scholarly journals Genetically-induced Redox Stress Occurs in a Yeast Model for Roberts Syndrome

2021 ◽  
Author(s):  
Michael G Mfarej ◽  
Robert V Skibbens
Keyword(s):  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Gene Mutations ◽  
Loss Of Function ◽  
Redox Stress ◽  
Cellular Processes ◽  
Transcriptional Dysregulation ◽  
Dna Damaging Agents ◽  
Roberts Syndrome ◽  
Mutant Cells

Abstract Roberts Syndrome (RBS) is a multi-spectrum developmental disorder characterized by severe limb, craniofacial, and organ abnormalities and often intellectual disabilities. The genetic basis of RBS is rooted in loss-of-function mutations in the essential N-acetyltransferase ESCO2 which is conserved from yeast (Eco1/Ctf7) to humans. ESCO2/Eco1 regulate many cellular processes that impact chromatin structure, chromosome transmission, gene expression, and repair of the genome. The etiology of RBS remains contentious with current models that include transcriptional dysregulation or mitotic failure. Here, we report evidence that supports an emerging model rooted in defective DNA damage responses. First, the results reveal that redox stress is elevated in both eco1 and cohesion factor Saccharomyces cerevisiae mutant cells. Second, we provide evidence that Eco1 and cohesion factors are required for the repair of oxidative DNA damage such that ECO1 and cohesin gene mutations result in reduced cell viability and hyperactivation of DNA damage checkpoints that occur in response to oxidative stress. Moreover, we show that mutation of ECO1 is solely sufficient to induce endogenous redox stress and sensitizes mutant cells to exogenous genotoxic challenges. Remarkably, antioxidant treatment desensitizes eco1 mutant cells to a range of DNA damaging agents, raising the possibility that modulating the cellular redox state may represent an important avenue of treatment for Roberts Syndrome and tumors that bear ESCO2 mutations.

scholarly journals Budding Yeast CTDK-I Is Required for DNA Damage-Induced Transcription

2003 ◽  
Vol 2 (2) ◽  
pp. 274-283 ◽  
Author(s):  
Denis Ostapenko ◽  
Mark J. Solomon
Keyword(s):  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Scale ◽  
Large Subunit ◽  
Growth Conditions ◽  
Dna Damaging Agents ◽  
Irradiation Treatment ◽  
Mutant Cells

ABSTRACT CTDK-I phosphorylates the C-terminal domain (CTD) of the large subunit of yeast RNA polymerase II in a reaction that stimulates transcription elongation. Mutations in CTDK-I subunits—Ctk1p, Ctk2p, and Ctk3p—confer conditional phenotypes. In this study, we examined the role of CTDK-I in the DNA damage response. We found that mutation of individual CTDK-I subunits rendered yeast sensitive to hydroxyurea (HU) and UV irradiation. Treatment with DNA-damaging agents increased phosphorylation of Ser2 within the CTD repeats in wild-type but not in ctk1Δ mutant cells. Using microarray hybridization, we identified genes whose transcription following DNA damage is Ctk1p dependent, including several DNA repair and stress response genes. Following HU treatment, the level of Ser2-phosphorylated RNA polymerase II increased both globally and on the CTDK-I-regulated genes. The pleiotropic phenotypes of ctk mutants suggest that CTDK-I activity is essential during large-scale transcriptional repatterning under stress and unfavorable growth conditions.

scholarly journals Hsp90 induces increased genomic instability toward DNA-damaging agents by tuning down RAD53 transcription

2016 ◽  
Vol 27 (15) ◽  
pp. 2463-2478 ◽  
Author(s):  
Nidhi Khurana ◽  
Shyamasree Laskar ◽  
Mrinal K. Bhattacharyya ◽  
Sunanda Bhattacharyya
Keyword(s):  
Dna Damage ◽  
Genomic Instability ◽  
Transcriptional Repression ◽  
Strand Break ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Checkpoint Activation ◽  
Dna Damage Signaling ◽  
Dna Damaging Agents ◽  
G1 Cells

It is well documented that elevated body temperature causes tumors to regress upon radiotherapy. However, how hyperthermia induces DNA damage sensitivity is not clear. We show that a transient heat shock and particularly the concomitant induction of Hsp90 lead to increased genomic instability under DNA-damaging conditions. Using Saccharomyces cerevisiae as a model eukaryote, we demonstrate that elevated levels of Hsp90 attenuate efficient DNA damage signaling and dictate preferential use of the potentially mutagenic double-strand break repair pathway. We show that under normal physiological conditions, Hsp90 negatively regulates RAD53 transcription to suppress DNA damage checkpoint activation. However, under DNA damaging conditions, RAD53 is derepressed, and the increased level of Rad53p triggers an efficient DNA damage response. A higher abundance of Hsp90 causes increased transcriptional repression on RAD53 in a dose-dependent manner, which could not be fully derepressed even in the presence of DNA damage. Accordingly, cells behave like a rad53 loss-of-function mutant and show reduced NHEJ efficiency, with a drastic failure to up-regulate RAD51 expression and manifestly faster accumulation of CLN1 and CLN2 in DNA-damaged G1, cells leading to premature release from checkpoint arrest. We further demonstrate that Rad53 overexpression is able to rescue all of the aforementioned deleterious effects caused by Hsp90 overproduction.

scholarly journals An ever-changing landscape in Roberts syndrome biology: Implications for macromolecular damage

PLoS Genetics ◽  
2020 ◽  
Vol 16 (12) ◽  
pp. e1009219
Author(s):  
Michael G. Mfarej ◽  
Robert V. Skibbens
Keyword(s):  
Genetic Basis ◽  
Developmental Disorder ◽  
Essential Gene ◽  
Loss Of Function ◽  
Cellular Processes ◽  
Roberts Syndrome ◽  
Chromatid Cohesion ◽  
Limb Malformations ◽  
Molecular Etiology ◽  
Macromolecular Damage

Roberts syndrome (RBS) is a rare developmental disorder that can include craniofacial abnormalities, limb malformations, missing digits, intellectual disabilities, stillbirth, and early mortality. The genetic basis for RBS is linked to autosomal recessive loss-of-function mutation of the establishment of cohesion (ESCO) 2 acetyltransferase. ESCO2 is an essential gene that targets the DNA-binding cohesin complex. ESCO2 acetylates alternate subunits of cohesin to orchestrate vital cellular processes that include sister chromatid cohesion, chromosome condensation, transcription, and DNA repair. Although significant advances were made over the last 20 years in our understanding of ESCO2 and cohesin biology, the molecular etiology of RBS remains ambiguous. In this review, we highlight current models of RBS and reflect on data that suggests a novel role for macromolecular damage in the molecular etiology of RBS.

scholarly journals Nse2, a Component of the Smc5-6 Complex, Is a SUMO Ligase Required for the Response to DNA Damage

2005 ◽  
Vol 25 (1) ◽  
pp. 185-196 ◽  
Author(s):  
Emily A. Andrews ◽  
Jan Palecek ◽  
John Sergeant ◽  
Elaine Taylor ◽  
Alan R. Lehmann ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ring Finger ◽  
Dependent Manner ◽  
Dna Damaging Agents ◽  
Sumo Ligase ◽  
Essential Function ◽  
Mutant Cells ◽  
Smc Proteins

ABSTRACT The Schizosaccharomyces pombe SMC proteins Rad18 (Smc6) and Spr18 (Smc5) exist in a high-M r complex which also contains the non-SMC proteins Nse1, Nse2, Nse3, and Rad62. The Smc5-6 complex, which is essential for viability, is required for several aspects of DNA metabolism, including recombinational repair and maintenance of the DNA damage checkpoint. We have characterized Nse2 and show here that it is a SUMO ligase. Smc6 (Rad18) and Nse3, but not Smc5 (Spr18) or Nse1, are sumoylated in vitro in an Nse2-dependent manner, and Nse2 is itself autosumoylated, predominantly on the C-terminal part of the protein. Mutations of C195 and H197 in the Nse2 RING-finger-like motif abolish Nse2-dependent sumoylation. nse2.SA mutant cells, in which nse2.C195S-H197A is integrated as the sole copy of nse2, are viable, whereas the deletion of nse2 is lethal. Smc6 (Rad18) is sumoylated in vivo: the sumoylation level is increased upon exposure to DNA damage and is drastically reduced in the nse2.SA strain. Since nse2.SA cells are sensitive to DNA-damaging agents and to exposure to hydroxyurea, this implicates the Nse2-dependent sumoylation activity in DNA damage responses but not in the essential function of the Smc5-6 complex.

scholarly journals PTRH2: an adhesion regulated molecular switch at the nexus of life, death, and differentiation

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Austin D. Corpuz ◽  
Joe W. Ramos ◽  
Michelle L. Matter
Keyword(s):  
Cell Survival ◽  
Developmental Delays ◽  
Gene Mutations ◽  
Pancreatic Disease ◽  
Loss Of Function ◽  
Multisystem Disease ◽  
Cellular Processes ◽  
State Of The Science ◽  
Bcl2 Expression

AbstractPeptidyl-tRNA hydrolase 2 (PTRH2; Bit-1; Bit1) is an underappreciated regulator of adhesion signals and Bcl2 expression. Its key roles in muscle differentiation and integrin-mediated signaling are central to the pathology of a recently identified patient syndrome caused by a cluster of Ptrh2 gene mutations. These loss-of-function mutations were identified in patients presenting with severe deleterious phenotypes of the skeletal muscle, endocrine, and nervous systems resulting in a syndrome called Infantile-onset Multisystem Nervous, Endocrine, and Pancreatic Disease (IMNEPD). In contrast, in cancer PTRH2 is a potential oncogene that promotes malignancy and metastasis. PTRH2 modulates PI3K/AKT and ERK signaling in addition to Bcl2 expression and thereby regulates key cellular processes in response to adhesion including cell survival, growth, and differentiation. In this Review, we discuss the state of the science on this important cell survival, anoikis and differentiation regulator, and opportunities for further investigation and translation. We begin with a brief overview of the structure, regulation, and subcellular localization of PTRH2. We discuss the cluster of gene mutations thus far identified which cause developmental delays and multisystem disease. We then discuss the role of PTRH2 and adhesion in breast, lung, and esophageal cancers focusing on signaling pathways involved in cell survival, cell growth, and cell differentiation.

scholarly journals 3′-Phosphodiesterase and 3′→5′ Exonuclease Activities of Yeast Apn2 Protein and Requirement of These Activities for Repair of Oxidative DNA Damage

2001 ◽  
Vol 21 (5) ◽  
pp. 1656-1661 ◽  
Author(s):  
Ildiko Unk ◽  
Lajos Haracska ◽  
Satya Prakash ◽  
Louise Prakash
Keyword(s):  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Dna Polymerases ◽  
Oxygen Species ◽  
Genetic Studies ◽  
Strand Breaks ◽  
Abasic Sites ◽  
Dna Damaging Agents ◽  
End Groups ◽  
Dna Strand

ABSTRACT In Saccharomyces cerevisiae, the AP endonucleases encoded by the APN1 and APN2 genes provide alternate pathways for the removal of abasic sites. Oxidative DNA-damaging agents, such as H2O2, produce DNA strand breaks which contain 3′-phosphate or 3′-phosphoglycolate termini. Such 3′ termini are inhibitory to synthesis by DNA polymerases. Here, we show that purified yeast Apn2 protein contains 3′-phosphodiesterase and 3′→5′ exonuclease activities, and mutation of the active-site residue Glu59 to Ala in Apn2 inactivates both these activities. Consistent with these biochemical observations, genetic studies indicate the involvement of APN2 in the repair of H2O2-induced DNA damage in a pathway alternate to APN1, and the Ala59 mutation inactivates this function of Apn2. From these results, we conclude that the ability of Apn2 to remove 3′-end groups from DNA is paramount for the repair of strand breaks arising from the reaction of DNA with reactive oxygen species.

How do cells sense DNA lesions?

2020 ◽  
Vol 48 (2) ◽  
pp. 677-691 ◽  
Author(s):  
Chiara Vittoria Colombo ◽  
Marco Gnugnoli ◽  
Elisa Gobbini ◽  
Maria Pia Longhese
Keyword(s):  
Dna Damage ◽  
Protein Complexes ◽  
Dna Lesions ◽  
Exogenous Dna ◽  
Cellular Targets ◽  
Cellular Processes ◽  
Dna Lesion ◽  
Dna Damaging Agents ◽  
Wide Range ◽  
Damage Response

DNA is exposed to both endogenous and exogenous DNA damaging agents that chemically modify it. To counteract the deleterious effects exerted by DNA lesions, eukaryotic cells have evolved a network of cellular pathways, termed DNA damage response (DDR). The DDR comprises both mechanisms devoted to repair DNA lesions and signal transduction pathways that sense DNA damage and transduce this information to specific cellular targets. These targets, in turn, impact a wide range of cellular processes including DNA replication, DNA repair and cell cycle transitions. The importance of the DDR is highlighted by the fact that DDR inactivation is commonly found in cancer and causes many different human diseases. The protein kinases ATM and ATR, as well as their budding yeast orthologs Tel1 and Mec1, act as master regulators of the DDR. The initiating events in the DDR entail both DNA lesion recognition and assembly of protein complexes at the damaged DNA sites. Here, we review what is known about the early steps of the DDR.

scholarly journals MasterPATH: network analysis of functional genomics screening data

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Natalia Rubanova ◽  
Guillaume Pinna ◽  
Jeremie Kropp ◽  
Anna Campalans ◽  
Juan Pablo Radicella ◽  
...  
Keyword(s):  
Dna Damage ◽  
Functional Genomics ◽  
Oxidative Dna Damage ◽  
Transcriptome Profiling ◽  
Molecular Pathways ◽  
Biological Processes ◽  
Loss Of Function ◽  
Damage Recognition ◽  
Dna Damage Recognition

Abstract Background Functional genomics employs several experimental approaches to investigate gene functions. High-throughput techniques, such as loss-of-function screening and transcriptome profiling, allow to identify lists of genes potentially involved in biological processes of interest (so called hit list). Several computational methods exist to analyze and interpret such lists, the most widespread of which aim either at investigating of significantly enriched biological processes, or at extracting significantly represented subnetworks. Results Here we propose a novel network analysis method and corresponding computational software that employs the shortest path approach and centrality measure to discover members of molecular pathways leading to the studied phenotype, based on functional genomics screening data. The method works on integrated interactomes that consist of both directed and undirected networks – HIPPIE, SIGNOR, SignaLink, TFactS, KEGG, TransmiR, miRTarBase. The method finds nodes and short simple paths with significant high centrality in subnetworks induced by the hit genes and by so-called final implementers – the genes that are involved in molecular events responsible for final phenotypic realization of the biological processes of interest. We present the application of the method to the data from miRNA loss-of-function screen and transcriptome profiling of terminal human muscle differentiation process and to the gene loss-of-function screen exploring the genes that regulates human oxidative DNA damage recognition. The analysis highlighted the possible role of several known myogenesis regulatory miRNAs (miR-1, miR-125b, miR-216a) and their targets (AR, NR3C1, ARRB1, ITSN1, VAV3, TDGF1), as well as linked two major regulatory molecules of skeletal myogenesis, MYOD and SMAD3, to their previously known muscle-related targets (TGFB1, CDC42, CTCF) and also to a number of proteins such as C-KIT that have not been previously studied in the context of muscle differentiation. The analysis also showed the role of the interaction between H3 and SETDB1 proteins for oxidative DNA damage recognition. Conclusion The current work provides a systematic methodology to discover members of molecular pathways in integrated networks using functional genomics screening data. It also offers a valuable instrument to explain the appearance of a set of genes, previously not associated with the process of interest, in the hit list of each particular functional genomics screening.

scholarly journals FANCI-FANCD2 Binds RNA, Which Stimulates Its Monoubiquitination

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 645-645
Author(s):  
Zhuobin Liang ◽  
Yaqun Teng ◽  
Jingchun Liu ◽  
Simonne Longerich ◽  
Xiaoyong Chen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Developmental Abnormalities ◽  
Replicative Stress ◽  
The Relationship ◽  
Mutant Cells

Abstract Fanconi anemia (FA) is characterized by developmental abnormalities, bone marrow failure, and a strong cancer predisposition. FA cells are hypersensitive to DNA replicative stress, and accumulate co-transcriptional R-loops. Previous work has demonstrated that BRCA2 binds to R loops, and increased R loops are noted in FA-D2 mutant cells. Additionally, it is understood that at least one FA protein, FANCA, binds RNA. The goal of this study was to understand the relationship between FANCD2 and RNA, especially with regard to manifestation of R loops as a part of the pathophysiology of FA. First, we confirmed the increased presence of R loops in FA mutant cells using the S9.6 monoclonal antibody immunofluorescence microscopy. RNAseH overexpression removes R loop signal and increases cell survival upon mitomycin C treatment. We also showed the presence of increased R loops in an actively transcribed region of the actin gene by bisulfite DNA sequencing. We used the Damage At RNA Transcription (DART) assay, which is designed to combine oxidative DNA damage and the genomic insertion of a hyper transcription site (Fig A). Coactivation of transcription and DNA damage results in colocalization of FANCD2 and S9.6/R loop signal at the transcriptional site (Fig B and C). Consistent with the S9.6 IF, wild type RNAseH overexpression resulted in the abrogation of FANCD2 colocalization. We then asked if FANCD2 binds RNA. FANCD2 in cell lysate bound to biotinylated RNA species, preferring GC rich RNAs. Using recombinant FANCI-FANCD2 (ID2) protein (Fig D), we found that ID2 binds preferably to single stranded RNA in a more robust manner than DNA (Fig E and F). Interestingly, an ID2 complex with a known DNA binding mutation in FANCI also was defective for RNA binding. Furthermore, ID2 bound to R loops but was mediated via the single stranded DNA component of the structure. Importantly, an in vitro monoubiquitination reconstitution system using FANCL as the E3 ligase demonstrated that monoubiquitination of ID2 was stimulated to an equal or greater degree by RNA versus DNA, with greater signal in presence of GC-rich, single-stranded RNA as well as R loops (Fig G and H and data not shown). Collectively, our results support a novel mechanism the ID2 complex suppresses the formation of pathogenic R-loops by binding RNA species, thereby activating the FA pathway (Fig I). Figure. Figure. Disclosures No relevant conflicts of interest to declare.

scholarly journals The secret identities of TMPRSS2: Fertility factor, virus trafficker, inflammation moderator, prostate protector and tumor suppressor

Tumor Biology ◽  
2021 ◽  
Vol 43 (1) ◽  
pp. 159-176
Author(s):  
Richard J. Epstein
Keyword(s):  
Prostate Cancer ◽  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Inflammatory Responses ◽  
Inducible Promoter ◽  
Loss Of Function ◽  
Coding Region ◽  
Antiinflammatory Drugs ◽  
Reading Frame ◽  
Secret Identities

The human TMPRSS2 gene is pathogenetically implicated in both coronaviral lung infection and prostate cancer, suggesting its potential as a drug target in both contexts. SARS-COV-2 spike polypeptides are primed by the host transmembrane TMPRSS2 protease, triggering virus fusion with epithelial cell membranes followed by an endocytotic internalisation process that bypasses normal endosomal activation of cathepsin-mediated innate immunity; viral co-opting of TMPRSS2 thus favors microbial survivability by attenuating host inflammatory responses. In contrast, most early hormone-dependent prostate cancers express TMPRSS2:ERG fusion genes arising from deletions that eliminate the TMPRSS2 coding region while juxtaposing its androgen-inducible promoter and the open reading frame of ERG, upregulating pro-inflammatory ERG while functionally disabling TMPRSS2. Moreover, inflammatory oxidative DNA damage selects for TMPRSS2:ERG-fused cancers, whereas patients treated with antiinflammatory drugs develop fewer of these fusion-dependent tumors. These findings imply that TMPRSS2 protects the prostate by enabling endosomal bypass of pathogens which could otherwise trigger inflammation-induced DNA damage that predisposes to TMPRSS2:ERG fusions. Hence, the high oncogenic selectability of TMPRSS2:ERG fusions may reflect a unique pro-inflammatory synergy between androgenic ERG gain-of-function and fusogenic TMPRSS2 loss-of-function, cautioning against the use of TMPRSS2-inhibitory drugs to prevent or treat early prostate cancer.

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