p53 Ser15 phosphorylation disrupts the p53–RPA70 complex and induces RPA70-mediated DNA repair in hypoxia

2012 ◽  
Vol 443 (3) ◽  
pp. 811-820 ◽  
Author(s):  
Esha Madan ◽  
Rajan Gogna ◽  
Uttam Pati
Keyword(s):  
Dna Repair ◽  
Cancer Cells ◽  
Excision Repair ◽  
Protein A ◽  
Cancer Therapeutics ◽  
End Joining ◽  
Nhej Repair ◽  
Dependent Protein ◽  
Non Homologous End Joining ◽  
Cellular Dna

Cellular stressors are known to inhibit the p53–RPA70 (replication protein A, 70 kDa subunit) complex, and RPA70 increases cellular DNA repair in cancer cells. We hypothesized that regulation of RPA70-mediated DNA repair might be responsible for the inhibition of apoptosis in hypoxic tumours. We have shown that, in cancer cells, hypoxia disrupts the p53–RPA70 complex, thereby enhancing RPA70-mediated NER (nucleotide excision repair)/NHEJ (non-homologous end-joining) repair. In normal cells, RPA70 binds to the p53-NTD (N-terminal domain), whereas this binding is disrupted in hypoxia. Phosphorylation of p53-NTD is a crucial event in dissociating both NTD–RPA70 and p53–RPA70 complexes. Serial mutations at serine and threonine residues in the NTD confirm that p53Ser15 phosphorylation induces dissociation of the p53–RPA70 complex in hypoxia. DNA-PK (DNA-dependent protein kinase) is shown to induce p53Ser15 phosphorylation, thus enhancing RPA70-mediated NER/NHEJ repair. Furthermore, RPA70 gene silencing induces significant increases in cellular apoptosis in the resistant hypoxic cancer cells. We have thus elucidated a novel pathway showing how DNA-PK-mediated p53Ser15 phosphorylation dissociates the p53–RPA70 complex, thus enhancing NER/NHEJ repair, which causes resistance to apoptosis in hypoxic cancer cells. This novel finding may open new strategies in developing cancer therapeutics on the basis of the regulation of RPA70-mediated NER/NHEJ repair.

scholarly journals Beta Human Papillomavirus 8E6 Attenuates Non-Homologous End Joining by Hindering DNA-PKcs Activity

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2356
Author(s):  
Changkun Hu ◽  
Taylor Bugbee ◽  
Monica Gamez ◽  
Nicholas A. Wallace
Keyword(s):  
Dna Repair ◽  
Viral Infections ◽  
Dna Lesions ◽  
Human Papillomaviruses ◽  
Molecular Evidence ◽  
Repair Pathway ◽  
End Joining ◽  
Dsb Repair ◽  
Dependent Protein ◽  
Non Homologous End Joining

Cutaneous viral infections occur in a background of near continual exposure to environmental genotoxins, like UV radiation in sunlight. Failure to repair damaged DNA is an established driver of tumorigenesis and substantial cellular resources are devoted to repairing DNA lesions. Beta-human papillomaviruses (β-HPVs) attenuate DNA repair signaling. However, their role in human disease is unclear. Some have proposed that β-HPV promotes tumorigenesis, while others suggest that β-HPV protects against skin cancer. Most of the molecular evidence that β-HPV impairs DNA repair has been gained via characterization of the E6 protein from β-HPV 8 (β-HPV 8E6). Moreover, β-HPV 8E6 hinders DNA repair by binding and destabilizing p300, a transcription factor for multiple DNA repair genes. By reducing p300 availability, β-HPV 8E6 attenuates a major double strand DNA break (DSB) repair pathway, homologous recombination. Here, β-HPV 8E6 impairs another DSB repair pathway, non-homologous end joining (NHEJ). Specifically, β-HPV 8E6 acts by attenuating DNA-dependent protein kinase (DNA-PK) activity, a critical NHEJ kinase. This includes DNA-PK activation and the downstream of steps in the pathway associated with DNA-PK activity. Notably, β-HPV 8E6 inhibits NHEJ through p300 dependent and independent means. Together, these data expand the known genome destabilizing capabilities of β-HPV 8E6.

scholarly journals ELL2 regulates DNA non-homologous end joining (NHEJ) repair in prostate cancer cells

2018 ◽  
Vol 415 ◽  
pp. 198-207 ◽  
Author(s):  
Yachen Zang ◽  
Laura E. Pascal ◽  
Yibin Zhou ◽  
Xiaonan Qiu ◽  
Leizhen Wei ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Cells ◽  
Prostate Cancer Cells ◽  
End Joining ◽  
Nhej Repair ◽  
Non Homologous End Joining

scholarly journals NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase

Retrovirology ◽  
2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Ekaterina Knyazhanskaya ◽  
Andrey Anisenko ◽  
Olga Shadrina ◽  
Anastasia Kalinina ◽  
Timofei Zatsepin ◽  
...  
Keyword(s):  
Dna Repair ◽  
Wild Type Virus ◽  
Dna Breaks ◽  
End Joining ◽  
Double Stranded Dna ◽  
Repair Efficiency ◽  
Non Homologous End Joining ◽  
Cellular Dna ◽  
Successful Replication ◽  
Hiv 1

Abstract Background HIV-1 integration results in genomic DNA gaps that are repaired by cellular DNA repair pathways. This step of the lentiviral life cycle remains poorly understood despite its crucial importance for successful replication. We and others reported that Ku70 protein of the non-homologous end joining pathway (NHEJ) directly binds HIV-1 integrase (IN). Here, we studied the importance of this interaction for post-integrational gap repair and the recruitment of NHEJ factors in this process. Results We engineered HIV-based pseudovirus with mutant IN defective in Ku70 binding and generated heterozygous Ku70, Ku80 and DNA-PKcs human knockout (KO) cells using CRISPR/Cas9. KO of either of these proteins or inhibition of DNA-PKcs catalytic activity substantially decreased the infectivity of HIV-1 with native IN but not with the mutant one. We used a recently developed qPCR assay for the measurement of gap repair efficiency to show that HIV-1 with mutant IN was defective in DNA post-integrational repair, whereas the wild type virus displayed such a defect only when NHEJ system was disrupted in any way. This effect was present in CRISPR/Cas9 modified 293T cells, in Jurkat and CEM lymphoid lines and in primary human PBMCs. Conclusions Our data provide evidence that IN recruits DNA-PK to the site of HIV-1 post-integrational repair due to Ku70 binding—a novel finding that explains the involvement of DNA-PK despite the absence of free double stranded DNA breaks. In addition, our data clearly indicate the importance of interactions between HIV-1 IN and Ku70 in HIV-1 replication at the post-integrational repair step.

Enhancement of non-homologous end joining DNA repair capacity confers cancer cells resistance to the novel selenophene compound, D-501036

2011 ◽  
Vol 309 (1) ◽  
pp. 110-118 ◽  
Author(s):  
Yung-Ning Yang ◽  
Kai-ming Chou ◽  
Wen-Yu Pan ◽  
Yih-wen Chen ◽  
Tsui-Chun Tsou ◽  
...  
Keyword(s):  
Dna Repair ◽  
Cancer Cells ◽  
The Novel ◽  
End Joining ◽  
Repair Capacity ◽  
Dna Repair Capacity ◽  
Non Homologous End Joining

scholarly journals Targeting MEK5 impairs non-homologous end-joining repair and sensitizes prostate cancer to DNA damaging agents

2019 ◽  
Author(s):  
Constantinos G. Broustas ◽  
Axel J. Duval ◽  
Kunal R. Chaudhary ◽  
Richard A. Friedman ◽  
Renu K. Virk ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Dna Repair ◽  
Cancer Cells ◽  
Genotoxic Stress ◽  
Clonogenic Survival ◽  
Prostate Cancer Cells ◽  
End Joining ◽  
Etoposide Treatment ◽  
Non Homologous End Joining ◽  
Repair Defect

AbstractRadiotherapy is commonly used to treat a variety of solid human tumors, including localized prostate cancer. However, treatment failure often ensues due to tumor intrinsic or acquired radioresistance. Here we find that the MEK5/ERK5 signaling pathway is associated with resistance to genotoxic stress in aggressive prostate cancer cells. MEK5 knockdown by RNA interference sensitizes prostate cancer cells to ionizing radiation (IR) and etoposide treatment, as assessed by clonogenic survival and short-term proliferation assays. Mechanistically, MEK5 downregulation impairs phosphorylation of the catalytic subunit of DNA-PK at serine 2056 in response to IR or etoposide treatment. Although MEK5 knockdown does not influence the initial appearance of radiation- and etoposide-induced γH2AX and 53BP1 foci, it markedly delays their resolution, indicating a DNA repair defect. A cell-based assay shows that non-homologous end joining (NHEJ) is compromised in cells with ablated MEK5 protein expression. Finally, MEK5 silencing combined with focal irradiation causes strong inhibition of tumor growth in mouse xenografts, compared with MEK5 depletion or radiation alone. These findings reveal a convergence between MEK5 signaling and DNA repair by NHEJ in conferring resistance to genotoxic stress in advanced prostate cancer and suggest targeting MEK5 as an effective therapeutic intervention in the management of this disease.

scholarly journals Discovery and Development of Novel DNA-PK Inhibitors by Targeting the unique Ku-DNA Interaction

2020 ◽  
Author(s):  
Navnath S. Gavande ◽  
Pamela S. VanderVere-Carozza ◽  
Katherine S. Pawelczak ◽  
Tyler L. Vernon ◽  
Leslyn A. Hanakahi ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Critical Role ◽  
Cancer Therapeutics ◽  
Dna Interaction ◽  
Direct Result ◽  
End Joining ◽  
Gene Insertion ◽  
Dependent Protein ◽  
Non Homologous End Joining

ABSTRACTDNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) repair pathway and the DNA damage response (DDR). DNA-PK has therefore been pursued for the development of anti-cancer therapeutics in combination with ionizing radiation (IR). We report the discovery of a new class of DNA-PK inhibitors that act via a novel mechanism of action, inhibition of the Ku-DNA interaction. We have developed a series of highly potent and specific Ku-DNA binding inhibitors (Ku-DBi’s) that block the Ku-DNA interaction and inhibit DNA-PK kinase activity. Ku-DBi’s directly interact with the Ku and inhibit in vitro NHEJ, cellular NHEJ, and potentiate the activity of IR and radiomimetics. Analysis of Ku-null cells demonstrates that Ku-DBi’s cellular activity is a direct result of Ku inhibition, as Ku-null cells are insensitive to Ku-DBi’s. The utility of Ku-DBi’s was also demonstrated in a CRISPR gene-editing model where we demonstrate that the efficiency of gene insertion events was increased in cells pre-treated with Ku-DBi’s, consistent with inhibition of NHEJ and activation of homologous recombination to facilitate gene insertion. These data demonstrate the discovery and application of new series of compounds that modulate DNA repair pathways via a unique mechanism of action.

scholarly journals CometChip Enables Parallel Analysis of Multiple DNA Repair Activities

2021 ◽  
Author(s):  
Jing Ge ◽  
Le P. Ngo ◽  
Simran Kaushal ◽  
Ian J. Tay ◽  
Elina Thadhani ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Comet Assay ◽  
Excision Repair ◽  
Parallel Analysis ◽  
End Joining ◽  
Dna Damaging Agents ◽  
Nucleotide Excision ◽  
Base Excision ◽  
Non Homologous End Joining

ABSTRACTDNA damage can be cytotoxic and mutagenic and is directly linked to aging, cancer, and heritable diseases. To counteract the deleterious effects of DNA damage, cells have evolved highly conserved DNA repair pathways. Many commonly used DNA repair assays are relatively low throughput and are limited to analysis of one protein or one pathway. Here, we have explored the capacity of the CometChip platform for parallel analysis of multiple DNA repair activities. Taking advantage of the versatility of the traditional comet assay and leveraging micropatterning techniques, the CometChip platform offers increased throughput and sensitivity compared to the traditional comet assay. By exposing cells to DNA damaging agents that create substrates of Base Excision Repair, Nucleotide Excision Repair, and Non-Homologous End Joining, we show that the CometChip is an effective method for assessing repair deficiencies in all three pathways. With these advanced applications of the CometChip platform, we expand the efficacy of the comet assay for precise, high-throughput, parallel analysis of multiple DNA repair activities.

scholarly journals PRMT5 promotes DNA repair through methylation of 53BP1 and is regulated by Src-mediated phosphorylation

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Jee Won Hwang ◽  
Su-Nam Kim ◽  
Nayeon Myung ◽  
Doona Song ◽  
Gyoonhee Han ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Apoptotic Cell ◽  
Negative Regulator ◽  
End Joining ◽  
Cellular Survival ◽  
Cellular Processes ◽  
Nhej Repair ◽  
Damage Process ◽  
Non Homologous End Joining

AbstractPRMT5 participates in various cellular processes, including transcription regulation, signal transduction, mRNA splicing, and DNA repair; however, its mechanism of regulation is poorly understood. Here, we demonstrate that PRMT5 is phosphorylated at residue Y324 by Src kinase, a negative regulator of its activity. Either phosphorylation or substitution of the Y324 residue suppresses PRMT5 activity by preventing its binding with the methyl donor S-adenosyl-L-methionine. Additionally, we show that PRMT5 activity is associated with non-homologous end joining (NHEJ) repair by methylating and stabilizing p53-binding protein 1 (53BP1), which promotes cellular survival after DNA damage. Src-mediated phosphorylation of PRMT5 and the subsequent inhibition of its activity during the DNA damage process blocks NHEJ repair, leading to apoptotic cell death. Altogether, our findings suggest that PRMT5 regulates DNA repair through Src-mediated Y324 phosphorylation in response to DNA damage.

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