scholarly journals Structural and functional interactions of the prostate cancer suppressor protein NKX3.1 with topoisomerase I

2013 ◽  
Vol 453 (1) ◽  
pp. 125-136 ◽  
Author(s):  
Liang-Nian Song ◽  
Cai Bowen ◽  
Edward P. Gelmann
Keyword(s):  
Dna Damage ◽  
Protein Interactions ◽  
Topoisomerase I ◽  
Cellular Response ◽  
Peptide Fragments ◽  
The Core ◽  
Tumour Suppressor Protein ◽  
Linker Domain ◽  
In Cells

NKX3.1 (NK3 homeobox 1) is a prostate tumour suppressor protein with a number of activities that are critical for its role in tumour suppression. NKX3.1 mediates the cellular response to DNA damage by interacting with ATM (ataxia telangiectasia mutated) and by activation of topoisomerase I. In the present study we characterized the interaction between NKX3.1 and topoisomerase I. The NKX3.1 homeodomain binds to a region of topoisomerase I spanning the junction between the core and linker domains. Loss of the topoisomerase I N-terminal domain, a region for frequent protein interactions, did not affect binding to NKX3.1 as was shown by the activation of Topo70 (N-terminal truncated topoisomerase I) in vitro. In contrast, NKX3.1 interacts with the enzyme reconstituted from peptide fragments of the core and linker active site domains, but inhibits the DNA-resolving activity of the reconstituted enzyme in vitro. The effect of NKX3.1 on both Topo70 and the reconstituted enzyme was seen in the presence and absence of camptothecin. Neither NKX3.1 nor CPT (camptothecin) had an effect on the interaction of the other with topoisomerase I. Therefore the interactions of NKX3.1 and CPT with the linker domain of topoisomerase I are mutually exclusive. However, in cells the effect of NKX3.1 on topoisomerase binding to DNA sensitized the cells to cellular toxicity and the induction of apoptosis by low doses of CPT. Lastly, topoisomerase I is important for the effect of NKX3.1 on cell survival after DNA damage as topoisomerase knockdown blocked the effect of NKX3.1 on clonogenicity after DNA damage. Therefore NKX3.1 and topoisomerase I interact in vitro and in cells to affect the CPT sensitivity and DNA-repair functions of NKX3.1.

scholarly journals The DNA damage inducible lncRNA SCAT7 regulates genomic integrity and topoisomerase 1 turnover in lung adenocarcinoma

NAR Cancer ◽  
2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Luisa Statello ◽  
Mohamad M Ali ◽  
Silke Reischl ◽  
Sagar Mahale ◽  
Subazini Thankaswamy Kosalai ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cancer Biology ◽  
Topoisomerase I ◽  
Cell Cycle Checkpoints ◽  
Topoisomerase 1 ◽  
Damage Response ◽  
Promote Cell Survival

Abstract Despite the rapid improvements in unveiling the importance of lncRNAs in all aspects of cancer biology, there is still a void in mechanistic understanding of their role in the DNA damage response. Here we explored the potential role of the oncogenic lncRNA SCAT7 (ELF3-AS1) in the maintenance of genome integrity. We show that SCAT7 is upregulated in response to DNA-damaging drugs like cisplatin and camptothecin, where SCAT7 expression is required to promote cell survival. SCAT7 silencing leads to decreased proliferation of cisplatin-resistant cells in vitro and in vivo through interfering with cell cycle checkpoints and DNA repair molecular pathways. SCAT7 regulates ATR signaling, promoting homologous recombination. Importantly, SCAT7 also takes part in proteasome-mediated topoisomerase I (TOP1) degradation, and its depletion causes an accumulation of TOP1–cc structures responsible for the high levels of intrinsic DNA damage. Thus, our data demonstrate that SCAT7 is an important constituent of the DNA damage response pathway and serves as a potential therapeutic target for hard-to-treat drug resistant cancers.

scholarly journals Cleavage and Inactivation of ATM during Apoptosis

1999 ◽  
Vol 19 (9) ◽  
pp. 6076-6084 ◽  
Author(s):  
Graeme C. M. Smith ◽  
Fabrizio d’adda di Fagagna ◽  
Nicholas D. Lakin ◽  
Stephen P. Jackson
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Caspase 3 ◽  
Cysteine Proteases ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Dna Damage Signalling ◽  
In Cells

ABSTRACT The activation of the cysteine proteases with aspartate specificity, termed caspases, is of fundamental importance for the execution of programmed cell death. These proteases are highly specific in their action and activate or inhibit a variety of key protein molecules in the cell. Here, we study the effect of apoptosis on the integrity of two proteins that have critical roles in DNA damage signalling, cell cycle checkpoint controls, and genome maintenance—the product of the gene defective in ataxia telangiectasia, ATM, and the related protein ATR. We find that ATM but not ATR is specifically cleaved in cells induced to undergo apoptosis by a variety of stimuli. We establish that ATM cleavage in vivo is dependent on caspases, reveal that ATM is an efficient substrate for caspase 3 but not caspase 6 in vitro, and show that the in vitro caspase 3 cleavage pattern mirrors that in cells undergoing apoptosis. Strikingly, apoptotic cleavage of ATM in vivo abrogates its protein kinase activity against p53 but has no apparent effect on the DNA binding properties of ATM. These data suggest that the cleavage of ATM during apoptosis generates a kinase-inactive protein that acts, through its DNA binding ability, in a trans-dominant-negative fashion to prevent DNA repair and DNA damage signalling.

scholarly journals In vivo analysis of DNA-protein interactions on the human erythropoietin enhancer.

1997 ◽  
Vol 17 (2) ◽  
pp. 851-856 ◽  
Author(s):  
B Hu ◽  
E Wright ◽  
L Campbell ◽  
K L Blanchard
Keyword(s):  
Oxygen Tension ◽  
Protein Interactions ◽  
Proximal Promoter ◽  
Low Oxygen ◽  
Initiation Rate ◽  
Cobalt Exposure ◽  
In Cells ◽  
Dna Protein Interactions

The erythropoietin (EPO) gene is one of the best examples of a mammalian gene controlled by oxygen tension. The DNA elements responsible for hypoxia-induced transcription consist of a short region of the proximal promoter and a <50-bp 3' enhancer. The elements act cooperatively to increase the transcriptional initiation rate approximately 100-fold in response to low oxygen tension in Hep3B cells. Two distinct types of transactivating proteins have been demonstrated to bind the response elements in the human EPO enhancer in vitro: one shows hypoxia-inducible DNA binding activity, while the other activity binds DNA under normoxic and hypoxic conditions. We have investigated the DNA-protein interactions on the human EPO enhancer in living tissue culture cells that produce EPO in a regulated fashion (Hep3B) and in cells that do not express EPO under any conditions tested (HeLa). We have identified in vivo DNA-protein interactions on the control elements in the human EPO enhancer by ligation-mediated PCR technology. We show that the putative protein binding sites in the EPO enhancer are occupied in vivo under conditions of normoxia, hypoxia, and cobalt exposure in EPO-producing cells. These sites are not occupied in cells that do not produce EPO. We also provide evidence for a conformational change in the topography of the EPO enhancer in response to hypoxia and cobalt exposure.

scholarly journals hSSB2 (NABP1) is required for the recruitment of RPA during the cellular response to DNA UV damage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Didier Boucher ◽  
Ruvini Kariawasam ◽  
Joshua Burgess ◽  
Adrian Gimenez ◽  
Tristan E. Ocampo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
Excision Repair ◽  
Protein A ◽  
Ultraviolet B ◽  
Repair System ◽  
Uvb Radiation ◽  
Exogenous Dna

AbstractMaintenance of genomic stability is critical to prevent diseases such as cancer. As such, eukaryotic cells have multiple pathways to efficiently detect, signal and repair DNA damage. One common form of exogenous DNA damage comes from ultraviolet B (UVB) radiation. UVB generates cyclobutane pyrimidine dimers (CPD) that must be rapidly detected and repaired to maintain the genetic code. The nucleotide excision repair (NER) pathway is the main repair system for this type of DNA damage. Here, we determined the role of the human Single-Stranded DNA Binding protein 2, hSSB2, in the response to UVB exposure. We demonstrate that hSSB2 levels increase in vitro and in vivo after UVB irradiation and that hSSB2 rapidly binds to chromatin. Depletion of hSSB2 results in significantly decreased Replication Protein A (RPA32) phosphorylation and impaired RPA32 localisation to the site of UV-induced DNA damage. Delayed recruitment of NER protein Xeroderma Pigmentosum group C (XPC) was also observed, leading to increased cellular sensitivity to UVB. Finally, hSSB2 was shown to have affinity for single-strand DNA containing a single CPD and for duplex DNA with a two-base mismatch mimicking a CPD moiety. Altogether our data demonstrate that hSSB2 is involved in the cellular response to UV exposure.

The Coffee Constituent Chlorogenic Acid Induces Cellular DNA Damage and Formation of Topoisomerase I– and II–DNA Complexes in Cells

2012 ◽  
Vol 60 (30) ◽  
pp. 7384-7391 ◽  
Author(s):  
Estefanía Burgos-Morón ◽  
José Manuel Calderón-Montaño ◽  
Manuel Luis Orta ◽  
Nuria Pastor ◽  
Concepción Pérez-Guerrero ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chlorogenic Acid ◽  
Topoisomerase I ◽  
Dna Complexes ◽  
Cellular Dna ◽  
In Cells

Control of genome stability by Slx protein complexes

2009 ◽  
Vol 37 (3) ◽  
pp. 495-510 ◽  
Author(s):  
John Rouse
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Protein Interactions ◽  
Cellular Response ◽  
Genome Stability ◽  
Molecular Mechanisms ◽  
Excision Repair ◽  
Protein Complexes ◽  
Alkylating Agents ◽  
Strand Break

The six Saccharomyces cerevisiae SLX genes were identified in a screen for factors required for the viability of cells lacking Sgs1, a member of the RecQ helicase family involved in processing stalled replisomes and in the maintenance of genome stability. The six SLX gene products form three distinct heterodimeric complexes, and all three have catalytic activity. Slx3–Slx2 (also known as Mus81–Mms4) and Slx1–Slx4 are both heterodimeric endonucleases with a marked specificity for branched replication fork-like DNA species, whereas Slx5–Slx8 is a SUMO (small ubiquitin-related modifier)-targeted E3 ubiquitin ligase. All three complexes play important, but distinct, roles in different aspects of the cellular response to DNA damage and perturbed DNA replication. Slx4 interacts physically not only with Slx1, but also with Rad1–Rad10 [XPF (xeroderma pigmentosum complementation group F)–ERCC1 (excision repair cross-complementing 1) in humans], another structure-specific endonuclease that participates in the repair of UV-induced DNA damage and in a subpathway of recombinational DNA DSB (double-strand break) repair. Curiously, Slx4 is essential for repair of DSBs by Rad1–Rad10, but is not required for repair of UV damage. Slx4 also promotes cellular resistance to DNA-alkylating agents that block the progression of replisomes during DNA replication, by facilitating the error-free mode of lesion bypass. This does not require Slx1 or Rad1–Rad10, and so Slx4 has several distinct roles in protecting genome stability. In the present article, I provide an overview of our current understanding of the cellular roles of the Slx proteins, paying particular attention to the advances that have been made in understanding the cellular roles of Slx4. In particular, protein–protein interactions and underlying molecular mechanisms are discussed and I draw attention to the many questions that have yet to be answered.

scholarly journals Regulation of cellular response to cisplatin-induced DNA damage and DNA repair in cells overexpressing p185erbB-2 is dependent on the ras signaling pathway

Oncogene ◽  
1997 ◽  
Vol 14 (15) ◽  
pp. 1827-1835 ◽  
Author(s):  
Lily Yen ◽  
Nie Zeng-Rong ◽  
Xiao-Li You ◽  
Stéphane Richard ◽  
Beatrice C Langton-Webster ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Signaling Pathway ◽  
Cellular Response ◽  
Ras Signaling ◽  
Ras Signaling Pathway ◽  
In Cells

scholarly journals Nuclear Accumulation of LAP1:TRF2 Complex during DNA Damage Response Uncovers a Novel Role for LAP1

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1804
Author(s):  
Cátia D. Pereira ◽  
Filipa Martins ◽  
Mariana Santos ◽  
Thorsten Müeller ◽  
Odete A. B. da Cruz e Silva ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cellular Response ◽  
Nuclear Accumulation ◽  
Protein Levels ◽  
Dna Damaging Agents ◽  
Physiological Properties ◽  
Damage Response

Lamina-associated polypeptide 1 (LAP1) is a nuclear envelope (NE) protein whose function remains poorly characterized. In a recent LAP1 protein interactome study, a putative regulatory role in the DNA damage response (DDR) has emerged and telomeric repeat-binding factor 2 (TRF2), a protein intimately associated with this signaling pathway, was among the list of LAP1 interactors. To gain insights into LAP1′s physiological properties, the interaction with TRF2 in human cells exposed to DNA-damaging agents was investigated. The direct LAP1:TRF2 binding was validated in vitro by blot overlay and in vivo by co-immunoprecipitation after hydrogen peroxide and bleomycin treatments. The regulation of this protein interaction by LAP1 phosphorylation was demonstrated by co-immunoprecipitation and mass spectrometry following okadaic acid exposure. The involvement of LAP1 and TRF2 in the DDR was confirmed by their increased nuclear protein levels after bleomycin treatment, evaluated by immunoblotting, as well as by their co-localization with DDR factors at the NE and within the nucleoplasm, assessed by immunocytochemistry. Effectively, we showed that the LAP1:TRF2 complex is established during a cellular response against DNA damage. This work proposes a novel functional role for LAP1 in the DDR, revealing a potential biological mechanism that may be disrupted in LAP1-associated pathologies.

The roles of hypoxia, PTEN, and Rad51 in mediating metastatic prostate cancer cells' responses to PARP inhibitor and topoisomerase 1 inhibitor.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e13564-e13564
Author(s):  
Jingsong Zhang ◽  
Minghui Wu ◽  
Xue Wang
Keyword(s):  
Prostate Cancer ◽  
Dna Damage ◽  
Topoisomerase I ◽  
Metastatic Prostate Cancer ◽  
Synthetic Lethality ◽  
Parp Inhibitor ◽  
Single Agent ◽  
Study Results

e13564 Background: With the recent success of poly (ADP-ribose) polymerase inhibitor (PARPi) in the treatment of BRCA1 or BRCA2 mutated cancers, there is increasing interest to explore synthetic lethality in cancers with defective DNA repair pathways. Rad51 is an essential protein in the homologous recombination repair (HRR) of DNA double strand breaks. Previous studies with non metastatic prostate cancer (mCaP) cells have reported low Rad51 levels in cells with loss of PTEN or under hypoxia, which then led to their sensitivity to PARPi. Given intra tumor hypoxia and loss of PTEN is common in mCaP, we test PAPRi, ABT888 and DNA damaging topoisomerase I inhibitor, CPT11, either alone or in combination in mCaP preclinical models. Methods: mCaP cell lines with functional PTEN (DU145) and loss of PTEN (PC3) were grew under normoxia (21% O2) or hypoxia (0.2% O2). DNA damage, HRR, apoptosis were assessed with comet assay, western blot, immunofluorescence and flowcytometry. The regulation of RAD51 was studied with quantitative RT-PCR and RAD51 promoter reporter assay. PC3 xenograft was used for in vivo study. Results: Despite of its low levels of expression under hypoxia, up regulation of Rad51 was observed soon after treating hypoxic PC3 and Du145 cells with ABT888 or SN38, an active metabolite of CPT11. Such Rad51 up regulation led to less DNA damage and apoptosis under hypoxia compared to normoxia. Inhibiting RAD51 expression with siRNA overcame PC3 and Du145’s resistance to SN38. Furthermore, ABT888 enhanced the activities of SN38 as detected by clonogenic assay and flowcytometry under both normoxia and hypoxia. Consistent with the in vitro data, ABT888 by itself had limited anti-tumor activities despite the loss of PTEN in PC3 xenografts. The anti-tumor activity of single agent CPT11 was significantly improved with the ABT888 and CPT11 combination (P<0.008). Conclusions: neither loss of PTEN nor hypoxia sensitized mCaP cells to PARPi or DNA damaging drugs. Such resistance under hypoxia was at least partly due to up regulation of Rad51. Combining ABT888 with CPT11 overcame the resistance to CPT11 under hypoxia and enhanced its anti-tumor activities both in vitro and in vivo.

scholarly journals A Dynamic Scaffold of Pre-snoRNP Factors Facilitates Human Box C/D snoRNP Assembly

2007 ◽  
Vol 27 (19) ◽  
pp. 6782-6793 ◽  
Author(s):  
Kenneth Scott McKeegan ◽  
Charles Maurice Debieux ◽  
Séverine Boulon ◽  
Edouard Bertrand ◽  
Nicholas James Watkins
Keyword(s):  
Protein Interactions ◽  
Core Protein ◽  
The Novel ◽  
Protein Protein Interactions ◽  
Core Complex ◽  
The Core ◽  
Snornp Biogenesis ◽  
The Individual ◽  
Vitro Protein

ABSTRACT The box C/D small nucleolar RNPs (snoRNPs) are essential for the processing and modification of rRNA. The core box C/D proteins are restructured during human U3 box C/D snoRNP biogenesis; however, the molecular basis of this is unclear. Here we show that the U8 snoRNP is also restructured, suggesting that this may occur with all box C/D snoRNPs. We have characterized four novel human biogenesis factors (BCD1, NOP17, NUFIP, and TAF9) which, along with the ATPases TIP48 and TIP49, are likely to be involved in the formation of the pre-snoRNP. We have analyzed the in vitro protein-protein interactions between the assembly factors and core box C/D proteins. Surprisingly, this revealed few interactions between the individual core box C/D proteins. However, the novel biogenesis factors and TIP48 and TIP49 interacted with one or more of the core box C/D proteins, implying that they mediate the assembly of the pre-snoRNP. Consistent with this, we show that NUFIP bridges interactions between the core box C/D proteins in a partially reconstituted pre-snoRNP. Restructuring of the core complex probably reflects the conversion of the pre-snoRNP, where core protein-protein interactions are maintained by the bridging biogenesis factors, to the mature snoRNP.

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