Autism-associated vigilin depletion impairs DNA damage repair

Vigilin (Vgl1) is essential for heterochromatin formation, chromosome segregation, mRNA stability and is associated with autism-spectrum disorders and cancer, vigilin, for example, can suppress proto-oncogene c-fms expression in breast cancer. Conserved from yeast to humans, vigilin is an RNA-binding protein with 14 tandemly arranged nonidentical hnRNP K type homology (KH) domains. Here we report that vigilin depletion increased cell sensitivity to cisplatin- or ionizing radiation (IR)-induced cell death and genomic instability due to defective DNA repair. Vigilin depletion delayed dephosphorylation of IR-induced γ-H2AX, elevated levels of residual 53BP1 and RIF1 foci, while reducing Rad51 and BRCA1 foci formation, DNA end resection and double strand break (DSB) repair. We show that vigilin interacts with the DNA damage response (DDR) proteins RAD51 and BRCA1, and vigilin depletion impairs their recruitment to DSB sites. Transient hydroxyurea (HU) induced replicative stress in vigilin-depleted cells increased replication fork stalling and blocked restart of DNA synthesis. Furthermore, histone acetylation promoted vigilin recruitment to DSBs preferentially in transcriptionally active genome. These findings uncover a novel vigilin role in DNA damage repair with implications for autism and cancer related disorders.

Download Full-text

Ablating Ku70 phosphorylation results in defective DNA damage repair and spontaneous induction of hepatocellular carcinoma

10.1101/2021.03.03.433432 ◽

2021 ◽

Author(s):

Janapriya Saha ◽

Jinsung Bae ◽

Shih-Ya Wang ◽

Lori J. Chappell ◽

Purva Gopal ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Dna Damage ◽

Genomic Instability ◽

Dna Damage Repair ◽

Strand Break ◽

Damage Repair ◽

Dna End Resection ◽

Non Homologous End Joining ◽

Transformed Cell Lines ◽

Dna Ends

SUMMARYMultiple pathways mediate the repair of DNA double-strand break (DSB), with numerous mechanisms responsible for driving choice between the pathways. Previously, we reported that phosphorylation of the non-homologous end joining (NHEJ) factor, Ku70, is required for the dissociation of the Ku heterodimer from DNA ends to allow DSB repair via homologous recombination (HR). A knock-in mouse, in which phosphorylation is ablated in the three conserved sites of Ku70 (Ku703A/3A), was generated in order to test the hypothesis that Ku70 phosphorylation is required for initiation of HR and that blocking this process results in enhanced genomic instability and tumorigenesis. Here, we show that Ku703A/3A mice develop spontaneous and have accelerated chemical-induced hepatocellular carcinoma (HCC) compared to wild-type (Ku70+/+) littermates. The HCC tumors from the Ku703A/3A mice have increased γH2AX and 8-oxo-G staining, suggesting DNA repair is decreased in these mice. Spontaneous transformed cell lines from Ku703A/3A mice are more radiosensitive, have a significant decrease in DNA end resection, and are more sensitive to the DNA cross-linking agent mitomycin C compared to cells from Ku70+/+ littermates. Collectively, these findings demonstrate that phosphorylation-mediated dissociation of Ku heterodimer from DNA ends is required for efficient DNA damage repair and disruption of this process results in genomic instability and accelerated development of HCC.

Download Full-text

Effect of enzalutamide on sensitivity in prostate cancer cells to radiation by inhibition of DNA double strand break repair.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.6_suppl.208 ◽

2017 ◽

Vol 35 (6_suppl) ◽

pp. 208-208 ◽

Cited By ~ 2

Author(s):

Maryam Ghashghaei ◽

Thierry Muanza ◽

Miltiadis Paliouras ◽

Tamim Niazi

Keyword(s):

Prostate Cancer ◽

Dna Damage ◽

Dna Damage Repair ◽

Strand Break ◽

Dependent Manner ◽

Castration Resistance ◽

Concurrent Administration ◽

Damage Repair ◽

Pathway Inhibition ◽

Approved Drugs

208 Background: Prostate cancer is the second leading cause of cancer-related deaths amongst men in North America. Data suggests that, following radiation therapy (XRT), androgen receptor (AR) enhances DNA damage repair and contributes to resistance of prostate cancer (PCa) cells to XRT. At present AR-pathway inhibition is the mainstay treatment of metastatic castration resistance prostate cancer (mCRPC). Enzalutamide (ENZA), a potent AR inhibitor is one of the approved drugs in this setting. The purpose of this study was to assess the potential radiosensitization of ENZA and its mechanism of action in hormone resistant PCa cells. Methods: The effect of ENZA alone or in combination with XRT was assessed on hormone-sensitive, (HS: LNCaP, PC3-T877A) and insensitive PCa cells (HI: PC3, PC3-AR V7, C4-2) using viability and clonogenic assays, cell cycle arrest and DNA damage analysis. Results: MTT assay demonstrates that ENZA significantly inhibits the proliferation of HS PCa cells in a dose dependent manner whereas CRPC required ENZA in combination with ADT (androgen deprivation therapy). Additionally, clonogenic assay proves that concurrent administration of ENZA or ADT+ENZA and XRT led to a supra-additive antitumor effect with the dose enhancement factor of 1.76±0.008 in LNCaP, 1.65±0.01 in PC3-T877A and 1.35±0.003 in C4-2 respectively at surviving fraction of 0.1. This effect was not observed in PC3 and PC3-AR V7 cells pre-treated with ENZA (in all cases DEF = 1 at SF = 0.1). Additionally, the level of γH2AX increased in HS cells and CRPC cells treated with ENZA/ADT+ENZA and XRT when compared to XRT alone. The enhanced H2AX activity remained unchanged up to 24 hours after combination treatment. Furthermore, there is an initial inhibition of DNA-PKcs in HS and CRPC cells treated with ENZA/ADT+ENZA administered before XRT. Conclusions: Our data suggest that the higher efficacy of ENZA/ENZA+ADT and XRT could be partially due to inhibition of DNA damage repair. Our results demonstrated a significant enhancement of XRT efficacy and confirms the rational for the ongoing combination clinical trials with XRT.

Download Full-text

Recruitment of the Type B Histone Acetyltransferase Hat1p to Chromatin Is Linked to DNA Double-Strand Breaks

Molecular and Cellular Biology ◽

10.1128/mcb.26.9.3649-3658.2006 ◽

2006 ◽

Vol 26 (9) ◽

pp. 3649-3658 ◽

Cited By ~ 48

Author(s):

Song Qin ◽

Mark R. Parthun

Keyword(s):

Dna Damage ◽

Molecular Mechanisms ◽

Dna Damage Repair ◽

Strand Break ◽

Double Strand Break ◽

Similar Distribution ◽

Tail Domain ◽

Type B ◽

Direct Role ◽

Damage Repair

ABSTRACT Type B histone acetyltransferases are thought to catalyze the acetylation of the NH2-terminal tails of newly synthesized histones. Although Hat1p has been implicated in cellular processes, such as telomeric silencing and DNA damage repair, the underlying molecular mechanisms by which it functions remain elusive. In an effort to understand how Hat1p is involved in the process of DNA double-strand break (DSB) repair, we examined whether Hat1p is directly recruited to sites of DNA damage. Following induction of the endonuclease HO, which generates a single DNA DSB at the MAT locus, we found that Hat1p becomes associated with chromatin near the site of DNA damage. The nuclear Hat1p-associated histone chaperone Hif1p is also recruited to an HO-induced DSB with a similar distribution. In addition, while the acetylation of all four histone H4 NH2-terminal tail domain lysine residues is increased following DSB formation, only the acetylation of H4 lysine 12, the primary target of Hat1p activity, is dependent on the presence of Hat1p. Kinetic analysis of Hat1p localization indicates that it is recruited after the phosphorylation of histone H2A S129 and concomitant with the recombinational-repair factor Rad52p. Surprisingly, Hat1p is still recruited to chromatin in strains that cannot repair an HO-induced double-strand break. These results indicate that Hat1p plays a direct role in DNA damage repair and is responsible for specific changes in histone modification that occur during the course of recombinational DNA repair.

Download Full-text

Persistent DNA Double-Strand Breaks After Repeated Diagnostic CT Scans in Breast Epithelial Cells and Lymphocytes

Frontiers in Oncology ◽

10.3389/fonc.2021.634389 ◽

2021 ◽

Vol 11 ◽

Author(s):

Natalia V. Bogdanova ◽

Nina Jguburia ◽

Dhanya Ramachandran ◽

Nora Nischik ◽

Katharina Stemwedel ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Repair ◽

Cell Types ◽

Strand Break ◽

Ct Scans ◽

Breast Epithelial Cell ◽

Damage Repair ◽

Breast Epithelial ◽

The Impact ◽

Diagnostic Ct

DNA double-strand break (DSB) induction and repair have been widely studied in radiation therapy (RT); however little is known about the impact of very low exposures from repeated computed tomography (CT) scans for the efficiency of repair. In our current study, DSB repair and kinetics were investigated in side-by-side comparison of RT treatment (2 Gy) with repeated diagnostic CT scans (≤20 mGy) in human breast epithelial cell lines and lymphoblastoid cells harboring different mutations in known DNA damage repair proteins. Immunocytochemical analysis of well known DSB markers γH2AX and 53BP1, within 48 h after each treatment, revealed highly correlated numbers of foci and similar appearance/disappearance profiles. The levels of γH2AX and 53BP1 foci after CT scans were up to 30% of those occurring 0.5 h after 2 Gy irradiation. The DNA damage repair after diagnostic CT scans was monitored and quantitatively assessed by both γH2AX and 53BP1 foci in different cell types. Subsequent diagnostic CT scans in 6 and/or 12 weeks intervals resulted in elevated background levels of repair foci, more pronounced in cells that were prone to genomic instability due to mutations in known regulators of DNA damage response (DDR). The levels of persistent foci remained enhanced for up to 6 months. This “memory effect” may reflect a radiation-induced long-term response of cells after low-dose x-ray exposure.

Download Full-text

ThermomiR-377-3p-induced Suppression of Cirbp Expression Is Required for Effective Elimination of Cancer Cells and Cancer Stem-like Cells by Hyperthermia

10.21203/rs.3.rs-917435/v1 ◽

2021 ◽

Author(s):

Tao-Yan Lin ◽

Jun-Shuang Jia ◽

Wei-Ren Luo ◽

Sheng-Jun Xiao ◽

Xiao-Lin Lin ◽

...

Keyword(s):

Dna Damage ◽

Rna Binding ◽

Dna Damage Repair ◽

Ectopic Expression ◽

Killing Effect ◽

Tumor Xenografts ◽

Damage Repair ◽

Tumor Killing ◽

Therapeutic Hyperthermia

Abstract In recent years, the development of adjunctive therapeutic hyperthermia for cancer therapy has received considerable attention. However, the mechanisms underlying hyperthermia resistance are still poorly understood. In this study, we investigated the roles of cold-inducible RNA binding protein (Cirbp) in regulating hyperthermia resistance and underlying mechanisms in nasopharyngeal carcinoma (NPC). Our results firstly revealed that hyperthermia significantly attenuated the stemness property of NPC cells, while combination treatment of hyperthermia and oridonin dramatically increased the killing effect on NPC cells and cancer stem cell (CSC)-like population. Moreover, hyperthermia substantially improved the sensitivity of radiation-resistant NPC cells and CSC-like cells to radiotherapy. Hyperthermia noticeably suppressed Cirbp expression in NPC cells and xenograft tumor tissues. Furthermore, Cirbp inhibition remarkably boosted anti-tumor-killing activity of hyperthermia against NPC cells and CSC-like cells, whereas ectopic expression of Cirbp compromised tumor-killing effect of hyperthermia on these cells, indicating that Cirbp overexpression induces hyperthermia resistance. ThermomiR-377–3p improved the sensitivity of NPC cells and cancer stem-like cells to hyperthermia in vitro by directly suppressing Cirbp expression. More importantly, our results displayed the significantly boosted sensitization of tumor xenografts to hyperthermia by Cirbp silencing in vivo, but ectopic expression of Cirbp nearly completely counteracted hyperthermia-mediated tumor cell-killing effect against tumor xenografts in vivo. Mechanistically, Cirbp silencing-induced inhibition of DNA damage repair by inactivating ATM-Chk2 and ATR-Chk1 pathways, decrease in stemness and increase in cell death contributed to hyperthermic sensitization; conversely, Cirbp overexpression-induced promotion of DNA damage repair, increase in stemness and decrease in cell apoptosis contributed to hyperthermia resistance. Taken together, these findings reveal a previously unrecognized role for Cirbp in positively regulating hyperthermia resistance and suggest that thermomiR-377–3p and its target gene Cirbp represent promising targets for therapeutic hyperthermia.

Download Full-text

Lamin A/C Depletion Enhances DNA Damage-Induced Stalled Replication Fork Arrest

Molecular and Cellular Biology ◽

10.1128/mcb.01676-12 ◽

2013 ◽

Vol 33 (6) ◽

pp. 1210-1222 ◽

Cited By ~ 64

Author(s):

Mayank Singh ◽

Clayton R. Hunt ◽

Raj K. Pandita ◽

Rakesh Kumar ◽

Chin-Rang Yang ◽

...

Keyword(s):

Dna Damage ◽

Replication Fork ◽

Dna Damage Repair ◽

Genomic Stability ◽

Replication Stress ◽

Lamin A ◽

Replicative Stress ◽

Damage Repair ◽

Repair Factor ◽

Recombination Pathway

The humanLMNAgene encodes the essential nuclear envelope proteins lamin A and C (lamin A/C). Mutations inLMNAresult in altered nuclear morphology, but how this impacts the mechanisms that maintain genomic stability is unclear. Here, we report that lamin A/C-deficient cells have a normal response to ionizing radiation but are sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with ICL agents (cisplatin, camptothecin, and mitomycin), lamin A/C-deficient cells displayed normal γ-H2AX focus formation but a higher frequency of cells with delayed γ-H2AX removal, decreased recruitment of the FANCD2 repair factor, and a higher frequency of chromosome aberrations. Similarly, following hydroxyurea-induced replication stress, lamin A/C-deficient cells had an increased frequency of cells with delayed disappearance of γ-H2AX foci and defective repair factor recruitment (Mre11, CtIP, Rad51, RPA, and FANCD2). Replicative stress also resulted in a higher frequency of chromosomal aberrations as well as defective replication restart. Taken together, the data can be interpreted to suggest that lamin A/C has a role in the restart of stalled replication forks, a prerequisite for initiation of DNA damage repair by the homologous recombination pathway, which is intact in lamin A/C-deficient cells. We propose that lamin A/C is required for maintaining genomic stability following replication fork stalling, induced by either ICL damage or replicative stress, in order to facilitate fork regression prior to DNA damage repair.

Download Full-text

Abstract 125: Proteome Analysis of a Regulatory Pathway of Pathologic Vascular Injury as Mediated by KLF5 and its Transcriptional Complexes

Circulation ◽

10.1161/circ.116.suppl_16.ii_2-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Kenichi Aizawa ◽

Toru Suzuki ◽

Takayoshi Mastumura ◽

Nanae Kada ◽

Daigo Sawaki ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Repair ◽

Proteome Analysis ◽

Strand Break ◽

Double Strand Break ◽

Chain Gene ◽

Repair System ◽

Peptide Mass ◽

Damage Repair ◽

A Chain

Background: Transcription factor Krüppel-like factor 5 (KLF5) is a key element linking external stress and cardiovascular remodeling by up-regulating platelet derived growth factor (PDGF)-A chain gene activity. However, the underlying mechanisms remain to be elucidated. The unambiguous and comprehensive identification of interacting proteins is crucial for understanding these mechanisms. In the present study, we identified interacting factors of KLF5 by proteomic analysis and characterized their regulation in the vascular pathogenic response. Methods&Results: Double-stranded oligonucleotide containing the binding sequence for KLF5 in the PDGF-A promoter was synthesized and attached to metal beads, to which cell nuclear extract was applied. SDS-PAGE visualized specific bands to the sequence, which were subjected to in-gel digestion and peptide mass fingerprinting by MALDI-TOF/MS spectrometry. Factors that are known to be important in the DNA damage/repair pathway were successively identified. We therefore examined the involvement of the complex in vascular pathologies. Double-strand break as determined by immunohistochemistry using γ-H2AX antibody, a marker of activation of the double-stranded DNA damage/repair response, was observed in pathogenically stimulated vascular endothelial cells (HUVEC) and neointimal tissues in rat carotid artery balloon injury model. Further, KLF5 was shown to mediate the response on γ-H2AX as shown by co-immunoprecipitation and confocal microscopy. Discussion: We show a hitherto unknown regulatory mechanism by DNA double-strand break/repair system involving KLF5 in the vascular pathogenic response. Our findings might provide a clue to understanding the initiation of pathological cell proliferation observed in atherosclerosis or restenosis after coronary intervention. This new pathway might also be a tempting target for therapeutic intervention aimed at modulating the activity of KLF5 upon PDGF-A chain and its associated pathologies in the cardiovascular system.

Download Full-text

Nuclear localization of Beclin 1 promotes radiation-induced DNA damage repair independent of autophagy

Scientific Reports ◽

10.1038/srep45385 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 13

Author(s):

Fei Xu ◽

Yixuan Fang ◽

Lili Yan ◽

Lan Xu ◽

Suping Zhang ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Repair ◽

Strand Break ◽

Beclin 1 ◽

Dsb Repair ◽

Dna Topoisomerase ◽

Damage Repair ◽

Dna Double Strand Break ◽

Radiation Induced

Abstract Beclin 1 is a well-established core mammalian autophagy protein that is embryonically indispensable and has been presumed to suppress oncogenesis via an autophagy-mediated mechanism. Here, we show that Beclin 1 is a prenatal primary cytoplasmic protein but rapidly relocated into the nucleus during postnatal development in mice. Surprisingly, deletion of beclin1 in in vitro human cells did not block an autophagy response, but attenuated the expression of several DNA double-strand break (DSB) repair proteins and formation of repair complexes, and reduced an ability to repair DNA in the cells exposed to ionizing radiation (IR). Overexpressing Beclin 1 improved the repair of IR-induced DSB, but did not restore an autophagy response in cells lacking autophagy gene Atg7, suggesting that Beclin 1 may regulate DSB repair independent of autophagy in the cells exposed to IR. Indeed, we found that Beclin 1 could directly interact with DNA topoisomerase IIβ and was recruited to the DSB sites by the interaction. These findings reveal a novel function of Beclin 1 in regulation of DNA damage repair independent of its role in autophagy particularly when the cells are under radiation insult.

Download Full-text

DNA Damage Repair and DNA Methylation in the Kidney

American Journal of Nephrology ◽

10.1159/000501356 ◽

2019 ◽

Vol 50 (2) ◽

pp. 81-91 ◽

Cited By ~ 4

Author(s):

Kaori Hayashi ◽

Akihito Hishikawa ◽

Hiroshi Itoh

Keyword(s):

Dna Methylation ◽

Dna Damage ◽

Dna Repair ◽

Methylation Status ◽

Dna Damage Repair ◽

Strand Break ◽

Repair System ◽

Damage Repair ◽

Dna Double Strand Break ◽

Cell Type Specific

The DNA repair system is essential for the maintenance of genome integrity and is mainly investigated in the areas of aging and cancer. The DNA repair system is strikingly cell-type specific, depending on the expression of DNA repair factors; therefore, different DNA repair systems may exist in each type of kidney cell. Importance of DNA repair in the kidney is suggested by renal phenotypes caused by both genetic mutations in the DNA repair pathway and increased stimuli of DNA damage. Recently, we reported the importance of DNA double-strand break repair in glomerular podocytes and its involvement in the alteration of DNA methylation status, which regulates podocyte phenotypes. In this review, we summarize the roles of the DNA repair system in the kidneys and possible associations with altered kidney DNA methylation, which have been infrequently reported together. Investigations of DNA damage repair and epigenetic changes in the kidneys may achieve a profound understanding of kidney aging and diseases.

Download Full-text

The nuclear interactome of DYRK1A reveals a functional role in DNA damage repair

10.1101/432757 ◽

2018 ◽

Author(s):

Steven E. Guard ◽

Zachary C. Poss ◽

Christopher C. Ebmeier ◽

Maria Pagratis ◽

Dylan J. Taatjes ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Repair ◽

Nuclear Interaction ◽

Autism Spectrum ◽

Transcriptional Elongation ◽

Loss Of Function ◽

Neurocognitive Deficits ◽

Dna Double Strand Breaks ◽

Homology Directed Repair ◽

Damage Repair

AbstractLoss of function mutations in the protein kinase DYRK1A lead to a syndromic form of autism spectrum disorder and intellectual disability. Conversely, increased DYRK1A dosage is implicated in atypical brain development and neurocognitive deficits in trisomy 21. DYRK1A regulates a diverse array of cellular processes through kinase dependent and independent interactions with substrates and binding partners. Recent evidence implicates DYRK1A in direct regulation of the transcriptional machinery, but many of the molecular details are not yet known. Furthermore, the landscape of DYRK1A interactions in the nucleus is incomplete, impeding progress toward understanding its function in transcription. Here, we used immunoaffinity purification and mass spectrometry to identify nuclear interaction partners of endogenous DYRK1A. These were enriched in DNA damage repair factors, transcriptional elongation factors and E3 ubiquitin ligases. We validated an interaction with RNF169, a factor that promotes homology directed repair upon DNA damage. We further show that knockout of DYRK1A or treatment with DYRK1A inhibitors in HeLa cells impaired efficient recruitment of 53BP1 to DNA double strand breaks induced by ionizing radiation. This nuclear interactome thus reveals a new role for DYRK1A in DNA damage repair and provides a resource for exploring new functions of DYRK1A in the nucleus.

Download Full-text