fastqsanger.gz |
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Project description:TrAEL-seq was performed on hydroxyurea-blocked and then released yeast cells to track replication fork stalling and replication fork restart, in wild-type and replisome mutant strains.
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Project description: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.
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Project description:Precise DNA replication is critical to the maintenance of genome stability, and the DNA replication machinery is a focal point of many current and upcoming chemotherapeutics. TrAEL-seq is a robust method for profiling DNA replication genome-wide that works in unsynchronised cells and does not require treatment with drugs or nucleotide analogues. Here, we provide an updated method for TrAEL-seq including multiplexing of up to 6 samples that dramatically improves sample quality and throughput, and we validate TrAEL-seq in multiple mammalian cell lines. The updated protocol is straightforward and robust yet provides excellent resolution comparable to OK-seq in mammalian cell samples, and does not require cell synchronisation, drug treatment, labelling or sorting. High resolution replication profiles can be obtained across large panels of samples and in dynamic systems, for example during the progressive onset of oncogene induced senescence. In addition to mapping zones where replication initiates and terminates, TrAEL-seq is sensitive to replication fork speed, revealing effects of both transcription and proximity to replication initiation zones on fork progression. Although forks move more slowly through transcribed regions, this does not have a significant impact on the broader dynamics of replication fork progression, which is dominated by rapid fork movement in long replication regions (>1Mb). Short and long replication regions are not intrinsically different, and instead replication forks accelerate across the first ~1 Mb of travel such that forks progress faster in the middle of regions lying between widely spaced initiation zones. We propose that this is a natural consequence of fewer replication forks being active later in S phase when these distal regions are replicated and there being less competition for replication factors.
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Project description:TrAEL-seq was used to assess the impact of Triapine (3AP), a RRM2 inhibitor, on replication fork accumulation and changes to the replication profile of IMR32 cells.
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Project description:During each cell division, tens of thousands of DNA replication origins are coordinately activated to ensure the complete duplication of the entire human genome. However, the progression of replication forks can be challenged by numerous factors. One such factor is transcription-replication conflict (TRC), which can either be co-directional or head-on and the latter has been revealed as more dangerous for genome integrity. In order to study the direction of replication fork movement and TRC, we developed a bioinformatics tool, called OKseqHMM, to direct measure the replication fork directionality (RFD) as well as replication initiation and termination, along human genome obtained by sequencing of Okazaki fragments (OK-Seq) and related techniques. We have gathered and analyzed OK-seq data from a large number of organisms including yeast, mouse and human, to generate high-quality RFD profiles and determine initiation zones and termination zones by using Hidden Markov Model (HMM) algorithm (all tools and data are available at https://github.com/CL-CHEN-Lab/OK-Seq). In addition, we have extended our analysis to data obtained by related techniques, such as eSPAN and TrAEL-seq, which also contain RFD information. Our works, therefore, provide an important tool and resource for the community to further study TRC and genome instability, in a wide range of cell line models and growth conditions, which is of prime importance for human health.
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Project description:The influence of mono-ubiquitylation of histone H2B (H2Bub) on transcription via nucleosome reassembly has been widely documented. Recently, it has also been shown that H2Bub promotes recovery from replication stress; however, the underling molecular mechanism remains unclear. Here, we show that H2B ubiquitylation coordinates activation of the intra-S replication checkpoint and chromatin re-assembly, in order to limit fork progression and DNA damage in the presence of replication stress. In particular, we show that the absence of H2Bub affects replication dynamics (enhanced fork progression and reduced origin firing), leading to γH2A accumulation and increased hydroxyurea sensitivity. Further genetic analysis indicates a role for H2Bub in transducing Rad53 phosphorylation. Concomitantly, we found that a change in replication dynamics is not due to a change in dNTP level, but is mediated by reduced Rad53 activation and destabilization of the RecQ helicase Sgs1 at the fork. Furthermore, we demonstrate that H2Bub facilitates the dissociation of the histone chaperone Asf1 from Rad53, and nucleosome reassembly behind the fork is compromised in cells lacking H2Bub. Taken together, these results indicate that the regulation of H2B ubiquitylation is a key event in the maintenance of genome stability, through coordination of intra-S checkpoint activation, chromatin assembly and replication fork progression. S.cerevisiae oligonucleotide microarrays were provided by Affymetrix (S.cerevisiae Tiling 1.0R, P/N 900645). BrdU and proteins ChIP-chip analyses were carried out as described (Fachinetti et al., M Cell, 2010).
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Project description:Analysis of topoisomerase function in bacterial replication fork movement: use of DNA microarrays. We used DNA microarrays of the Escherichia coli genome to trace the progression of chromosomal replication forks in synchronized cells. We found that both DNA gyrase and topoisomerase IV (topo IV) promote replication fork progression. When both enzymes were inhibited, the replication fork stopped rapidly. The elongation rate with topo IV alone was 1/3 of normal. Genetic data confirmed and extended these results. Inactivation of gyrase alone caused a slow stop of replication. Topo IV activity was sufficient to prevent accumulation of (+) supercoils in plasmid DNA in vivo, suggesting that topo IV can promote replication by removing (+) supercoils in front of the chromosomal fork. This study is detailed in Khodursky AB et al.(2000) Proc Natl Acad Sci U S A 97:9419-24 Keywords: other
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Project description:1) The Pgal-3HA cup1 yeast strain, in which all CUP1 ORFs are replaced with 3HA and all CUP1 promoters are replaced by GAL1-10 promoters (PMID: 28654659, which carries ~17 tandem repeats of the modified Pgal-3HA cup1 repeat) undergoes extensive CNV when shifted to galactose. Here we use TrAEL-seq to look for replication fork stalling in this strain with and without induction. 2) We also profile the impact on replication of integrating an ARSH4 origin upstream of the RSC30 gene (so outside the CUP1 locus), using the 3xCUP1 strain which carries 3 repeats of CUP1 (PMID: 28654659). The origin was integrated with a URA3 marker, the control strain has the URA3 marker but no origin.
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