scholarly journals Mechanism of efficient double-strand break repair by a long non-coding RNA

2020 ◽  
Vol 48 (19) ◽  
pp. 10953-10972 ◽  
Author(s):  
Roopa Thapar ◽  
Jing L Wang ◽  
Michal Hammel ◽  
Ruiqiong Ye ◽  
Ke Liang ◽  
...  
Keyword(s):  
Dna Repair ◽  
Strand Break ◽  
End Joining ◽  
Development Of Resistance ◽  
Non Coding Rna ◽  
Repair Protein ◽  
Structured Motifs ◽  
Dna Repair Protein ◽  
Non Homologous End Joining ◽  
Synaptic Event

Abstract Mechanistic studies in DNA repair have focused on roles of multi-protein DNA complexes, so how long non-coding RNAs (lncRNAs) regulate DNA repair is less well understood. Yet, lncRNA LINP1 is over-expressed in multiple cancers and confers resistance to ionizing radiation and chemotherapeutic drugs. Here, we unveil structural and mechanistic insights into LINP1’s ability to facilitate non-homologous end joining (NHEJ). We characterized LINP1 structure and flexibility and analyzed interactions with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes that direct NHEJ. LINP1 self-assembles into phase-separated condensates via RNA–RNA interactions that reorganize to form filamentous Ku-containing aggregates. Structured motifs in LINP1 bind Ku, promoting Ku multimerization and stabilization of the initial synaptic event for NHEJ. Significantly, LINP1 acts as an effective proxy for PAXX. Collective results reveal how lncRNA effectively replaces a DNA repair protein for efficient NHEJ with implications for development of resistance to cancer therapy.

scholarly journals Role of the yeast DNA repair protein Nej1 in end processing during the repair of DNA double strand breaks by non-homologous end joining

DNA Repair ◽  
2015 ◽  
Vol 31 ◽  
pp. 1-10 ◽  
Author(s):  
Hui Yang ◽  
Yoshihiro Matsumoto ◽  
Kelly M. Trujillo ◽  
Susan P. Lees-Miller ◽  
Mary Ann Osley ◽  
...  
Keyword(s):  
Dna Repair ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Repair Protein ◽  
Dna Repair Protein ◽  
Non Homologous End Joining

Abstract 265: Assessment Of Oxidative DNA Double Strand Break And Repair In Cardiomyocytes

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Bo Ye ◽  
Ning Hou ◽  
Lu Xiao ◽  
Haodong Xu ◽  
Faqian Li
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Dna Repair ◽  
Stress Responses ◽  
Strand Break ◽  
Neonatal Rat ◽  
Rat Cardiomyocytes ◽  
Repair Protein ◽  
Dna Repair Protein

Backgrounds: DNA damage occurs in cardiomyocytes during normal cellular metabolism and is significantly increased under cardiac stresses. How cardiomyocytes repair their DNA damage, especially DNA double strand breaks (DSBs), remains undetermined. We assessed DSBs caused by oxidative stress. More importantly, we investigated the spatiotemporal dynamics of DNA repair protein assembly/disassembly in DNA damage sites. Methods: Cultured neonatal rat cardiomyocytes were treated with different doses of hydrogen peroxide (H2O2) for 30 minutes to assess DNA damage response (DDR). To investigate the dynamics of DDR, cells were treated with 200 uM H2O2 and followed up to 72 hours. DSBs were evaluated by counting DNA damage foci after staining with antibody against histone H2AX phosphorylation at serine 139 (g-H2AX). The dynamics and posttranslational modification of DNA repair proteins were determined by Western blotting, immunolabeling, and confocal microscopy. Result: g-H2AX was proportionally increased to H2O2 dosage. Discrete nuclear g-H2AX foci were seen 30 minutes after hydrogen peroxide treatment with 50 uM, but became pannuclear when H2O2 was above 400 uM. At 200 uM of hydrogen peroxide, g-H2AX started to increase at 15 minutes and reached to highest levels at 60 minutes with up to 70 nuclear foci, started to decline at 2 hours, and returned to basal levels at 24 hours. DDR transducer kinase, ataxia telangiectasia mutated (ATM) was activated at 5 minutes with increased phosphorylation at serine 1981 (pATM) which started to decrease at 24 hours, but remained elevated up to 48 hours. Another DDR transducer kinase, ATM and Rad3-related (ATR) showed a biphasic activation at 30 minutes and 8 hours. ATM and ATR colocalized with g-H2AX. DNA damage mediator proteins such as MRN complex and p53BP1 were also recruited to sites of DNA damage at g-H2AX foci. Conclusions: DSBs and their repair have emerged as a new frontier of stress responses. Newly developed methods for studying g-H2AX and DNA repair protein dynamics can be explored to investigate DDR to oxidative stress in cardiomyocytes.

Thymocyte Selection-Associated High-Mobility Group Box Protein (TOX) Induces Genomic Instability in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 475-475
Author(s):  
Riadh Lobbardi ◽  
Jordan Pinder ◽  
Barbara Martinez-Pastor ◽  
Jessica S Blackburn ◽  
Nouran Abdelfattah ◽  
...  
Keyword(s):  
Dna Repair ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Genomic Instability ◽  
Lymphoblastic Leukemia ◽  
T Cell Development ◽  
Strand Break ◽  
End Joining ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Non Homologous End Joining

Abstract MYC and NOTCH are major oncogenic drivers in T-cell Acute Lymphoblastic Leukemia (T-ALL), yet additional collaborating genetic lesions likely collaborate to induce frank malignancy. To identify these factors, a large-scale transgenic screen was completed where 38 amplified and over-expressed genes found in human T-ALL were assessed for accelerating leukemia onset in the zebrafish transgenic model. From this analysis, Thymocyte selection-associated homeobox protein (TOX) synergized with both MYC and NOTCH to induce T-ALL. TOX is dynamically regulated in T cell development with peak expression occurring when thymocytes are actively undergoing T cell receptor (TCR) recombination. TOX is best known for regulating the specification of the mature CD4+ T cells. Despite TOX being genomically amplified in a subset of human and mouse T-ALL and being overexpressed in 100% of human T-ALL, a role for TOX in regulating leukemogenesis has not been reported. Characterization of zebrafish T-ALLs revealed that TOX expands the overall number of malignant T-ALL clones and promoted genomic instability as assessed by changes in DNA content. To identify TOX binding partners, antibody immunoprecipitation studies were performed followed by Tandem Mass Spectrometry. TOX was found to interact with KU70/KU80 but not other DNA repair enzymes including LigaseIV, DNA-PKC, or XRCC4. These results were verified by Western blot analysis and reciprocal immunoprecipitation studies using antibodies specific to KU70/KU80 both in the absence and presence of DNAseI treatment. Given that TOX elevated genomic instability in the zebrafish model and bound specifically to KU70/KU80 – the initiating factors required for Non-Homologous End Joining (NHEJ) repair - we hypothesized that TOX is a negative regulator of double-strand break repair. Fluorescent repair assays were completed in 3T3 fibroblasts and confirmed that TOX inhibits Non-Homologous End Joining (NHEJ). Both the nuclear localization signal and HMG-box were required for the ability of TOX to inhibit double-strand break repair. Dynamic real-time imaging studies confirmed that TOX suppresses recruitment of fluorescent-tagged KU70 to DNA breaks. Importantly, TOX loss of function increased NHEJ in human T-ALL cells and reduced time to DNA repair as assessed by fluorescent Traffic Light Reporter assays and quantitative assessment of 53BP1 and γH2A.X foci resolution following irradiation. Given the prominent role TOX has in T cell development and its coordinated regulation during active TCRβ and TCRα rearrangement, it is likely that the normal function of TOX is to transiently suppress the NHEJ pathway during Recombination-Activating Gene (RAG)-mediated recombination. Prolonging the time to DNA repair would likely facilitate long-range repair across VDJ segments. In the setting of T-ALL, TOX is aberrantly re-activated, thereby suppressing KU70/KU80 function to promote genomic instability and ultimately elevating rates at which acquired mutations and rearrangements are amassed in developing pre-malignant T cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Impact of hypoxia on the double-strand break repair after photon and carbon ion irradiation of radioresistant HNSCC cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Anne-Sophie Wozny ◽  
Gersende Alphonse ◽  
Audrey Cassard ◽  
Céline Malésys ◽  
Safa Louati ◽  
...  
Keyword(s):  
Dna Repair ◽  
Ion Irradiation ◽  
Strand Break ◽  
Carbon Ions ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Photon Irradiation ◽  
Hypoxic Conditions ◽  
Non Homologous End Joining

AbstractDNA double-strand breaks (DSBs) induced by photon irradiation are the most deleterious damage for cancer cells and their efficient repair may contribute to radioresistance, particularly in hypoxic conditions. Carbon ions (C-ions) act independently of the oxygen concentration and trigger complex- and clustered-DSBs difficult to repair. Understanding the interrelation between hypoxia, radiation-type, and DNA-repair is therefore essential for overcoming radioresistance. The DSBs signaling and the contribution of the canonical non-homologous end-joining (NHEJ-c) and homologous-recombination (HR) repair pathways were assessed by immunostaining in two cancer-stem-cell (CSCs) and non-CSCs HNSCC cell lines. Detection and signaling of DSBs were lower in response to C-ions than photons. Hypoxia increased the decay-rate of the detected DSBs (γH2AX) in CSCs after photons and the initiation of DSB repair signaling (P-ATM) in CSCs and non-CSCs after both radiations, but not the choice of DSB repair pathway (53BP1). Additionally, hypoxia increased the NHEJ-c (DNA-PK) and the HR pathway (RAD51) activation only after photons. Furthermore, the involvement of the HR seemed to be higher in CSCs after photons and in non-CSCs after C-ions. Taken together, our results show that C-ions may overcome the radioresistance of HNSCC associated with DNA repair, particularly in CSCs, and independently of a hypoxic microenvironment.

scholarly journals Mechanistic modelling and Bayesian inference elucidates the variable dynamics of double-strand break repair

2015 ◽  
Author(s):  
M. Woods ◽  
C.P. Barnes
Keyword(s):  
Dna Repair ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Data Sets ◽  
End Joining ◽  
Strand Breaks ◽  
Alternative End Joining ◽  
Intermediate Repair ◽  
Non Homologous End Joining

AbstractDNA double-strand breaks are lesions that form during metabolism, DNA replication and exposure to mutagens. When a double-strand break occurs one of a number of repair mechanisms is recruited, all of which have differing propensities for mutational events. Despite DNA repair being of crucial importance, the relative contribution of these mechanisms and their regulatory interactions remain to be fully elucidated. Understanding these mutational processes will have a profound impact on our knowledge of genomic instability, with implications across health, disease and evolution. Here we present a new method to model the combined activation of non-homologous end joining, single strand annealing and alternative end joining, following exposure to ionizing radiation. We use Bayesian statistics to integrate eight biological data sets of double-strand break repair curves under varying genetic knockouts and confirm that our model is predictive by re-simulating and comparing to additional data. Analysis of the model suggests that there are at least three disjoint modes of repair, which we assign as fast, slow and intermediate. Our results show that when multiple data sets are combined, the rate for intermediate repair is variable amongst genetic knockouts. Further analysis suggests that the ratio between slow and intermediate repair depends on the presence or absence of DNA-PKcs and Ku70, which implies that non-homologous end joining and alternative end joining are not independent. Finally, we consider the proportion of double-strand breaks within each mechanism as a time series and predict activity as a function of repair rate. We outline how our insights can be directly tested using imaging and sequencing techniques and conclude that there is evidence of variable dynamics in alternative repair pathways. Our approach is an important step towards providing a unifying theoretical framework for the dynamics of DNA repair processes.

scholarly journals Microarray screening reveals a non-conventional SUMO-binding mode linked to DNA repair by non-homologous end-joining

2021 ◽  
Author(s):  
Maria Jose Cabello-Lobato ◽  
Matthew Jenner ◽  
Christian M. Loch ◽  
Stephen P. Jackson ◽  
Qian Wu ◽  
...  
Keyword(s):  
Dna Repair ◽  
Binding Mode ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Cellular Processes ◽  
Dna Repair Protein ◽  
Microarray Screening ◽  
Non Homologous End Joining

SUMOylation is critical for a plethora of cellular signalling pathways including the repair of DNA double-strand breaks (DSBs). If misrepaired, DSBs can lead to cancer, neurodegeneration, immunodeficiency and premature ageing. Based on systematic proteome microarray screening combined with widely applicable carbene footprinting and high-resolution structural profiling, we define two non-conventional SUMO2-binding modules on XRCC4, a DNA repair protein important for DSB repair by non-homologous end-joining (NHEJ). Mechanistically, interaction of SUMO2 with XRCC4 is incompatible with XRCC4 binding to at least two other NHEJ proteins – XLF and DNA ligase 4 (LIG4). These findings are consistent with SUMO2 interactions of XRCC4 acting as backup pathways at different stages of NHEJ, in the absence of these factors or their dysfunctioning. Such scenarios are not only relevant for carcinogenesis, but also for the design of precision anti-cancer medicines and the optimisation of CRISPR/Cas9-based gene editing. This work reveals insights into topology-specific SUMO recognition and its potential for modulating DSB repair by NHEJ. Moreover, it provides a rich resource on binary SUMO receptors that can be exploited for uncovering regulatory layers in a wide array of cellular processes.

scholarly journals The (Lack of) DNA Double-Strand Break Repair Pathway Choice During V(D)J Recombination

2022 ◽  
Vol 12 ◽  
Author(s):  
Alice Libri ◽  
Timea Marton ◽  
Ludovic Deriano
Keyword(s):  
Dna Repair ◽  
Gene Diversity ◽  
Receptor Gene ◽  
Strand Break ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Homology Directed Repair ◽  
Pathway Choice ◽  
Non Homologous End Joining

DNA double-strand breaks (DSBs) are highly toxic lesions that can be mended via several DNA repair pathways. Multiple factors can influence the choice and the restrictiveness of repair towards a given pathway in order to warrant the maintenance of genome integrity. During V(D)J recombination, RAG-induced DSBs are (almost) exclusively repaired by the non-homologous end-joining (NHEJ) pathway for the benefit of antigen receptor gene diversity. Here, we review the various parameters that constrain repair of RAG-generated DSBs to NHEJ, including the peculiarity of DNA DSB ends generated by the RAG nuclease, the establishment and maintenance of a post-cleavage synaptic complex, and the protection of DNA ends against resection and (micro)homology-directed repair. In this physiological context, we highlight that certain DSBs have limited DNA repair pathway choice options.

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