Role of topoisomerase inhibition and DNA repair mechanisms in the genotoxicity of alternariol and altertoxin-II

2013 ◽  
Vol 6 (3) ◽  
pp. 233-244 ◽  
Author(s):  
C. Tiessen ◽  
H. Gehrke ◽  
C. Kropat ◽  
C. Schwarz ◽  
S. Bächler ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Topoisomerase I ◽  
Topoisomerase Ii ◽  
Dna Lesions ◽  
Nuclear Antigen ◽  
Substantial Impact ◽  
Ht29 Cells ◽  
Repair Pathways

Alternariol (AOH) and altertoxin-II (ALTX-II) have been demonstrated to possess genotoxic properties. However, the underlying mechanisms of action have not been fully elucidated yet. AOH has recently been shown to act as a topoisomerase I and II poison, contributing to its genotoxic properties. The topoisomerase-specific repair factor tyrosyl-DNA-phosphodiesterase-1 (TDP1) is involved in the respective repair processes of damaged DNA induced by topoisomerase II poison. In the present study, we investigated the role of DNA repair pathways for the extent of DNA damage by AOH and addressed the question whether interference with topoisomerase II might play a role in the genotoxicity of ALTX-II. Under cell-free conditions, AOH and ALTX-II suppressed the activity of topoisomerase II at a comparable concentration range. In HT29 cells, AOH enhanced the level of covalent DNA-topoisomerase II complexes, thus acting as a topoisomerase poison in DNA damaging concentrations. In contrast, ALTX-II in genotoxic concentrations did not show any effect on the stability of these complexes, indicating that interference with topoisomerases does not play a relevant role in genotoxicity. The differences in genotoxic mechanisms seem to be reflected in the activation of p53. AOH was found to increase p53 phosphorylation in HT29 cells in DNA damaging concentrations. In contrast, incubation with ALTX-II did not affect p53 phosphorylation despite substantial increase in tail intensity in the comet assay, suggesting that the DNA lesions formed by ALTX-II are not detected by the DNA-repair machinery of HT29 cells. These results are supported by differences in persistence of DNA damage, still maintained after 24 h for ALTX-II but nearly vanished already after 3 h for AOH. Furthermore, microarray and qPCR analysis did not indicate any substantial impact of AOH on the transcription of key elements of DNA repair pathways. However, siRNA-approaches indicate that, in addition to TDP1, the expression of other elements of the DNA repair machinery exemplified by the 70 kDa Ku autoantigen and the proliferating cell nuclear antigen are relevant for AOH-mediated DNA damage.

scholarly journals Regulation of multiple DNA repair pathways by the Fanconi anemia protein SLX4

Blood ◽  
2013 ◽  
Vol 121 (1) ◽  
pp. 54-63 ◽  
Author(s):  
Yonghwan Kim ◽  
Gabriella S. Spitz ◽  
Uma Veturi ◽  
Francis P. Lach ◽  
Arleen D. Auerbach ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Cell Line ◽  
Fanconi Anemia ◽  
Partial Resistance ◽  
Topoisomerase I ◽  
Dna Lesions ◽  
Holliday Junction ◽  
Cross Linking ◽  
Repair Pathways

Abstract SLX4, the newly identified Fanconi anemia protein, FANCP, is implicated in repairing DNA damage induced by DNA interstrand cross-linking (ICL) agents, topoisomerase I (TOP1) inhibitors, and in Holliday junction resolution. It interacts with and enhances the activity of XPF-ERCC1, MUS81-EME1, and SLX1 nucleases, but the requirement for the specific nucleases in SLX4 function is unclear. Here, by complementing a null FA-P Fanconi anemia cell line with SLX4 mutants that specifically lack the interaction with each of the nucleases, we show that the SLX4-dependent XPF-ERCC1 activity is essential for ICL repair but is dispensable for repairing TOP1 inhibitor-induced DNA lesions. Conversely, MUS81-SLX4 interaction is critical for resistance to TOP1 inhibitors but is less important for ICL repair. Mutation of SLX4 that abrogates interaction with SLX1 results in partial resistance to both cross-linking agents and TOP1 inhibitors. These results demonstrate that SLX4 modulates multiple DNA repair pathways by regulating appropriate nucleases.

Endometrial DNA damage response is modulated in endometriosis

2020 ◽  
Author(s):  
Kashmira Bane ◽  
Junita Desouza ◽  
Diksha Shetty ◽  
Prakash Choudhary ◽  
Shalaka Kadam ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Small Sample ◽  
Nuclear Antigen ◽  
Dna Repair Genes ◽  
Eutopic Endometrium ◽  
Damage Response ◽  
Repair Genes

Abstract STUDY QUESTION Is the DNA damage response (DDR) dysregulated in the eutopic endometrium of women with endometriosis? SUMMARY ANSWER Endometrial expression of genes involved in DDR is modulated in women with endometriosis, compared to those without the disease. WHAT IS KNOWN ALREADY Ectopic endometriotic lesions are reported to harbour somatic mutations, thereby hinting at dysregulation of DDR and DNA repair pathways. However, it remains inconclusive whether the eutopic endometrium also manifests dysregulated DDR in endometriosis. STUDY DESIGN, SIZE, DURATION For this case–control study conducted between 2015 and 2019, eutopic endometrial (E) samples (EE- from women with endometriosis, CE- from women without endometriosis) were collected in either mid-proliferative (EE-MP, n = 23; CE-MP, n = 17) or mid-secretory (EE-MS, n = 17; CE-MS, n = 9) phases of the menstrual cycle. This study compares: (i) DNA damage marker localization, (ii) expression of DDR genes and (iii) expression of DNA repair genes in eutopic endometrial samples from women with and without endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS The study included (i) 40 women (aged 31.9 ± 0.81 years) with endometriosis and (ii) 26 control women (aged 31.4 ± 1.02 years) without endometriosis. Eutopic endometrial samples from the two groups were divided into different parts for histological analysis, immunohistochemistry, RNA extraction, protein extraction and comet assays. Eighty-four genes of relevance in the DNA damage signalling pathway were evaluated for their expression in eutopic endometrial samples, using RT2 Profiler PCR arrays. Validations of the expression of two GADD (Growth Arrest DNA Damage Inducible) proteins - GADD45A and GADD45G were carried out by immunoblotting. DNA damage was assessed by immunohistochemical localization of γ-H2AFX (a phosphorylated variant of histone H2AX) and 8-OHdG (8-hydroxy-2′-deoxyguanosine). RNA sequencing data from mid-proliferative (EE-MP, n = 4; CE-MP, n = 3) and mid-secretory phase (EE-MS and CE-MS, n = 4 each) endometrial samples were scanned to compare the expression status of all the genes implicated in human DNA repair. PCNA (Proliferating Cell Nuclear Antigen) expression was determined to assess endometrial proliferation. Residual DNA damage in primary endometrial cells was checked by comet assays. Public datasets were also scanned for the expression of DDR and DNA repair genes as our RNASeq data were limited by small sample size. All the comparisons were made between phase-matched endometrial samples from women with and without endometriosis. MAIN RESULTS AND THE ROLE OF CHANCE Endometrial expression of DDR genes and intensity of immunolocalized γ-H2AFX were significantly (P < 0.05) higher in EE, compared to CE samples. DDR proteins, especially those belonging to the GADD family, were found to be differentially abundant in EE, as compared to CE. These patterns were evident in both mid-proliferative and mid-secretory phases. Intriguingly, higher DDR was associated with increased cell proliferation in EE-MP, compared to CE-MP. Furthermore, among the differentially expressed transcripts (DETs) encoded by DNA repair genes, the majority showed up-regulation in EE-MP, compared to CE-MP. Interestingly, CE-MP and EE-MP had a comparable percentage (P > 0.05) of cells with residual DNA damage. However, unlike the mid-proliferative phase data, many DETs encoded by DNA repair genes were down-regulated in EE-MS, compared to CE-MS. An analysis of the phase-matched control and endometriosis samples included in the GSE51981 dataset available in the Gene Expression Omnibus database also revealed significant (P < 0.05) alterations in the expression of DDR and DNA repair genes in EE, compared to CE. LARGE-SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION The study was conducted on a limited number of endometrial samples. Also, the study does not reveal the causes underlying dysregulated DDR in the eutopic endometrium of women with endometriosis. WIDER IMPLICATIONS OF THE FINDINGS Alterations in the expression of DDR and DNA repair genes indirectly suggest that eutopic endometrium, as compared to its healthy counterpart, encounters DNA damage-inducing stimuli, either of higher strength or for longer duration in endometriosis. It will be worthwhile to identify the nature of such stimuli and also explore the role of higher genomic insults and dysregulated DDR/DNA repair in the origin and/or progression of endometriosis. STUDY FUNDING/COMPETING INTEREST(S) The study was supported by the Department of Biotechnology and Indian Council of Medical Research, Government of India. No conflict of interest is declared.

scholarly journals DNA Repair Functions That Control Sensitivity to Topoisomerase-Targeting Drugs

2004 ◽  
Vol 3 (1) ◽  
pp. 82-90 ◽  
Author(s):  
Mobeen Malik ◽  
John L. Nitiss
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Topoisomerase I ◽  
Topoisomerase Ii ◽  
Excision Repair ◽  
Dna Strand Breaks ◽  
Dna Topology ◽  
Strand Breaks ◽  
Wide Range ◽  
Dna Strand

ABSTRACT DNA topoisomerases play critical roles in a wide range of cellular processes by altering DNA topology to facilitate replication, transcription, and chromosome segregation. Topoisomerases alter DNA topology by introducing transient DNA strand breaks that involve a covalent protein DNA intermediate. Many agents have been found to prevent the religation of DNA strand breaks induced by the enzymes, thereby converting the enzymes into DNA-damaging agents. Repair of the DNA damage induced by topoisomerases is significant in understanding drug resistance arising following treatment with topoisomerase-targeting drugs. We have used the fission yeast Schizosaccharomyces pombe to identify DNA repair pathways that are important for cell survival following drug treatment. S. pombe strains carrying mutations in genes required for homologous recombination such as rad22A or rad32 (homologues of RAD52 and MRE11) are hypersensitive to drugs targeting either topoisomerase I or topoisomerase II. In contrast to results observed with Saccharomyces cerevisiae, S. pombe strains defective in nucleotide excision repair are also hypersensitive to topoisomerase-targeting agents. The loss of DNA replication or DNA damage checkpoints also sensitizes cells to both topoisomerase I and topoisomerase II inhibitors. Finally, repair genes (such as the S. pombe rad8+ gene) with no obvious homologs in other systems also play important roles in causing sensitivity to topoisomerase drugs. Since the pattern of sensitivity is distinct from that seen with other systems (such as the S. cerevisiae system), our results highlight the usefulness of S. pombe in understanding how cells deal with the unique DNA damage induced by topoisomerases.

scholarly journals DNA damage-specific effects of Tel1/ATM and γH2A/γH2AX on checkpoint signaling inSaccharomyces cerevisiae

2019 ◽  
Author(s):  
Jasmine Siler ◽  
Na Guo ◽  
Zhengfeng Liu ◽  
Yuhua Qin ◽  
Xin Bi
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Topoisomerase I ◽  
Cell Cycle Progression ◽  
Dynamic Balance ◽  
Dna Lesions ◽  
Atm Kinase ◽  
Checkpoint Signaling ◽  
Specific Effects ◽  
Damage Repair

AbstractDNA lesions trigger the activation of DNA damage checkpoints (DDCs) that stop cell cycle progression and promote DNA damage repair.Saccharomyces cerevisiaeTel1 is a homolog of mammalian ATM kinase that plays an auxiliary role in DDC signaling. γH2A, equivalent to γH2AX in mammals, is an early chromatin mark induced by DNA damage that is recognized by a group of DDC and DNA repair factors. We find that both Tel1 and γH2A negatively impact G2/M checkpoint in response to DNA topoisomerase I poison camptothecin independently of each other. γH2A also negatively regulates DDC induced by DNA alkylating agent methyl methanesulfonate. These results, together with prior findings demonstrating positive or no roles of Tel1 and γH2A in DDC in response to other DNA damaging agents such as phleomycin and ionizing radiation, suggest that Tel1 and γH2A have DNA damage-specific effects on DDC. We present data indicating that Tel1 acts in the same pathway as Mre11-Rad50-Xrs2 complex to suppress CPT induced DDC possibly by repairing topoisomerase I-DNA crosslink. On the other hand, we find evidence consistent with the notion that γH2A regulates DDC by mediating the competitive recruitment of DDC mediator Rad9 and DNA repair factor Rtt107 to sites of DNA damage. We propose that γH2A serves to create a dynamic balance between DDC and DNA repair that is influenced by the nature of DNA damage.

scholarly journals Dianhydrogalactitol synergizes with topoisomerase poisons to overcome DNA repair activity in tumor cells

2020 ◽  
Vol 11 (7) ◽  
Author(s):  
Beibei Zhai ◽  
Yue Li ◽  
Sudha Sravanti Kotapalli ◽  
Jeffrey Bacha ◽  
Dennis Brown ◽  
...  
Keyword(s):  
Dna Repair ◽  
Topoisomerase I ◽  
Topoisomerase Ii ◽  
Dna Lesions ◽  
Γh2ax Foci ◽  
Topoisomerase Poisons ◽  
Repair Activity ◽  
Functional Dna ◽  
Combination Regimens ◽  
The Impact

Abstract 1,2:5,6-Dianhydrogalactitol (DAG) is a bi-functional DNA-targeting agent currently in phase II clinical trial for treatment of temozolomide-resistant glioblastoma (GBM). In the present study, we investigated the cytotoxic activity of DAG alone or in combination with common chemotherapy agents in GBM and prostate cancer (PCa) cells, and determined the impact of DNA repair pathways on DAG-induced cytotoxicity. We found that DAG produced replication-dependent DNA lesions decorated with RPA32, RAD51, and γH2AX foci. DAG-induced cytotoxicity was unaffected by MLH1, MSH2, and DNA-PK expression, but was enhanced by knockdown of BRCA1. Acting in S phase, DAG displayed selective synergy with topoisomerase I (camptothecin and irinotecan) and topoisomerase II (etoposide) poisons in GBM, PCa, and lung cancer cells with no synergy observed for docetaxel. Importantly, DAG combined with irinotecan treatment enhanced tumor responses and prolonged survival of tumor-bearing mice. This work provides mechanistic insight into DAG cytotoxicity in GBM and PCa cells and offers a rational for exploring combination regimens with topoisomerase I/II poisons in future clinical trials.

scholarly journals ADAR-mediated RNA editing of DNA:RNA hybrids is required for DNA double strand break repair

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sonia Jimeno ◽  
Rosario Prados-Carvajal ◽  
María Jesús Fernández-Ávila ◽  
Sonia Silva ◽  
Domenico Alessandro Silvestris ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Rna Editing ◽  
Genomic Stability ◽  
Strand Break ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Dna Double Strand Break ◽  
Editing Enzyme

AbstractThe maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.

The role of DNA damage in laminopathy progeroid syndromes

2011 ◽  
Vol 39 (6) ◽  
pp. 1715-1718 ◽  
Author(s):  
Christopher J. Hutchison
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Excision Repair ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Therapeutic Approaches ◽  
Strand Breaks ◽  
Progeroid Syndromes ◽  
Repair Pathways

Progeroid laminopathies are characterized by the abnormal processing of lamin A, the appearance of misshapen nuclei, and the accumulation and persistence of DNA damage. In the present article, I consider the contribution of defective DNA damage pathways to the pathology of progeroid laminopathies. Defects in DNA repair pathways appear to be caused by a combination of factors. These include abnormal epigenetic modifications of chromatin that are required to recruit DNA repair pathways to sites of DNA damage, abnormal recruitment of DNA excision repair proteins to sites of DNA double-strand breaks, and unrepairable ROS (reactive oxygen species)-induced DNA damage. At least two of these defective processes offer the potential for novel therapeutic approaches.

Histone marks: repairing DNA breaks within the context of chromatin

2012 ◽  
Vol 40 (2) ◽  
pp. 370-376 ◽  
Author(s):  
Kyle M. Miller ◽  
Stephen P. Jackson
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Genome Stability ◽  
Nuclear Dna ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Dna Dsbs

Inherited or acquired defects in detecting, signalling or repairing DNA damage are associated with various human pathologies, including immunodeficiencies, neurodegenerative diseases and various forms of cancer. Nuclear DNA is packaged into chromatin and therefore the true in vivo substrate of damaged DNA occurs within the context of chromatin. Our work aims to decipher the mechanisms by which cells detect DNA damage and signal its presence to the DNA-repair and cell-cycle machineries. In particular, much of our work has focused on DNA DSBs (double-strand breaks) that are generated by ionizing radiation and radiomimetic chemicals, and which can also arise when the DNA replication apparatus encounters other DNA lesions. In the present review, we describe some of our recent work, as well as the work of other laboratories, that has identified new chromatin proteins that mediate DSB responses, control SDB processing or modulate chromatin structure at DNA-damage sites. We also aim to survey several recent advances in the field that have contributed to our understanding of how particular histone modifications and involved in DNA repair. It is our hope that by understanding the role of chromatin and its modifications in promoting DNA repair and genome stability, this knowledge will provide opportunities for developing novel classes of drugs to treat human diseases, including cancer.

scholarly journals Is there a role of phase partitioning in coordinating DNA damage response?

2020 ◽  
Author(s):  
D. Tosolini ◽  
G. Antoniali ◽  
E. Dalla ◽  
G. Tell
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Phase Separation ◽  
Dna Damage Response ◽  
Rna Binding ◽  
Dna Lesions ◽  
Functional Enrichment ◽  
Starting Point ◽  
Damage Response

AbstractDNA repair pathways are critical processes that need both spatial and temporal fine regulation. Liquid-liquid phase separation (LLPS) is a way to concentrate biochemical reactions, while excluding non-interacting components. Protein’s disordered domains, as well as RNA, favor condensation to modulate this process. Recent insights about phase-separation mechanisms pointed to new fascinating models that could explain how cells could cope with DNA damage responses. In this context, it is emerging that RNA-processing pathways and PARylation events, through the addition of an ADP-ribose moiety to both proteins and DNA, participate in different aspects of the DNA Damage Response (DDR). Remarkably, defects in these regulatory connections are associated with genomic instability and human pathologies. In addition, it has been recently noticed that several DNA repair enzymes, such as 53BP1 and APE1, are endowed with RNA binding abilities. APE1 is a multifunctional protein belonging to the Base Excision Repair (BER) pathway of non-distorting DNA lesions, bearing additional ‘non-canonical’ DNA-repair functions associated with processes coping with RNA metabolism. In this work, after reviewing the recent literature supporting a role of LLPS in DDR, we analyze, as a proof of principle, the interactome of APE1 using a bioinformatics approach to look for clues of LLPS in BER. Some of the APE1 interactors are associated with cellular processes in which LLPS has been either proved or proposed and are involved in several tumorigenic and amyloidogenic events. This work represents a paradigmatical pipeline for evaluating the relevance of LLPS in DDR.Statement of significanceIn this work, we aimed to test the hypothesis of an involvement of phase-separation in regulating the molecular mechanisms of the multifunctional enzyme APE1 starting from the analysis of its recently-characterized protein-protein interactome (PPI). We compared APE1-PPI to phase-separation databases and we performed functional enrichment analysis, uncovering links between APE1 and already known demixing factors, establishing an association with liquidliquid phase separation. This analysis could represent a starting point for implementing downstream experimental validations, using in vitro and in vivo approaches, to assess actual demixing.

scholarly journals Unpicking the Roles of DNA Damage Protein Kinases in Trypanosomatids

2021 ◽  
Vol 9 ◽  
Author(s):  
Gabriel L. A. Silva ◽  
Luiz R. O. Tosi ◽  
Richard McCulloch ◽  
Jennifer Ann Black
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Protein Kinases ◽  
Genetic Manipulation ◽  
Excision Repair ◽  
Dna Lesions ◽  
Wide Range ◽  
Damage Response ◽  
Repair Pathways ◽  
Signal Relays

To preserve genome integrity when faced with DNA lesions, cells activate and coordinate a multitude of DNA repair pathways to ensure timely error correction or tolerance, collectively called the DNA damage response (DDR). These interconnecting damage response pathways are molecular signal relays, with protein kinases (PKs) at the pinnacle. Focused efforts in model eukaryotes have revealed intricate aspects of DNA repair PK function, including how they direct DDR pathways and how repair reactions connect to wider cellular processes, including DNA replication and transcription. The Kinetoplastidae, including many parasites like Trypanosoma spp. and Leishmania spp. (causative agents of debilitating, neglected tropical infections), exhibit peculiarities in several core biological processes, including the predominance of multigenic transcription and the streamlining or repurposing of DNA repair pathways, such as the loss of non-homologous end joining and novel operation of nucleotide excision repair (NER). Very recent studies have implicated ATR and ATM kinases in the DDR of kinetoplastid parasites, whereas DNA-dependent protein kinase (DNA-PKcs) displays uncertain conservation, questioning what functions it fulfills. The wide range of genetic manipulation approaches in these organisms presents an opportunity to investigate DNA repair kinase roles in kinetoplastids and to ask if further kinases are involved. Furthermore, the availability of kinase inhibitory compounds, targeting numerous eukaryotic PKs, could allow us to test the suitability of DNA repair PKs as novel chemotherapeutic targets. Here, we will review recent advances in the study of trypanosomatid DNA repair kinases.

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