Activation of the Prereplication Complex Is Blocked by Mimosine through Reactive Oxygen Species-activated Ataxia Telangiectasia Mutated (ATM) Protein without DNA Damage

Kubota, Shoichi; Fukumoto, Yasunori; Ishibashi, Keiichiro; Soeda, Shuhei; Kubota, Sho; Yuki, Ryuzaburo; Nakayama, Yuji; Aoyama, Kazumasa; Yamaguchi, Noritaka; Yamaguchi, Naoto

doi:10.1074/jbc.m113.546655

Cited by 35 publications

(37 citation statements)

References 72 publications

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“…Briefly, template nuclei are isolated from human cells synchronised in the late G1 phase of the cell cycle by the iron-chelating compound mimosine, which inhibits the transition of pre-replication to pre-initiation complexes at replication origins (27,29,30). DNA replication can rapidly be initiated in these template nuclei by adding a soluble extract from proliferating human cells, which contains all essential replication factors (27,31,32).…”

Section: Introductionmentioning

confidence: 99%

Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq)

Langley¹,

Gräf²,

Smith³

et al. 2016

Nucleic Acids Res

135

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Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq.

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Section: Introductionmentioning

confidence: 99%

Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq)

Langley¹,

Gräf²,

Smith³

et al. 2016

Nucleic Acids Res

135

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“…The DDR preserves genetic stability by detecting DNA lesions, activating cell cycle checkpoints and promoting DNA damage repair [18][19][20][21]. The common denominator integrating these forms of genotoxic stresses and eliciting the signalling cascade is increased production of ROS as harbinger to DNA damage [22][23][24][25][26][27]. Chemical agents such as drugs used in cancer chemotherapy are also able to induce some form of DNA lesions [28].…”

Section: Introductionmentioning

confidence: 99%

NRF2 inhibition causes repression of ATM and ATR expression leading to aberrant DNA Damage Response

Khalil¹,

Deeni²

2015

Biodiscovery

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Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a master regulator of the antioxidant response (AR) pathway and functions as a transcription factor for basal and oxidative stress-induced expression of a battery of detoxification enzymes and cytoprotective genes. Recent evidence has also demonstrated a role of NRF2 in driving resistance of numerous cancers to chemotherapeutic agents. ATM and ATR are serine/threonine kinases that are activated following DNA damage and function as central components of DNA Damage Response (DDR) pathway. Activities of these kinases cause cell cycle arrest and activate DNA repair signals leading to cytoprotection against genotoxic agents. In this study, we elucidated the roles of ATM-and ATR-dependent DDR and NRF2-mediated AR pathways in promoting cytoprotection following cisplatin challenge in ovarian cancer cell line models. We also determined whether these pathways were inter-dependent for full activation following genotoxic insults and as such demonstrated crosstalk in their signaling mechanism to elicit cytoprotective pathways. Treatment with cisplatin caused NRF2 induction and generation of reactive oxygen species (ROS) that caused cytotoxicity in ovarian cancer cells. This was attenuated by the ROS scavenger N-Acetyl cysteine, implicating NRF2 function in cytoprotection against cisplatin. Treatment with retinoic acid (RA) caused down regulation of NRF2, disruption of AR pathway, significant accumulation of ROS and enhanced cisplatin cytotoxicity. Interestingly, RA treatment also led to repression of total ATM and ATR proteins and aberrant DDR activation following cisplatin challenge. In order to determine whether the RA induced ATM and ATR repression was dependent on NRF2 inhibition, we silenced NRF2 using SiRNA. This caused transcriptional repression of both ATM and ATR expression as determined by their promoter driven luciferase assays. Thus, NRF2 inhibition led to DDR suppression by down-regulating ATM and ATR that led to enhanced cytotoxicity. These findings demonstrate mechanism of crosstalk between the AR and the DDR pathways and extend the scope of NRF2 in promoting cancer therapeutic resistance.

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“…Immunodetection was performed by enhanced chemiluminescence (Millipore) as described (38,42). Results were analyzed using a ChemiDoc XRS-Plus image analyzer (BioRad).…”

Section: Methodsmentioning

confidence: 99%

“…The EBNA1-based episomal pEBMulti-H1 vector, which encodes the H1 promoter and a neomycin-resistant gene, was generated from the pEBMulti vector (Wako Pure Chemical Industries, Osaka, Japan) by replacing the CAG promoter with the H1 promoter as described (37). The oligonucleotides used for shRNA were annealed and subcloned into the pEBMulti-H1 vector (37)(38)(39). To generate AKAP8-knockdown cells, HCT116 cells were transfected with pEBMulti-neo/shAKAP8 selected in 600 g/ml G418.…”

Section: Methodsmentioning

confidence: 99%

Role for Tyrosine Phosphorylation of A-kinase Anchoring Protein 8 (AKAP8) in Its Dissociation from Chromatin and the Nuclear Matrix

Kubota

Morii

Yuki

et al. 2015

Journal of Biological Chemistry

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Background: Tyrosine kinases are active in the cell nucleus and involved in global nuclear structure. Results: Phosphorylation of AKAP8 at multiple tyrosine residues by several nucleus-localized tyrosine kinases, including c-Src, induces AKAP8's dissociation from nuclear structures. Conclusion: Nuclear tyrosine phosphorylation of AKAP8 is involved in global nuclear structure changes. Significance: These findings highlight the importance of nuclear tyrosine phosphorylation in dynamic chromatin regulation.

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Activation of the Prereplication Complex Is Blocked by Mimosine through Reactive Oxygen Species-activated Ataxia Telangiectasia Mutated (ATM) Protein without DNA Damage

Cited by 35 publications

References 72 publications

Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq)

Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq)

NRF2 inhibition causes repression of ATM and ATR expression leading to aberrant DNA Damage Response

Role for Tyrosine Phosphorylation of A-kinase Anchoring Protein 8 (AKAP8) in Its Dissociation from Chromatin and the Nuclear Matrix

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