aniFOUND: analysing the associated proteome and genomic landscape of the repaired nascent non-replicative chromatin

Stefos, Georgios C; Szantai, Eszter; Konstantopoulos, Dimitris; Samiotaki, Martina; Fousteri, Maria

doi:10.1093/nar/gkab144

Cited by 6 publications

(3 citation statements)

References 66 publications

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“…The well-known drawback of iPOND assay is the difficulty in detection of high molecular weight proteins, high background, fuzzy bands, etc. [ 37 ]. We replaced the original iPOND lysis buffer with RIPA buffer, reduced the SDS concentration and solved this problem.…”

Section: Discussionmentioning

confidence: 99%

Modified iPOND revealed the role of mutant p53 in promoting helicase function and telomere maintenance

Wang,

Hou,

Yang

et al. 2023

Aging

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The G-rich DNA, such as telomere, tends to form G-quadruplex (G4) structure, which slows down the replication fork progression, induces replication stress, and becomes the chromosome fragile sites. Here we described a molecular strategy that cells developed to overcome the DNA replication stress via DNA helicase regulation. The p53N236S (p53S) mutation has been found in the Werner syndrome mouse embryo fibroblast (MEFs) escaped from senescence, could be the driving force for cell escaping senescence. We revealed that the p53S could transcriptionally up-regulate DNA helicases expression, including Wrn, Blm, Timeless, Ddx, Mcm, Gins, Fanc, as well as telomere specific proteins Terf1, Pot1, through which p53S promoted the unwinding of G4 structures, and protected the cells from DNA replication stress induced by G4 stabilizer. By modified iPOND (isolation of proteins on nascent DNA) assay and telomere assay, we demonstrated that the p53S could promote the recruitment of those helicases to the DNA replication forks, facilitated the maintenance of telomere, and prevent the telomere dysfunction induced by G4 stabilizer. Interestingly, we did not observe the function of promoting G4 resolving and facilitating telomere lengthening in the cells with Li-Fraumeni Syndrome mutation-p53R172H (p53H), which suggests that this is the specific gain of function for p53S. Together our data suggest that the p53S could gain the new function of releasing the replication stress via regulating the helicase function and G4 structure, which benefits telomere lengthening. This strategy could be applied to the treatment of diseases caused by telomere replication stress.

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Section: Discussionmentioning

confidence: 99%

Modified iPOND revealed the role of mutant p53 in promoting helicase function and telomere maintenance

Wang,

Hou,

Yang

et al. 2023

Aging

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“…This unscheduled DNA synthesis (UDS) often occur as a repairing mechanism of UV-induced DNA lesions. aniFOUND was developed based on nucleotide excision repair (NER) mechanisms and click chemistry-based protocols [49]. aniFOUND has been successfully tested in immortalized human skin fibroblast cell lines (1BR.3, VH10 normal, and NERdeficient XPA hTert) exposed to UVC irradiation.…”

Section: Non-replicative Chromatin-associated Proteome Anifound Approachmentioning

confidence: 99%

Current Analytical Strategies in Studying Chromatin-Associated-Proteome (Chromatome)

Khan¹,

Shahid²,

Asif³

2021

Molecules

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Chromatin is a dynamic structure comprising of DNA and proteins. Its unique nature not only help to pack the DNA tightly within the cell but also is pivotal in regulating gene expression DNA replication. Furthermore it also protects the DNA from being damaged. Various proteins are involved in making a specific complex within a chromatin and the knowledge about these interacting partners is helpful to enhance our understanding about the pathophysiology of various chromatin associated diseases. Moreover, it could also help us to identify new drug targets and design more effective remedies. Due to the existence of chromatin in different forms under various physiological conditions it is hard to develop a single strategy to study chromatin associated proteins under all conditions. In our current review, we tried to provide an overview and comparative analysis of the strategies currently adopted to capture the DNA bounded protein complexes and their mass spectrometric identification and quantification. Precise information about the protein partners and their function in the DNA-protein complexes is crucial to design new and more effective therapeutic molecules against chromatin associated diseases.

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“…1 A ), which can be coupled with fluorescent- or biotin-labeled azides for fast, sensitive, and high-throughput detection without DNA denaturation. Since 2008, EdU has been widely used in the analysis of DNA replication ( 5 – 9 ), cell proliferation and differentiation ( 10 ), DNA combing (DNA fiber analysis), and measuring nucleotide excision repair synthesis in the form of “unscheduled DNA synthesis” ( 11 – 14 ). Despite its utility and widespread use, EdU is actually highly toxic ( 15 , 16 ), causing more cell death than the other thymidine analogs used to study replication, and the underlying molecular mechanism of toxicity is not known.…”

mentioning

confidence: 99%

Nucleotide excision repair removes thymidine analog 5-ethynyl-2′-deoxyuridine from the mammalian genome

Wang

Cao

Yang

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Nucleotide excision repair is the principal mechanism for removing bulky DNA adducts from the mammalian genome, including those induced by environmental carcinogens such as UV radiation, and anticancer drugs such as cisplatin. Surprisingly, we found that the widely used thymidine analog EdU is a substrate for excision repair when incorporated into the DNA of replicating cells. A number of thymidine analogs were tested, and only EdU was a substrate for excision repair. EdU excision was absent in repair-deficient cells, and in vitro, DNA duplexes bearing EdU were also substrates for excision by mammalian cell-free extracts. We used the excision repair sequencing (XR-seq) method to map EdU repair in the human genome at single-nucleotide resolution and observed that EdU was excised throughout the genome and was subject to transcription-coupled repair as evidenced by higher repair rates in the transcribed strand (TS) relative to the nontranscribed strand (NTS) in transcriptionally active genes. These properties of EdU, combined with its cellular toxicity and ability to cross the blood–brain barrier, make it a potential candidate for treating cancers of the brain, a tissue that typically demonstrates limited replication in adults.

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aniFOUND: analysing the associated proteome and genomic landscape of the repaired nascent non-replicative chromatin

Cited by 6 publications

References 66 publications

Modified iPOND revealed the role of mutant p53 in promoting helicase function and telomere maintenance

Modified iPOND revealed the role of mutant p53 in promoting helicase function and telomere maintenance

Current Analytical Strategies in Studying Chromatin-Associated-Proteome (Chromatome)

Nucleotide excision repair removes thymidine analog 5-ethynyl-2′-deoxyuridine from the mammalian genome

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