TrAEL-seq for WRN inhibitors
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1064407 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR27536356.fastq.gz | Fastqsanger.gz | |||
| SRR27536357.fastq.gz | Fastqsanger.gz | |||
| SRR27536358.fastq.gz | Fastqsanger.gz | |||
| SRR27536359.fastq.gz | Fastqsanger.gz | |||
| SRR27536360.fastq.gz | Fastqsanger.gz |
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Project description:Cancers with microsatellite instability (MSI) depend on the WRN helicase enzyme to manage issues during DNA replication caused by long stretches of (TA) repeats in the DNA. Targeting WRN is a promising strategy for treating MSI cancers, and drugs that inhibit WRN are being developed. Through a process called fragment-based screening, we developed powerful and specific drugs that block WRN's action. These drugs effectively slowed down the growth of MSI cancer models in lab and animal studies by acting like WRN is absent, leading to DNA breaks at the long TA repeats and causing DNA damage. The development of these potent and specific drugs targeting WRN in MSI cancers proves that this approach can work and also helps us understand more about WRN's role in biology.
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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:We have applied the TrAEL-seq method to profile DNA replication in wild type and Snf2h knockoutmouse embryonic stem cells.
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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: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: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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