Responses of genes of DNA repair, alternative oxidase, and pro-/antioxidant state in Arabidopsis thaliana with altered expression of AOX1a to gamma irradiation

Background: PARP inhibitors (PARPi) kill cancer cells by stalling DNA replication and preventing DNA repair, resulting in a critical accumulation of DNA damage. Resistance to PARPi is a growing clinical problem in the treatment of high grade serous ovarian carcinoma (HGSOC). Acetylation of histone H3 lysine 14 (H3K14ac) and associated histone acetyltransferases (HATs) have known functions in DNA repair and replication, but their expression and activities have not been examined in the context of PARPi-resistant HGSOC. Results: Using mass spectrometry profiling of histone modifications, we observed altered H3K14ac enrichment in PARPi-resistant HGSOC cells relative to isogenic PARPi-sensitive lines. By RT-qPCR and RNA-Seq, we also observed altered expression of numerous HATs in PARPi-resistant HGSOC cells and a PARPi-resistant PDX model. Knockdown of HATs only modestly altered PARPi response, although knockdown and inhibition of PCAF significantly increased resistance. Pharmacologic inhibition of HBO1 severely depleted H3K14ac but did not affect PARPi response. However, knockdown and inhibition of BRPF3, which is known to interact in a complex with HBO1, did reduce PARPi resistance. Conclusions: This study demonstrates that severe depletion of H3K14ac does not affect PARPi response in HGSOC. Our data suggest that bromodomain functions of HAT proteins such as PCAF, or accessory proteins such as BRPF3, may play a greater role in PARPi response than acetyltransferase functions.

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Arabidopsis Mediator subunit 17 connects transcription with DNA repair after UV-B exposure

10.1101/2021.08.02.454780 ◽

2021 ◽

Author(s):

Marisol Giustozzi ◽

Santiago Freytes ◽

Aime Jaskolowski ◽

Micaela Lichy ◽

Julieta L. Mateos ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Transcription Initiation ◽

Plant Responses ◽

Repair System ◽

Root Tips ◽

Direct Role ◽

Altered Expression ◽

Eukaryotic Organisms

Mediator 17 (MED17) is a subunit of the Mediator complex that regulates transcription initiation in eukaryotic organisms. In yeast and humans, MED17 also participates in DNA repair, physically interacting with proteins of the Nucleotide Excision DNA Repair system. We here analyzed the role of MED17 in Arabidopsis plants exposed to UV-B radiation, which role has not been previously described. Comparison of med17 mutant transcriptome to that of WT plants showed that almost one third of transcripts with altered expression in med17 plants are also changed by UV-B exposure in WT plants. To validate the role of MED17 in UV-B irradiated plants, plant responses to UV-B were analyzed, including flowering time, DNA damage accumulation and programmed cell death in the meristematic cells of the root tips. Our results show that med17 and OE MED17 plants have altered responses to UV-B; and that MED17 participates in various aspects of the DNA damage response (DDR). Increased sensitivity to DDR after UV-B in med17 plants can be due to altered regulation of UV-B responsive transcripts; but additionally MED17 physically interacts with DNA repair proteins, suggesting a direct role of this Mediator subunit during repair. Finally, we here also show that MED17 is necessary to regulate the DDR activated by ATR, and that PDCD5 overexpression reverts the deficiencies in DDR shown in med17 mutants. Together, the data presented demonstrates that MED17 is an important regulator of the DDR after UV-B radiation in Arabidopsis plants.

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Characterization of Suspension Cultures of Arabidopsis thaliana (L.) Heynh Cells with Altered Expression of the Gene of Alternative Mitochondrial Oxidase Aox1a and Analysis of their Frost Resistance

The Bulletin of Irkutsk State University Series Biology Ecology ◽

10.26516/2073-3372.2021.35.3 ◽

2021 ◽

Vol 35 ◽

pp. 3-18

Author(s):

A. V. Stepanov ◽

◽

S. A. Kashin ◽

N. S. Zabanova ◽

O. A. Fedotova ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Suspension Culture ◽

Mitochondrial Respiration ◽

Frost Resistance ◽

Alternative Oxidase ◽

Uncoupling Protein ◽

Living Cells ◽

Subzero Temperature ◽

Wild Type ◽

Culture Cells

The enzyme alternative cyanide-resistant oxidase (AOX) localized in mitochondria is involved in the processes of plant adaptation to various unfavorable biotic and abiotic factors. Transfer of electrons from ubiquinone to oxygen by alternative oxidase has a nonprotonmotive character and, by bypassing two sites of H+ pumping in complexes III and IV, lowers the energy efficiency of respiration and energy of electron flow through AOX is released as heat. In this work, we characterized heterotrophic suspension cultures of Arabidopsis thaliana (L.) Heynh cells obtained from seeds of plants with altered (reduced (AS-12 line) and increased (XX-2 line)) expression of the alternative oxidase gene AOX1a and studied their viability under subzero temperature (-10 °С for 3, 6, 9 hours). Cell viability and reactive oxygen species (ROS) production were assessed using fluorescence microscopy with fluorescein diacetate (FDA) and propidium iodide (PI) for cell viability measurement and H2DCF-DA for ROS measurement. The proportion of living cells was calculated as the proportion of FDApositive and PI-negative cells. Differences between the studied lines were determined in the content of mitochondrial proteins of the respiratory chain (AOX, COXII, NDB) and uncoupling protein (UCP), as well as in the intensity of formation of ROS and frost resistance. The obtained results confirmed the higher content of the AOX protein and its high contribution to mitochondrial respiration in line XX-2. Suspension culture cells of the AS-12 line showed a decrease in the AOX protein content and its contribution to mitochondrial respiration, compared to the wild type (Col-0) and line XX-2. Simultaneously with a decrease in the AOX protein content in the AS-12 cell culture, an increase in the content of the uncoupling protein UCP and subunit II of cytochrome oxidase (COXII) was observed. ROS generation was reduced in cell cultures of both XX-2 and AS-12. The obtained results indicate that the cells of the wildtype (Col-0) suspension culture were subjected to the most significant effect of subzero temperature. Long-term exposure (for 9 h) under -10 °С revealed significant differences in the viability of wild-type culture cells and lines with altered AOX1a gene expression. Cells of line XX-2 with an increased content of AOX turned out to be more resistant to subzero temperature compared to wild-type and AS-12 cells. However, while the proportion of living cells in the culture of the AS-12 line 48 h after exposure remained at the same level as immediately after it, in the suspension culture of the wild type cell death developed over time. The obtained results indicate the importance of alternative oxidase in the development of frost resistance in plant cell.

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The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7222-7231.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7222-7231

Author(s):

V F Liu ◽

D T Weaver

Keyword(s):

Dna Repair ◽

Ionizing Radiation ◽

Gamma Irradiation ◽

Ataxia Telangiectasia ◽

Protein A ◽

Replication Protein A ◽

Replication Protein ◽

Inherited Disease ◽

Dna Replication And Repair ◽

In Cells

Replication protein A (RPA), the trimeric single-stranded DNA-binding protein complex of eukaryotic cells, is important to DNA replication and repair. Phosphorylation of the p34 subunit of RPA is modulated by the cell cycle, occurring during S and G2 but not during G1. The function of phosphorylated p34 remains unknown. We show that RPA p34 phosphorylation is significantly induced by ionizing radiation. The phosphorylated form, p36, is similar if not identical to the phosphorylated S/G2 form. gamma-Irradiation-induced phosphorylation occurs without new protein synthesis and in cells in G1. Mutation of cdc2-type protein kinase phosphorylation sites in p34 eliminates the ionizing radiation response. The gamma-irradiation-induced phosphorylation of RPA p34 is delayed in cells from ataxia telangiectasia, a human inherited disease conferring DNA repair defects and early-onset tumorigenesis. UV-induced phosphorylation of RPA p34 occurs less rapidly than gamma-irradiation-induced phosphorylation but is kinetically similar between ataxia telangiectasia and normal cells. This is the first time that modification of a repair protein, RPA, has been linked with a DNA damage response and suggests that phosphorylation may play a role in regulating DNA repair pathways.

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Azoospermic infertility is associated with altered expression of DNA repair genes

DNA Repair ◽

10.1016/j.dnarep.2019.01.006 ◽

2019 ◽

Vol 75 ◽

pp. 39-47 ◽

Cited By ~ 6

Author(s):

Vertika Singh ◽

Deepika Jaiswal ◽

Kanhaiya Singh ◽

Sameer Trivedi ◽

Neeraj K Agrawal ◽

...

Keyword(s):

Dna Repair ◽

Dna Repair Genes ◽

Altered Expression ◽

Repair Genes

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The Absence of Alternative Oxidase AOX1A Results in Altered Response of Photosynthetic Carbon Assimilation to Increasing CO2 in Arabidopsis thaliana

Plant and Cell Physiology ◽

10.1093/pcp/pcs107 ◽

2012 ◽

Vol 53 (9) ◽

pp. 1627-1637 ◽

Cited By ~ 32

Author(s):

Anthony Gandin ◽

Claire Duffes ◽

David A. Day ◽

Asaph B. Cousins

Keyword(s):

Arabidopsis Thaliana ◽

Alternative Oxidase ◽

Carbon Assimilation ◽

Photosynthetic Carbon ◽

Altered Response ◽

Photosynthetic Carbon Assimilation

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Involvement of Death-Associated Protein Kinases In DNA Damage Responses of B-ALL Cells.

Blood ◽

10.1182/blood.v116.21.3369.3369 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3369-3369

Author(s):

Magali Humbert ◽

Michaela Medova ◽

Barbara Geering ◽

Wieslawa Blank-Liss ◽

Hans-Uwe Simon ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Repair ◽

Gamma Irradiation ◽

Protein Kinases ◽

Dna Damage Response ◽

Lymphoblastic Leukemia ◽

Damage Response ◽

Dna Damage Responses

Abstract Abstract 3369 Intact DNA damage response pathways are important for genomic fidelity of cells in order to avoid tumor formation. On the other hand, inhibition of DNA repair provides an important mechanism to enhance the therapeutic efficacy of DNA damaging agents such as gamma-irradiation. Thus, it is important to identify novel players in DNA damage response that might represent novel targets for combination therapies. Death-associated protein kinases (DAPK) are serine/threonine kinases believed to be involved in cell death and autophagy mechanisms, whereby particularly the role of DAPK1 has previously been investigated. The DAPK family is composed of five members: DAPK1, DAPK2 (or DRP-1), DAPK3 (or ZIP kinase), DRAK1 and DRAK2. DAPK1 and DAPK2 share 80% homology in the catalytic domain. Generally, the role of DAPK in DNA damage responses is not well studied. To analyze the role of DAPK1 and DAPK2 in response to gamma-irradiation, we used p53 wild-type REH B-cell acute lymphoblastic leukemia (B-ALL) cells as a model. In response to irradiation, DAPK1 protein expression increased paralleled by an increased of total p53, phospho-Ser20-p53 and p21WAF1/CIP1. DAPK2 expression, however, did not increase. Since upregulation of p21WAF1/CIP1, a classical p53 target in response to DNA damage leads to cell cycle arrest, we asked whether knocking down DAPK1 or DAPK2 might affect the cell cycle. Interestingly, knocking down DAPK2 but not DAPK1 led to a significant increase of S-phase cells upon irradiation. Moreover, knocking down DAPK2 attenuated the induction of DAPK1 upon irradiation indicating a DAPK2-DAPK1 cascade in DNA damage responses. Next, given the significant role of p21WAF1/CIP1 and p53 in DNA damage responses, we tested if DAPK2 might directly participate in a novel signaling pathway by interacting with these proteins. Indeed, pull down assays revealed that p21WAF1/CIP1 and p53 are novel DAPK2 interacting proteins. Clearly, further experiments are needed to define the DAPK2-DAPK1-p53- p21WAF1/CIP1 network in DNA repair pathways. In conclusion, we identified a novel role for DAPK1 and DAPK2 in DNA damage responses of B-ALL cells and propose a novel DAPK2/DAPK1/p53/ p21WAF1/CIP1 DNA damage regulatory pathway. Disclosures: No relevant conflicts of interest to declare.

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