scholarly journals A Role for RAGE in DNA Double Strand Breaks (DSBs) Detected in Pathological Placentas and Trophoblast Cells

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 857
Author(s):  
Kary Y.F. Tsai ◽  
Benton Tullis ◽  
Katrina L. Breithaupt ◽  
Rylan Fowers ◽  
Nelson Jones ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Pregnancy Complications ◽  
Cell Model ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Trophoblast Cells ◽  
Dsb Repair ◽  
Dna Dsbs ◽  
The Impact

Impaired DNA damage responses are associated with several diseases, including pregnancy complications. Recent research identified an ATM-kinase dependent function for the nuclear isoform of the receptor for advanced glycation end-products (RAGE) during double strand break (DSB)-repair. RAGE contributes to end-resectioning of broken DNA sites by binding with the MRE11-Rad50-Nbs1 (MRN) complex. Placental research is limited regarding the impact of genomic instability and the mechanism for potential repair. We tested the hypothesis regarding the involvement of RAGE during the repair of placental DNA-DSBs. We first identified that the pregnancy complications of PE and preterm labor (PTL) experience loss of genomic integrity and an in vitro trophoblast cell model was used to characterize trophoblast DSBs. Colocalized immunofluorescence of γ-H2AX and RAGE support the potential involvement of RAGE in cellular responses to DNA-DSBs. Immunoblotting for both molecules in PE and PTL placenta samples and in trophoblast cells validated a connection. Co-immunoprecipitation studies revealed interactions between RAGE and pATM and MRE11 during DNA-DSBs. Reduced cellular invasion confirmed the role of genomic instability in trophoblastic function. Collectively, these experiments identified genomic instability in pregnancy complications, the impact of defective DNA on trophoblast function, and a possible RAGE-mediated mechanism during DNA-DSB repair.

scholarly journals DNA double-strand break repair: a theoretical framework and its application

2016 ◽  
Vol 13 (114) ◽  
pp. 20150679 ◽  
Author(s):  
Philip J. Murray ◽  
Bart Cornelissen ◽  
Katherine A. Vallis ◽  
S. Jon Chapman
Keyword(s):  
Dna Damage ◽  
Auger Electron ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Histone H2ax ◽  
Dna Double Strand Break ◽  
A Cell

DNA double-strand breaks (DSBs) are formed as a result of genotoxic insults, such as exogenous ionizing radiation, and are among the most serious types of DNA damage. One of the earliest molecular responses following DSB formation is the phosphorylation of the histone H2AX, giving rise to γ H2AX. Many copies of γ H2AX are generated at DSBs and can be detected in vitro as foci using well-established immuno-histochemical methods. It has previously been shown that anti- γ H2AX antibodies, modified by the addition of the cell-penetrating peptide TAT and a fluorescent or radionuclide label, can be used to visualize and quantify DSBs in vivo . Moreover, when labelled with a high amount of the short-range, Auger electron-emitting radioisotope, 111 In, the amount of DNA damage within a cell can be increased, leading to cell death. In this report, we develop a mathematical model that describes how molecular processes at individual sites of DNA damage give rise to quantifiable foci. Equations that describe stochastic mean behaviours at individual DSB sites are derived and parametrized using population-scale, time-series measurements from two different cancer cell lines. The model is used to examine two case studies in which the introduction of an antibody (anti- γ H2AX-TAT) that targets a key component in the DSB repair pathway influences system behaviour. We investigate: (i) how the interaction between anti- γ H2AX-TAT and γ H2AX effects the kinetics of H2AX phosphorylation and DSB repair and (ii) model behaviour when the anti- γ H2AX antibody is labelled with Auger electron-emitting 111 In and can thus instigate additional DNA damage. This work supports the conclusion that DSB kinetics are largely unaffected by the introduction of the anti- γ H2AX antibody, a result that has been validated experimentally, and hence the hypothesis that the use of anti- γ H2AX antibody to quantify DSBs does not violate the image tracer principle. Moreover, it provides a novel model of DNA damage accumulation in the presence of Auger electron-emitting 111 In that is supported qualitatively by the available experimental data.

scholarly journals FKBP25 participates in DNA double-strand break repair

2020 ◽  
Vol 98 (1) ◽  
pp. 42-49 ◽  
Author(s):  
David Dilworth ◽  
Fade Gong ◽  
Kyle Miller ◽  
Christopher J. Nelson
Keyword(s):  
Homologous Recombination ◽  
Strand Break ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Peptide Bonds ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Fk506 Binding Proteins

FK506-binding proteins (FKBPs) alter the conformation of proteins via cis–trans isomerization of prolyl-peptide bonds. While this activity can be demonstrated in vitro, the intractability of detecting prolyl isomerization events in cells has limited our understanding of the biological processes regulated by FKBPs. Here we report that FKBP25 is an active participant in the repair of DNA double-strand breaks (DSBs). FKBP25 influences DSB repair pathway choice by promoting homologous recombination (HR) and suppressing single-strand annealing (SSA). Consistent with this observation, cells depleted of FKBP25 form fewer Rad51 repair foci in response to etoposide and ionizing radiation, and they are reliant on the SSA repair factor Rad52 for viability. We find that FKBP25’s catalytic activity is required for promoting DNA repair, which is the first description of a biological function for this enzyme activity. Consistent with the importance of the FKBP catalytic site in HR, rapamycin treatment also impairs homologous recombination, and this effect is at least in part independent of mTor. Taken together these results identify FKBP25 as a component of the DNA DSB repair pathway.

scholarly journals O-GlcNAcylation Affects the Pathway Choice of DNA Double-Strand Break Repair

2021 ◽  
Vol 22 (11) ◽  
pp. 5715
Author(s):  
Sera Averbek ◽  
Burkhard Jakob ◽  
Marco Durante ◽  
Nicole B. Averbeck
Keyword(s):  
Strand Break ◽  
Protein Accumulation ◽  
Dna Double Strand Breaks ◽  
Post Translational Modification ◽  
Radiation Quality ◽  
Dsb Repair ◽  
Γh2ax Foci ◽  
Additional Level ◽  
Glcnac Transferase ◽  
The Impact

Exposing cells to DNA damaging agents, such as ionizing radiation (IR) or cytotoxic chemicals, can cause DNA double-strand breaks (DSBs), which are crucial to repair to maintain genetic integrity. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification (PTM), which has been reported to be involved in the DNA damage response (DDR) and chromatin remodeling. Here, we investigated the impact of O-GlcNAcylation on the DDR, DSB repair and chromatin status in more detail. We also applied charged particle irradiation to analyze differences of O-GlcNAcylation and its impact on DSB repair in respect of spatial dose deposition and radiation quality. Various techniques were used, such as the γH2AX foci assay, live cell microscopy and Fluorescence Lifetime Microscopy (FLIM) to detect DSB rejoining, protein accumulation and chromatin states after treating the cells with O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) inhibitors. We confirmed that O-GlcNAcylation of MDC1 is increased upon irradiation and identified additional repair factors related to Homologous Recombination (HR), CtIP and BRCA1, which were increasingly O-GlcNAcyated upon irradiation. This is consistent with our findings that the function of HR is affected by OGT inhibition. Besides, we found that OGT and OGA activity modulate chromatin compaction states, providing a potential additional level of DNA-repair regulation.

scholarly journals CRL4ADTL degrades DNA-PKcs to modulate NHEJ repair and induce genomic instability and subsequent malignant transformation

Oncogene ◽  
2021 ◽  
Author(s):  
Maoxiao Feng ◽  
Yunshan Wang ◽  
Lei Bi ◽  
Pengju Zhang ◽  
Huaizhi Wang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genomic Instability ◽  
Malignant Transformation ◽  
Precancerous Lesions ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Normal Cells ◽  
Nhej Repair ◽  
Cullin 4A

AbstractGenomic instability induced by DNA damage and improper DNA damage repair is one of the main causes of malignant transformation and tumorigenesis. DNA double strand breaks (DSBs) are the most detrimental form of DNA damage, and nonhomologous end-joining (NHEJ) mechanisms play dominant and priority roles in initiating DSB repair. A well-studied oncogene, the ubiquitin ligase Cullin 4A (CUL4A), is reported to be recruited to DSB sites in genomic DNA, but whether it regulates NHEJ mechanisms of DSB repair is unclear. Here, we discovered that the CUL4A-DTL ligase complex targeted the DNA-PKcs protein in the NHEJ repair pathway for nuclear degradation. Overexpression of either CUL4A or DTL reduced NHEJ repair efficiency and subsequently increased the accumulation of DSBs. Moreover, we demonstrated that overexpression of either CUL4A or DTL in normal cells led to genomic instability and malignant proliferation. Consistent with the in vitro findings, in human precancerous lesions, CUL4A expression gradually increased with increasing malignant tendency and was negatively correlated with DNA-PKcs and positively correlated with γ-H2AX expression. Collectively, this study provided strong evidence that the CUL4A-DTL axis increases genomic instability and enhances the subsequent malignant transformation of normal cells by inhibiting NHEJ repair. These results also suggested that CUL4A may be a prognostic marker of precancerous lesions and a potential therapeutic target in cancer.

scholarly journals DNA Double-Strand Break Repair: All Roads Lead to HeterochROMAtin Marks

2021 ◽  
Vol 12 ◽  
Author(s):  
Pierre Caron ◽  
Enrico Pobega ◽  
Sophie E. Polo
Keyword(s):  
De Novo ◽  
Current Knowledge ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Genome Integrity ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
The Impact

In response to DNA double-strand breaks (DSBs), chromatin modifications orchestrate DNA repair pathways thus safeguarding genome integrity. Recent studies have uncovered a key role for heterochromatin marks and associated factors in shaping DSB repair within the nucleus. In this review, we present our current knowledge of the interplay between heterochromatin marks and DSB repair. We discuss the impact of heterochromatin features, either pre-existing in heterochromatin domains or de novo established in euchromatin, on DSB repair pathway choice. We emphasize how heterochromatin decompaction and mobility further support DSB repair, focusing on recent mechanistic insights into these processes. Finally, we speculate about potential molecular players involved in the maintenance or the erasure of heterochromatin marks following DSB repair, and their implications for restoring epigenome function and integrity.

scholarly journals Actin-Related Protein 6 (Arp6) Influences Double-Strand Break Repair in Yeast

2021 ◽  
Vol 1 (2) ◽  
pp. 225-238
Author(s):  
Mohsen Hooshyar ◽  
Daniel Burnside ◽  
Maryam Hajikarimlou ◽  
Katayoun Omidi ◽  
Alexander Jesso ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Remodeling ◽  
Genetic Interaction ◽  
Interaction Analysis ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Dna Damaging Agents ◽  
Repair Efficiency ◽  
Chromosomal Breaks

DNA double-strand breaks (DSBs) are the most deleterious form of DNA damage and are repaired through non-homologous end-joining (NHEJ) or homologous recombination (HR). Repair initiation, regulation and communication with signaling pathways require several histone-modifying and chromatin-remodeling complexes. In budding yeast, this involves three primary complexes: INO80-C, which is primarily associated with HR, SWR1-C, which promotes NHEJ, and RSC-C, which is involved in both pathways as well as the general DNA damage response. Here we identify ARP6 as a factor involved in DSB repair through an RSC-C-related pathway. The loss of ARP6 significantly reduces the NHEJ repair efficiency of linearized plasmids with cohesive ends, impairs the repair of chromosomal breaks, and sensitizes cells to DNA-damaging agents. Genetic interaction analysis indicates that ARP6, MRE11 and RSC-C function within the same pathway, and the overexpression of ARP6 rescues rsc2∆ and mre11∆ sensitivity to DNA-damaging agents. Double mutants of ARP6, and members of the INO80 and SWR1 complexes, cause a significant reduction in repair efficiency, suggesting that ARP6 functions independently of SWR1-C and INO80-C. These findings support a novel role for ARP6 in DSB repair that is independent of the SWR1 chromatin remodeling complex, through an apparent RSC-C and MRE11-associated DNA repair pathway.

scholarly journals Localization matters: nuclear-trapped Survivin sensitizes glioblastoma cells to temozolomide by elevating cellular senescence and impairing homologous recombination

2021 ◽  
Author(s):  
Thomas R. Reich ◽  
Christian Schwarzenbach ◽  
Juliana Brandstetter Vilar ◽  
Sven Unger ◽  
Fabian Mühlhäusler ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Nuclear Export ◽  
Cell Model ◽  
Chromosome Instability ◽  
Direct Interaction ◽  
High Grade Glioma ◽  
Strand Break ◽  
Γh2ax Foci

AbstractTo clarify whether differential compartmentalization of Survivin impacts temozolomide (TMZ)-triggered end points, we established a well-defined glioblastoma cell model in vitro (LN229 and A172) and in vivo, distinguishing between its nuclear and cytoplasmic localization. Expression of nuclear export sequence (NES)-mutated Survivin (SurvNESmut-GFP) led to impaired colony formation upon TMZ. This was not due to enhanced cell death but rather due to increased senescence. Nuclear-trapped Survivin reduced homologous recombination (HR)-mediated double-strand break (DSB) repair, as evaluated by γH2AX foci formation and qPCR-based HR assay leading to pronounced induction of chromosome aberrations. Opposite, clones, expressing free-shuttling cytoplasmic but not nuclear-trapped Survivin, could repair TMZ-induced DSBs and evaded senescence. Mass spectrometry-based interactomics revealed, however, no direct interaction of Survivin with any of the repair factors. The improved TMZ-triggered HR activity in Surv-GFP was associated with enhanced mRNA and stabilized RAD51 protein expression, opposite to diminished RAD51 expression in SurvNESmut cells. Notably, cytoplasmic Survivin could significantly compensate for the viability under RAD51 knockdown. Differential Survivin localization also resulted in distinctive TMZ-triggered transcriptional pathways, associated with senescence and chromosome instability as shown by global transcriptome analysis. Orthotopic LN229 xenografts, expressing SurvNESmut exhibited diminished growth and increased DNA damage upon TMZ, as manifested by PCNA and γH2AX foci expression, respectively, in brain tissue sections. Consequently, those mice lived longer. Although tumors of high-grade glioma patients expressed majorly nuclear Survivin, they exhibited rarely NES mutations which did not correlate with survival. Based on our in vitro and xenograft data, Survivin nuclear trapping would facilitate glioma response to TMZ.

scholarly journals Regulatory and Functional Involvement of Long Non-Coding RNAs in DNA Double-Strand Break Repair Mechanisms

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1506
Author(s):  
Angelos Papaspyropoulos ◽  
Nefeli Lagopati ◽  
Ioanna Mourkioti ◽  
Andriani Angelopoulou ◽  
Spyridon Kyriazis ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Repair Mechanisms ◽  
Non Coding Rnas ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
Living Organisms

Protection of genome integrity is vital for all living organisms, particularly when DNA double-strand breaks (DSBs) occur. Eukaryotes have developed two main pathways, namely Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR), to repair DSBs. While most of the current research is focused on the role of key protein players in the functional regulation of DSB repair pathways, accumulating evidence has uncovered a novel class of regulating factors termed non-coding RNAs. Non-coding RNAs have been found to hold a pivotal role in the activation of DSB repair mechanisms, thereby safeguarding genomic stability. In particular, long non-coding RNAs (lncRNAs) have begun to emerge as new players with vast therapeutic potential. This review summarizes important advances in the field of lncRNAs, including characterization of recently identified lncRNAs, and their implication in DSB repair pathways in the context of tumorigenesis.

scholarly journals A stapled peptide mimetic of the CtIP tetramerization motif interferes with double-strand break repair and replication fork protection

2021 ◽  
Vol 7 (8) ◽  
pp. eabc6381
Author(s):  
Anika Kuster ◽  
Nour L. Mozaffari ◽  
Oliver J. Wilkinson ◽  
Jessica L. Wojtaszek ◽  
Christina Zurfluh ◽  
...  
Keyword(s):  
Strand Break ◽  
Amino Acid Residues ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Stapled Peptide ◽  
Patients With Cancer ◽  
Peptide Mimetic ◽  
Dna Damaging Agents ◽  
Stalled Replication Forks

Cancer cells display high levels of DNA damage and replication stress, vulnerabilities that could be exploited by drugs targeting DNA repair proteins. Human CtIP promotes homology-mediated repair of DNA double-strand breaks (DSBs) and protects stalled replication forks from nucleolytic degradation, thus representing an attractive candidate for targeted cancer therapy. Here, we establish a peptide mimetic of the CtIP tetramerization motif that inhibits CtIP activity. The hydrocarbon-stapled peptide encompassing amino acid residues 18 to 28 of CtIP (SP18–28) stably binds to CtIP tetramers in vitro and facilitates their aggregation into higher-order structures. Efficient intracellular uptake of SP18–28 abrogates CtIP localization to damaged chromatin, impairs DSB repair, and triggers extensive fork degradation. Moreover, prolonged SP18–28 treatment causes hypersensitivity to DNA-damaging agents and selectively reduces the viability of BRCA1-mutated cancer cell lines. Together, our data provide a basis for the future development of CtIP-targeting compounds with the potential to treat patients with cancer.

Protective Effect of Butylphthalide on Neuronal Apoptosis in Parkinson Rats and Its Effect on miR-146a-5p Expression and Phosphatidylinositide 3-Kinases/Protein Kinase B Pathway

2021 ◽  
Vol 11 (10) ◽  
pp. 1908-1917
Author(s):  
Rongkang Mai ◽  
Yiyao Cao ◽  
Huitian Yu ◽  
Yong Zheng ◽  
Juke Huang
Keyword(s):  
Cell Model ◽  
Morphological Changes ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cell Line ◽  
Vehicle Group ◽  
Human Neuroblastoma Cell ◽  
Human Neuroblastoma ◽  
Oil Emulsion ◽  
The Impact

80 male Wistar rats were stochastically assigned to Sham + Vehicle group, Sham + BUT group, PD + Vehicle group and PD + BUT group. Rotenone PD model rats were prepared by subcutaneous injection of rotenone sunflower oil emulsion 2 mg/(kg · d) for 5 consecutive weeks. Butylphthalide 80 mg/(kg · d) were given to the rats in Sham + BUT group and PD + BUT group by gavage from the first day of rotenone injection for 5 weeks. Subsequently, the motor retardation ability and the morphological changes of the substantia nigra (SN) of each group were evaluated. Meanwhile, the levels of neuronal injury, apoptosis, inflammation and oxidative stress in each group of rats were assayed. The impact of BUT treatment on miR-146a-5p expression and PI3K/AKT signal pathway in rat brain tissue was assayed. Finally, by constructing a PD cell model of the neurotoxin 6-hydroxydopamine (6-OHDA)-treated human neuroblastoma cell line SH-SY5Y, the in vitro anti-PD pharmacological effect of BUT was further verified.

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