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: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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Project description:Intracellular levels of deoxyribonucleoside triphosphate (dNTP) must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools have recently been associated with increased mutagenesis, genomic instability and tumorigenesis. However, the mechanisms by which low or imbalanced dNTP pools affect DNA replication remain poorly understood. Here, we have modulated the activity of ribonucleotide reductase (RNR), a key enzyme catalyzing a rate-limiting step of dNTP production, to monitor the effect of altered dNTP levels on replication dynamics in budding yeast. We show that dNTP pools are limiting for normal DNA synthesis as upregulation of RNR activity increases replication fork speed. In contrast, inhibition of RNR activity with hydroxyurea (HU) induces a sharp transition from a regular- to a slow-replication mode within minutes after S-phase entry. Interestingly, we found that upregulation of RNR activity delays this transition and that dNTP levels modulate both fork speed and origin usage under replication stress. Moreover, we report that chromosomal instability (CIN) mutants show increased dNTP pools and enhanced DNA synthesis in the presence of HU. Since upregulation of RNR allows forks to progress faster in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replication stress by upregulating dNTP pools.
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