scholarly journals Topoisomerase 2β Induces DNA Breaks To Regulate Human Papillomavirus Replication

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Paul Kaminski ◽  
Shiyuan Hong ◽  
Takeyuki Kono ◽  
Paul Hoover ◽  
Laimonis Laimins
Keyword(s):  
Dna Damage ◽  
Human Papillomavirus ◽  
Genome Replication ◽  
Type Ii ◽  
Torsional Stress ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Damage Repair ◽  
Topologically Associated Domains ◽  
Repair Pathways

ABSTRACT Topoisomerases regulate higher-order chromatin structures through the transient breaking and religating of one or both strands of the phosphodiester backbone of duplex DNA. TOP2β is a type II topoisomerase that induces double-strand DNA breaks at topologically associated domains (TADS) to relieve torsional stress arising during transcription or replication. TADS are anchored by CCCTC-binding factor (CTCF) and SMC1 cohesin proteins in complexes with TOP2β. Upon DNA cleavage, a covalent intermediate DNA-TOP2β (TOP2βcc) is transiently generated to allow for strand passage. The tyrosyl-DNA phosphodiesterase TDP2 can resolve TOP2βcc, but failure to do so quickly can lead to long-lasting DNA breaks. Given the role of CTCF/SMC1 proteins in the human papillomavirus (HPV) life cycle, we investigated whether TOP2β proteins contribute to HPV pathogenesis. Our studies demonstrated that levels of both TOP2β and TDP2 were substantially increased in cells with high-risk HPV genomes, and this correlated with large amounts of DNA breaks. Knockdown of TOP2β with short hairpin RNAs (shRNAs) reduced DNA breaks by over 50% as determined through COMET assays. Furthermore, this correlated with substantially reduced formation of repair foci such as phosphorylated H2AX (γH2AX), phosphorylated CHK1 (pCHK1), and phosphorylated SMC1 (pSMC1) indicative of impaired activation of DNA damage repair pathways. Importantly, knockdown of TOP2β also blocked HPV genome replication. Our previous studies demonstrated that CTCF/SMC1 factors associate with HPV genomes at sites in the late regions of HPV31, and these correspond to regions that also bind TOP2β. This study identifies TOP2β as responsible for enhanced levels of DNA breaks in HPV-positive cells and as a regulator of viral replication. IMPORTANCE High-risk human papillomaviruses (HPVs) infect epithelial cells and induce viral genome amplification upon differentiation. HPV proteins activate DNA damage repair pathways by inducing high numbers of DNA breaks in both viral and cellular DNAs. This activation is required for HPV genome replication. TOP2β is a type II topoisomerase that induces double-strand DNA breaks at topologically associated domains (TADS) to relieve torsional stress arising during transcription or replication. Our studies demonstrate that TOP2β levels are increased in HPV-positive cells and that this is required for HPV replication. Importantly, our studies further show that knockdown of TOP2β reduces the number of breaks by over 50% in HPV-positive cells and that this correlates with substantially impaired activation of DNA repair pathways. This study identifies a critical mechanism by which HPV replication is regulated by the topoisomerase TOP2β through DNA break formation.

scholarly journals Topoisomerase 2b induces DNA breaks to regulate human papillomavirus replication

2021 ◽  
Author(s):  
Paul Kaminski ◽  
Shiyuan Hong ◽  
Takeyuki Kono ◽  
Paul Hoover ◽  
Laimonis A. Laimins
Keyword(s):  
Dna Cleavage ◽  
Genome Replication ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Damage Repair ◽  
High Risk Hpv ◽  
Strand Passage ◽  
Covalent Intermediate ◽  
Do So

Topoisomerases regulate higher order chromatin structures through the transient breaking and re-ligating of one or both strands of the phosphodiester backbone of duplex DNA. TOP2b is a type II topoisomerase that induces double strand DNA breaks at topological-associated domains (TADS) to relieve torsional stress arising during transcription or replication. TADS are anchored by CTCF and SMC1 cohesin proteins in complexes with TOP2b. Upon DNA cleavage a covalent intermediate DNA-TOP2b (TOP2bcc) is transiently generated to allow for strand passage. The tyrosyl-DNA phosphodiesterase TDP2 can resolve TOP2bcc but failure to do so quickly can lead to long-lasting DNA breaks. Given the role of CTCF/SMC1 proteins in the HPV life cycle we investigated if TOP2b proteins contribute to HPV pathogenesis. Our studies demonstrated that levels of both TOP2b and TDP2 were substantially increased in cells with high risk HPV genomes and this correlated with high amounts of DNA breaks. Knockdown of TOP2b with shRNAs reduced DNA breaks by over 50% as determined through COMET assays.  Furthermore this correlated with substantially reduced formation of repair foci such as gH2AX, pCHK1 and pSMC1 indicative of impaired activation of DNA damage repair pathways. Importantly, knockdown of TOP2b also blocked HPV genome replication. Our previous studies demonstrated that CTCF /SMC1 factors associate with HPV genomes at sites in the late regions of HPV31 and these correspond to regions that also bind TOP2b. This study identifies TOP2b as responsible for enhanced levels of DNA breaks in HPV positive cells and as a regulator of viral replication.

366-OR: TCF19 Promotes Functional Beta-Cell Mass and Proliferative Capacity by Regulating DNA Damage Repair Pathways

Diabetes ◽  
2019 ◽  
Vol 68 (Supplement 1) ◽  
pp. 366-OR
Author(s):  
GRACE H. YANG ◽  
JEE YOUNG HAN ◽  
SUKANYA LODH ◽  
JOSEPH T. BLUMER ◽  
DANIELLE FONTAINE ◽  
...  
Keyword(s):  
Dna Damage ◽  
Beta Cell ◽  
Dna Damage Repair ◽  
Cell Mass ◽  
Beta Cell Mass ◽  
Proliferative Capacity ◽  
Damage Repair ◽  
Repair Pathways

scholarly journals Targeting DNA Replication Stress and DNA Double-Strand Break Repair for Optimizing SCLC Treatment

Cancers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1289 ◽  
Author(s):  
Xing Bian ◽  
Wenchu Lin
Keyword(s):  
Lung Cancer ◽  
Dna Damage ◽  
Dna Replication ◽  
Dna Damage Repair ◽  
Predictive Biomarkers ◽  
Replication Stress ◽  
Genotoxic Stress ◽  
Repair System ◽  
Damage Repair ◽  
Repair Pathways

Small cell lung cancer (SCLC), accounting for about 15% of all cases of lung cancer worldwide, is the most lethal form of lung cancer. Despite an initially high response rate of SCLC to standard treatment, almost all patients are invariably relapsed within one year. Effective therapeutic strategies are urgently needed to improve clinical outcomes. Replication stress is a hallmark of SCLC due to several intrinsic factors. As a consequence, constitutive activation of the replication stress response (RSR) pathway and DNA damage repair system is involved in counteracting this genotoxic stress. Therefore, therapeutic targeting of such RSR and DNA damage repair pathways will be likely to kill SCLC cells preferentially and may be exploited in improving chemotherapeutic efficiency through interfering with DNA replication to exert their functions. Here, we summarize potentially valuable targets involved in the RSR and DNA damage repair pathways, rationales for targeting them in SCLC treatment and ongoing clinical trials, as well as possible predictive biomarkers for patient selection in the management of SCLC.

Roles of Caenorhabditis elegans WRN Helicase in DNA Damage Responses, and a Comparison with Its Mammalian Homolog: A Mini-Review

Gerontology ◽  
2015 ◽  
Vol 62 (3) ◽  
pp. 296-303 ◽  
Author(s):  
Jin-Sun Ryu ◽  
Hyeon-Sook Koo
Keyword(s):  
Dna Damage ◽  
Caenorhabditis Elegans ◽  
Werner Syndrome ◽  
Model Organisms ◽  
Current Status ◽  
Helicase Domain ◽  
Replication Forks ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Double Strand Dna

Werner syndrome protein (WRN) is unusual among RecQ family DNA helicases in having an additional exonuclease activity. WRN is involved in the repair of double-strand DNA breaks via the homologous recombination and nonhomologous end joining pathways, and also in the base excision repair pathway. In addition, the protein promotes the recovery of stalled replication forks. The helicase activity is thought to unwind DNA duplexes, thereby moving replication forks or Holliday junctions. The targets of the exonuclease could be the nascent DNA strands at a replication fork or the ends of double-strand DNA breaks. However, it is not clear which enzyme activities are essential for repairing different types of DNA damage. Model organisms such as mice, flies, and worms deficient in WRN homologs have been investigated to understand the physiological results of defects in WRN activity. Premature aging, the most remarkable characteristic of Werner syndrome, is also seen in the mutant mice and worms, and hypersensitivity to DNA damage has been observed in WRN mutants of all three model organisms, pointing to conservation of the functions of WRN. In the nematode Caenorhabditis elegans, the WRN homolog contains a helicase domain but no exonuclease domain, so that this animal is very useful for studying the in vivo functions of the helicase without interference from the activity of the exonuclease. Here, we review the current status of investigations of C. elegans WRN-1 and discuss its functional differences from the mammalian homologs.

scholarly journals TDP-43 mutations link Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage

PLoS Genetics ◽  
2020 ◽  
Vol 16 (12) ◽  
pp. e1009260
Author(s):  
Marta Giannini ◽  
Aleix Bayona-Feliu ◽  
Daisy Sproviero ◽  
Sonia I. Barroso ◽  
Cristina Cereda ◽  
...  
Keyword(s):  
Dna Damage ◽  
Amyotrophic Lateral Sclerosis ◽  
Rna Binding ◽  
Neurodegenerative Disorder ◽  
Genome Instability ◽  
Neuronal Model ◽  
Dna Breaks ◽  
Dna And Rna ◽  
Double Strand Dna Breaks ◽  
Lateral Sclerosis

TDP-43 is a DNA and RNA binding protein involved in RNA processing and with structural resemblance to heterogeneous ribonucleoproteins (hnRNPs), whose depletion sensitizes neurons to double strand DNA breaks (DSBs). Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, in which 97% of patients are familial and sporadic cases associated with TDP-43 proteinopathies and conditions clearing TDP-43 from the nucleus, but we know little about the molecular basis of the disease. After showing with the non-neuronal model of HeLa cells that TDP-43 depletion increases R loops and associated genome instability, we prove that mislocalization of mutated TDP-43 (A382T) in transfected neuronal SH-SY5Y and lymphoblastoid cell lines (LCLs) from an ALS patient cause R-loop accumulation, R loop-dependent increased DSBs and Fanconi Anemia repair centers. These results uncover a new role of TDP-43 in the control of co-transcriptional R loops and the maintenance of genome integrity by preventing harmful R-loop accumulation. Our findings thus link TDP-43 pathology to increased R loops and R loop-mediated DNA damage opening the possibility that R-loop modulation in TDP-43-defective cells might help develop ALS therapies.

scholarly journals A Novel Role for Cathepsin S as a Potential Biomarker in Triple Negative Breast Cancer

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Richard D. A. Wilkinson ◽  
Roberta E. Burden ◽  
Sara H. McDowell ◽  
Darragh G. McArt ◽  
Stephen McQuaid ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Dna Damage ◽  
Adjuvant Therapy ◽  
Dna Damage Repair ◽  
Cathepsin S ◽  
Damage Repair ◽  
Potential Biomarker ◽  
Repair Pathways ◽  
Improved Outcome

Cathepsin S (CTSS) has previously been implicated in a number of cancer types, where it is associated with poor clinical features and outcome. To date, patient outcome in breast cancer has not been examined with respect to this protease. Here, we carried out immunohistochemical (IHC) staining of CTSS using a breast cancer tissue microarray in patients who received adjuvant therapy. We scored CTSS expression in the epithelial and stromal compartments and evaluated the association of CTSS expression with matched clinical outcome data. We observed differences in outcome based on CTSS expression, with stromal-derived CTSS expression correlating with a poor outcome and epithelial CTSS expression associated with an improved outcome. Further subtype characterisation revealed high epithelial CTSS expression in TNBC patients with improved outcome, which remained consistent across two independent TMA cohorts. Furtherin silicogene expression analysis, using both in-house and publicly available datasets, confirmed these observations and suggested high CTSS expression may also be beneficial to outcome in ER-/HER2+ cancer. Furthermore, high CTSS expression was associated with the BL1 Lehmann subgroup, which is characterised by defects in DNA damage repair pathways and correlates with improved outcome. Finally, analysis of matching IHC analysis reveals an increased M1 (tumour destructive) polarisation in macrophage in patients exhibiting high epithelial CTSS expression. In conclusion, our observations suggest epithelial CTSS expression may be prognostic of improved outcome in TNBC. Improved outcome observed with HER2+ at the gene expression level furthermore suggests CTSS may be prognostic of improved outcome in ER- cancers as a whole. Lastly, from the context of these patients receiving adjuvant therapy and as a result of its association with BL1 subgroup CTSS may be elevated in patients with defects in DNA damage repair pathways, indicating it may be predictive of tumour sensitivity to DNA damaging agents.

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