scholarly journals Genomic patterns of transcription-replication interactions in mouse primary B cells

2021 ◽  
Author(s):  
Commodore P St Germain ◽  
Hongchang Zhao ◽  
Vrishti Sinha ◽  
Lionel A Sanz ◽  
Frederic Chedin ◽  
...  
Keyword(s):  
B Cells ◽  
Genome Stability ◽  
Protein A ◽  
Replication Stress ◽  
Tumor Formation ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Age Related ◽  
Novel Method ◽  
Genomic Locations

Conflicts between transcription and replication machinery are a potent source of replication stress and genome stability; however, no technique currently exists to identify endogenous genomic locations prone to transcription-replication interactions. Here, we report a novel method to identify genomic loci prone to transcription-replication interactions termed transcription-replication immunoprecipitation on nascent DNA sequencing, TRIPn-Seq. TRIPn-Seq employs the sequential immunoprecipitation of RNA polymerase 2 phosphorylated at serine 5 (RNAP2s5) followed by enrichment of nascent DNA previously labeled with bromodeoxyuridine. Using TRIPn-Seq, we mapped 1,009 unique transcription-replication interactions (TRIs) in mouse primary B cells characterized by a bimodal pattern of RNAP2s5, bidirectional transcription, an enrichment of RNA:DNA hybrids, and a high probability of forming G-quadruplexes. While TRIs themselves map to early replicating regions, they exhibit enhanced Replication Protein A association and replication fork termination, marks of replication stress. TRIs colocalize with double-strand DNA breaks, are enriched for deletions, and accumulate mutations in tumors. We propose that replication stress at TRIs induces mutations potentially contributing to age-related disease, as well as tumor formation and development.

scholarly journals Inhibition of tryptophan 2,3-dioxygenase impairs DNA damage tolerance and repair in glioma cells

2020 ◽  
Author(s):  
Megan R. Reed ◽  
Leena Maddukuri ◽  
Amit Ketkar ◽  
Stephanie D. Byrum ◽  
Maroof K. Zafar ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genome Stability ◽  
Protein A ◽  
Treatment Strategies ◽  
Glioma Cells ◽  
Replication Stress ◽  
Dna Breaks ◽  
Aberrant Expression ◽  
Aryl Hydrocarbon ◽  
Proliferative Effect

ABSTRACTAberrant expression of tryptophan 2,3-dioxygenase (TDO) is a determinant of malignancy and immune response in gliomas in part through kynurenine (KYN)-mediated activation of the aryl hydrocarbon receptor (AhR). In the current study, we investigated the hypothesis that TDO activation in gliomas has a broad impact upon genome maintenance - promoting tolerance of replication stress (RS) and repair of DNA damage. We report that inhibition of TDO activity attenuated recovery from hydroxyurea (HU)-induced RS and increased the genotoxic effects of bis-chloroethylnitrosourea (BCNU), as fork progress was impeded when TDO-deficient glioma cells were treated with BCNU. Activation of the Chk1 arm of the replication stress response (RSR) was reduced when TDO activity was blocked prior to treatment with BCNU, whereas phosphorylation of serine 33 (pS33) on replication protein A (RPA) was enhanced – indicative of increased fork collapse. Restoration of KYN levels protected against some replication-associated effects of BCNU. Inhibition of TDO activity had a strong anti-proliferative effect on glioma-derived cells – enhancing the cytotoxic effects of BCNU. Analysis of results obtained using quantitative proteomics revealed TDO-dependent changes in several signaling pathways – including down-regulation of DNA repair factors and sirtuin signaling. Consistent with these observations, inhibition of TDO diminished SIRT7 recruitment to chromatin, which increased histone H3K18 acetylation – a key mark involved in 53BP1 recruitment to sites of DNA damage. Cells lacking TDO activity exhibited defective recruitment of 53BP1 to gH2AX foci, which corresponded with delayed repair of BCNU-induced DNA breaks. Addition of exogenous KYN increased the rate of break repair. The discovery that TDO activity modulates sensitivity to DNA damage by fueling SIRT7/53BP1 localization to chromatin and repair of BCNU-induced DNA damage highlights the potential for tumor-specific metabolic changes to influence genome stability and may have implications for glioma biology and treatment strategies.

Merlin-Deficient Schwann Cells Are More Susceptible to Radiation Injury than Normal Schwann Cells In Vitro

2021 ◽  
Author(s):  
Erin Cohen ◽  
Stefanie Pena ◽  
Christine Mei ◽  
Olena Bracho ◽  
Brian Marples ◽  
...  
Keyword(s):  
Schwann Cells ◽  
Protein A ◽  
Gene Mutations ◽  
Tumor Control ◽  
Radiation Toxicity ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Adjacent Structures ◽  
Dna Repair Protein

Abstract Objectives Vestibular schwannomas (VS) are intracranial tumors, which are caused by NF2 gene mutations that lead to loss of merlin protein. A treatment for VS is stereotactic radiosurgery, a form of radiation. To better understand the radiobiology of VS and radiation toxicity to adjacent structures, our main objectives were (1) investigate effects of single fraction (SF) radiation on viability, cytotoxicity, and apoptosis in normal Schwann cells (SCs) and merlin-deficient Schwann cells (MD-SCs) in vitro, and (2) analyze expression of double strand DNA breaks (γ-H2AX) and DNA repair protein Rad51 following irradiation. Study Design This is a basic science study. Setting This study is conducted in a research laboratory. Participants Patients did not participate in this study. Main Outcome Measures In irradiated normal SCs and MD-SCs (0–18 Gy), we measured (1) viability, cytotoxicity, and apoptosis using cell-based assays, and (2) percentage of cells with γ-H2AX and Rad51 on immunofluorescence. Results A high percentage of irradiated MD-SCs expressed γ-H2AX, which may explain the dose-dependent losses in viability in rodent and human cell lines. In comparison, the viabilities of normal SCs were only compromised at higher doses of radiation (>12 Gy, human SCs), which may be related to less Rad51 repair. There were no further reductions in viability in human MD-SCs beyond 9 Gy, suggesting that <9 Gy may be insufficient to initiate maximal tumor control. Conclusion The MD-SCs are more susceptible to radiation than normal SCs, in part through differential expression of γ-H2AX and Rad51. Understanding the radiobiology of MD-SCs and normal SCs is important for optimizing radiation protocols to maximize tumor control while limiting radiation toxicity in VS patients.

scholarly journals DDX5 resolves R-loops at DNA double-strand breaks to promote DNA repair and avoid chromosomal deletions

NAR Cancer ◽  
2020 ◽  
Vol 2 (3) ◽  
Author(s):  
Zhenbao Yu ◽  
Sofiane Y Mersaoui ◽  
Laure Guitton-Sert ◽  
Yan Coulombe ◽  
Jingwen Song ◽  
...  
Keyword(s):  
Dna Repair ◽  
Protein A ◽  
Helicase Domain ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Rna Transcripts ◽  
Chromosomal Deletions ◽  
Double Strand Dna Breaks ◽  
Dna Strand

Abstract R-loops are three-stranded structures consisting of a DNA/RNA hybrid and a displaced DNA strand. The regulatory factors required to process this fundamental genetic structure near double-strand DNA breaks (DSBs) are not well understood. We previously reported that cellular depletion of the ATP-dependent DEAD box RNA helicase DDX5 increases R-loops genome-wide causing genomic instability. In this study, we define a pivotal role for DDX5 in clearing R-loops at or near DSBs enabling proper DNA repair to avoid aberrations such as chromosomal deletions. Remarkably, using the non-homologous end joining reporter gene (EJ5-GFP), we show that DDX5-deficient U2OS cells exhibited asymmetric end deletions on the side of the DSBs where there is overlap with a transcribed gene. Cross-linking and immunoprecipitation showed that DDX5 bound RNA transcripts near DSBs and required its helicase domain and the presence of DDX5 near DSBs was also shown by chromatin immunoprecipitation. DDX5 was excluded from DSBs in a transcription- and ATM activation-dependent manner. Using DNA/RNA immunoprecipitation, we show DDX5-deficient cells had increased R-loops near DSBs. Finally, DDX5 deficiency led to delayed exonuclease 1 and replication protein A recruitment to laser irradiation-induced DNA damage sites, resulting in homologous recombination repair defects. Our findings define a role for DDX5 in facilitating the clearance of RNA transcripts overlapping DSBs to ensure proper DNA repair.

scholarly journals Direct, sensitive and specific detection of individual single- or double-strand DNA breaks by fluorescence microscopy

2019 ◽  
Author(s):  
Magdalena Kordon ◽  
Mirosław Zarębski ◽  
Kamil Solarczyk ◽  
Hanhui Ma ◽  
Thoru Pederson ◽  
...  
Keyword(s):  
Dna Damage ◽  
Fluorescence Microscopy ◽  
Single Cells ◽  
Primary Cultures ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Damage Site ◽  
Double Strand Dna Breaks ◽  
Age Related ◽  
Double Strand Dna

ABSTRACTWe here describe a technique termed STRIDE (SensiTive Recognition of Individual DNA Ends), which enables highly sensitive, specific, direct in situ detection of single- or double-strand DNA breaks (sSTRIDE or dSTRIDE), in nuclei of single cells, using fluorescence microscopy. Sensitivity of STRIDE was tested using specially developed CRISPR/Cas9 DNA damage induction system, capable of inducing small clusters or individual single- or double-strand breaks. STRIDE exhibits significantly higher sensitivity and specificity of detection of DNA breaks than the commonly used TUNEL assay or methods based on monitoring of recruitment of repair proteins or histone modifications at the damage site (e.g. γH2AX). Even individual genome site-specific DNA double-strand cuts induced by CRISPR/Cas9, as well as individual single-strand DNA scissions induced by the nickase version of Cas9, can be detected by STRIDE and precisely localized within the cell nucleus. We further show that STRIDE can detect low-level spontaneous DNA damage, including age-related DNA lesions, DNA breaks induced by several agents (bleomycin, doxorubicin, topotecan, hydrogen peroxide, UV, photosensitized reactions), and fragmentation of DNA in human spermatozoa. STRIDE methods are potentially useful in studies of mechanisms of DNA damage induction and repair in cell lines and primary cultures, including cells with impaired repair mechanisms.

scholarly journals Phospho-dependent recruitment of the yeast NuA4 acetyltransferase complex by MRX at DNA breaks regulates RPA dynamics during resection

2018 ◽  
Vol 115 (40) ◽  
pp. 10028-10033 ◽  
Author(s):  
Xue Cheng ◽  
Olivier Jobin-Robitaille ◽  
Pierre Billon ◽  
Rémi Buisson ◽  
Hengyao Niu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genome Stability ◽  
Protein A ◽  
Chromatin Modification ◽  
Cell Renewal ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Nonhistone Proteins ◽  
Ssdna Binding ◽  
Stem Cell Renewal

The KAT5 (Tip60/Esa1) histone acetyltransferase is part of NuA4, a large multifunctional complex highly conserved from yeast to mammals that targets lysines on H4 and H2A (X/Z) tails for acetylation. It is essential for cell viability, being a key regulator of gene expression, cell proliferation, and stem cell renewal and an important factor for genome stability. The NuA4 complex is directly recruited near DNA double-strand breaks (DSBs) to facilitate repair, in part through local chromatin modification and interplay with 53BP1 during the DNA damage response. While NuA4 is detected early after appearance of the lesion, its precise mechanism of recruitment remains to be defined. Here, we report a stepwise recruitment of yeast NuA4 to DSBs first by a DNA damage-induced phosphorylation-dependent interaction with the Xrs2 subunit of the Mre11-Rad50-Xrs2 (MRX) complex bound to DNA ends. This is followed by a DNA resection-dependent spreading of NuA4 on each side of the break along with the ssDNA-binding replication protein A (RPA). Finally, we show that NuA4 can acetylate RPA and regulate the dynamics of its binding to DNA, hence targeting locally both histone and nonhistone proteins for lysine acetylation to coordinate repair.

scholarly journals 53BP1 and BRCA1 control pathway choice for stalled replication restart

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yixi Xu ◽  
Shaokai Ning ◽  
Zheng Wei ◽  
Ran Xu ◽  
Xinlin Xu ◽  
...  
Keyword(s):  
Genome Stability ◽  
Dna Breaks ◽  
Dsb Repair ◽  
Independent Manner ◽  
Double Strand Dna Breaks ◽  
Replication Restart ◽  
Tumor Prevention ◽  
Pathway Choice ◽  
Break Induced Replication ◽  
Stalled Replication Forks

The cellular pathways that restart stalled replication forks are essential for genome stability and tumor prevention. However, how many of these pathways exist in cells and how these pathways are selectively activated remain unclear. Here, we describe two major fork restart pathways, and demonstrate that their selection is governed by 53BP1 and BRCA1, which are known to control the pathway choice to repair double-strand DNA breaks (DSBs). Specifically, 53BP1 promotes a fork cleavage-free pathway, whereas BRCA1 facilitates a break-induced replication (BIR) pathway coupled with SLX-MUS complex-mediated fork cleavage. The defect in the first pathway, but not DSB repair, in a 53BP1 mutant is largely corrected by disrupting BRCA1, and vice versa. Moreover, PLK1 temporally regulates the switch of these two pathways through enhancing the assembly of the SLX-MUS complex. Our results reveal two distinct fork restart pathways, which are antagonistically controlled by 53BP1 and BRCA1 in a DSB repair-independent manner.

scholarly journals Polζ ablation in B cells impairs the germinal center reaction, class switch recombination, DNA break repair, and genome stability

2009 ◽  
Vol 206 (2) ◽  
pp. 477-490 ◽  
Author(s):  
Dominik Schenten ◽  
Sven Kracker ◽  
Gloria Esposito ◽  
Sonia Franco ◽  
Ulf Klein ◽  
...  
Keyword(s):  
B Cells ◽  
Genome Stability ◽  
Somatic Hypermutation ◽  
Class Switch Recombination ◽  
Dna Breaks ◽  
Dsb Repair ◽  
Direct Role ◽  
Class Switch

Polζ is an error-prone DNA polymerase that is critical for embryonic development and maintenance of genome stability. To analyze its suggested role in somatic hypermutation (SHM) and possible contribution to DNA double-strand break (DSB) repair in class switch recombination (CSR), we ablated Rev3, the catalytic subunit of Polζ, selectively in mature B cells in vivo. The frequency of somatic mutation was reduced in the mutant cells but the pattern of SHM was unaffected. Rev3-deficient B cells also exhibited pronounced chromosomal instability and impaired proliferation capacity. Although the data thus argue against a direct role of Polζ in SHM, Polζ deficiency directly interfered with CSR in that activated Rev3-deficient B cells exhibited a reduced efficiency of CSR and an increased frequency of DNA breaks in the immunoglobulin H locus. Based on our results, we suggest a nonredundant role of Polζ in DNA DSB repair through nonhomologous end joining.

scholarly journals Hepatitis C Virus E2-CD81 Interaction Induces Hypermutation of the Immunoglobulin Gene in B Cells

2005 ◽  
Vol 79 (13) ◽  
pp. 8079-8089 ◽  
Author(s):  
Keigo Machida ◽  
Kevin T.-H. Cheng ◽  
Nicole Pavio ◽  
Vicky M.-H. Sung ◽  
Michael M. C. Lai
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
B Cells ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Immunoglobulin Gene ◽  
Dna Breaks ◽  
Necrosis Factor Alpha ◽  
Double Strand Dna Breaks ◽  
Double Strand Dna

ABSTRACT Hepatitis C virus (HCV) is one of the leading causes of chronic liver diseases and B-lymphocyte proliferative disorders, including mixed cryoglobulinemia and B-cell lymphoma. It has been suggested that HCV infects human cells through the interaction of its envelope glycoprotein E2 with a tetraspanin molecule CD81, the putative viral receptor. Here, we show that the engagement of B cells by purified E2 induced double-strand DNA breaks specifically in the variable region of immunoglobulin (VH ) gene locus, leading to hypermutation in the VH genes of B cells. Other gene loci were not affected. Preincubation with the anti-CD81 monoclonal antibody blocked this effect. E2-CD81 interaction on B cells triggered the enhanced expression of activation-induced cytidine deaminase (AID) and also stimulated the production of tumor necrosis factor alpha. Knockdown of AID by the specific small interfering RNA blocked the E2-induced double-strand DNA breaks and hypermutation of the VH gene. These findings suggest that HCV infection, through E2-CD81 interaction, may modulate host's innate or adaptive immune response by activation of AID and hypermutation of immunoglobulin gene in B cells, leading to HCV-associated B-cell lymphoproliferative diseases.

scholarly journals The shrinking arterial genome: role of double strand DNA breaks in age‐related DNA deletion (912.9)

2014 ◽  
Vol 28 (S1) ◽  
Author(s):  
Richard Morgan ◽  
Stephen Ives ◽  
Richard Cawthon ◽  
Robert Andtbacka ◽  
Dirk Noyes ◽  
...  
Keyword(s):  
Dna Breaks ◽  
Dna Deletion ◽  
Double Strand Dna Breaks ◽  
Age Related ◽  
Double Strand Dna

scholarly journals Ubiquitylation at the Fork: Making and Breaking Chains to Complete DNA Replication

2018 ◽  
Vol 19 (10) ◽  
pp. 2909 ◽  
Author(s):  
Maïlyn Yates ◽  
Alexandre Maréchal
Keyword(s):  
Replication Fork ◽  
Genome Stability ◽  
Protein A ◽  
Genetic Material ◽  
Replication Stress ◽  
Genome Maintenance ◽  
Post Translational Modifications ◽  
Replication Fork Progression ◽  
Ubiquitin System ◽  
Stalled Replication Forks

The complete and accurate replication of the genome is a crucial aspect of cell proliferation that is often perturbed during oncogenesis. Replication stress arising from a variety of obstacles to replication fork progression and processivity is an important contributor to genome destabilization. Accordingly, cells mount a complex response to this stress that allows the stabilization and restart of stalled replication forks and enables the full duplication of the genetic material. This response articulates itself on three important platforms, Replication Protein A/RPA-coated single-stranded DNA, the DNA polymerase processivity clamp PCNA and the FANCD2/I Fanconi Anemia complex. On these platforms, the recruitment, activation and release of a variety of genome maintenance factors is regulated by post-translational modifications including mono- and poly-ubiquitylation. Here, we review recent insights into the control of replication fork stability and restart by the ubiquitin system during replication stress with a particular focus on human cells. We highlight the roles of E3 ubiquitin ligases, ubiquitin readers and deubiquitylases that provide the required flexibility at stalled forks to select the optimal restart pathways and rescue genome stability during stressful conditions.

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