scholarly journals A multiplexed bioluminescent reporter for sensitive and non-invasive tracking of DNA double strand break repair dynamics in vitro and in vivo

2020 ◽  
Vol 48 (17) ◽  
pp. e100-e100 ◽  
Author(s):  
Jasper Che-Yung Chien ◽  
Elie Tabet ◽  
Kelsey Pinkham ◽  
Cintia Carla da Hora ◽  
Jason Cheng-Yu Chang ◽  
...  
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Dsb Repair ◽  
Non Invasive ◽  
Longitudinal Tracking ◽  
Dna Double Strand Break ◽  
Significant Difference ◽  
Therapeutic Development

Abstract Tracking DNA double strand break (DSB) repair is paramount for the understanding and therapeutic development of various diseases including cancers. Herein, we describe a multiplexed bioluminescent repair reporter (BLRR) for non-invasive monitoring of DSB repair pathways in living cells and animals. The BLRR approach employs secreted Gaussia and Vargula luciferases to simultaneously detect homology-directed repair (HDR) and non-homologous end joining (NHEJ), respectively. BLRR data are consistent with next-generation sequencing results for reporting HDR (R2 = 0.9722) and NHEJ (R2 = 0.919) events. Moreover, BLRR analysis allows longitudinal tracking of HDR and NHEJ activities in cells, and enables detection of DSB repairs in xenografted tumours in vivo. Using the BLRR system, we observed a significant difference in the efficiency of CRISPR/Cas9-mediated editing with guide RNAs only 1–10 bp apart. Moreover, BLRR analysis detected altered dynamics for DSB repair induced by small-molecule modulators. Finally, we discovered HDR-suppressing functions of anticancer cardiac glycosides in human glioblastomas and glioma cancer stem-like cells via inhibition of DNA repair protein RAD51 homolog 1 (RAD51). The BLRR method provides a highly sensitive platform to simultaneously and longitudinally track HDR and NHEJ dynamics that is sufficiently versatile for elucidating the physiology and therapeutic development of DSB repair.

scholarly journals A multiplexed bioluminescent reporter for sensitive and non-invasive tracking of DNA double strand break repair dynamics in vitro and in vivo

2020 ◽  
Author(s):  
Jasper Che-Yung Chien ◽  
Elie Tabet ◽  
Kelsey Pinkham ◽  
Cintia Carla da Hora ◽  
Jason Cheng-Yu Chang ◽  
...  
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Dsb Repair ◽  
Non Invasive ◽  
Longitudinal Tracking ◽  
Dna Double Strand Break ◽  
Significant Difference ◽  
Therapeutic Development

ABSTRACTTracking DNA double strand break (DSB) repair is paramount for the understanding and therapeutic development of various diseases including cancers. Herein, we describe a multiplexed bioluminescent repair reporter (BLRR) for non-invasive monitoring of DSB repair pathways in living cells and animals. The BLRR approach employs secreted Gaussia and Vargula luciferases to simultaneously detect homology-directed repair (HDR) and non-homologous end joining (NHEJ), respectively. BLRR data are consistent with next-generation sequencing results for reporting HDR (R2 = 0.9722) and NHEJ (R2 = 0.919) events. Moreover, BLRR analysis allows longitudinal tracking of HDR and NHEJ activities in cells, and enables detection of DSB repairs in xenografted tumours in vivo. Using the BLRR system, we observed a significant difference in the efficiency of CRISPR/Cas9-mediated editing with guide RNAs only 1-10 bp apart. Moreover, BLRR analysis detected altered dynamics for DSB repair induced by small-molecule modulators. Finally, we discovered HDR-suppressing functions of anticancer cardiac glycosides in human glioblastomas and glioma cancer stem-like cells via inhibition of DNA repair protein RAD51 homolog 1 (RAD51). The BLRR method provides a highly sensitive platform to simultaneously and longitudinally track HDR and NHEJ dynamics that is sufficiently versatile for elucidating the physiology and therapeutic development of DSB repair.

The PARP inhibitor olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo

Blood ◽  
2010 ◽  
Vol 116 (22) ◽  
pp. 4578-4587 ◽  
Author(s):  
Victoria J. Weston ◽  
Ceri E. Oldreive ◽  
Anna Skowronska ◽  
David G. Oscier ◽  
Guy Pratt ◽  
...  
Keyword(s):  
Cell Line ◽  
Tumor Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Prolymphocytic Leukemia ◽  
Dna Double Strand Break ◽  
Break Repair

Abstract The Ataxia Telangiectasia Mutated (ATM) gene is frequently inactivated in lymphoid malignancies such as chronic lymphocytic leukemia (CLL), T-prolymphocytic leukemia (T-PLL), and mantle cell lymphoma (MCL) and is associated with defective apoptosis in response to alkylating agents and purine analogues. ATM mutant cells exhibit impaired DNA double strand break repair. Poly (ADP-ribose) polymerase (PARP) inhibition that imposes the requirement for DNA double strand break repair should selectively sensitize ATM-deficient tumor cells to killing. We investigated in vitro sensitivity to the poly (ADP-ribose) polymerase inhibitor olaparib (AZD2281) of 5 ATM mutant lymphoblastoid cell lines (LCL), an ATM mutant MCL cell line, an ATM knockdown PGA CLL cell line, and 9 ATM-deficient primary CLLs induced to cycle and observed differential killing compared with ATM wildtype counterparts. Pharmacologic inhibition of ATM and ATM knockdown confirmed the effect was ATM-dependent and mediated through mitotic catastrophe independently of apoptosis. A nonobese diabetic/severe combined immunodeficient (NOD/SCID) murine xenograft model of an ATM mutant MCL cell line demonstrated significantly reduced tumor load and an increased survival of animals after olaparib treatment in vivo. Addition of olaparib sensitized ATM null tumor cells to DNA-damaging agents. We suggest that olaparib would be an appropriate agent for treating refractory ATM mutant lymphoid tumors.

scholarly journals DNA double-strand break repair: a theoretical framework and its application

2016 ◽  
Vol 13 (114) ◽  
pp. 20150679 ◽  
Author(s):  
Philip J. Murray ◽  
Bart Cornelissen ◽  
Katherine A. Vallis ◽  
S. Jon Chapman
Keyword(s):  
Dna Damage ◽  
Auger Electron ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Histone H2ax ◽  
Dna Double Strand Break ◽  
A Cell

DNA double-strand breaks (DSBs) are formed as a result of genotoxic insults, such as exogenous ionizing radiation, and are among the most serious types of DNA damage. One of the earliest molecular responses following DSB formation is the phosphorylation of the histone H2AX, giving rise to γ H2AX. Many copies of γ H2AX are generated at DSBs and can be detected in vitro as foci using well-established immuno-histochemical methods. It has previously been shown that anti- γ H2AX antibodies, modified by the addition of the cell-penetrating peptide TAT and a fluorescent or radionuclide label, can be used to visualize and quantify DSBs in vivo . Moreover, when labelled with a high amount of the short-range, Auger electron-emitting radioisotope, 111 In, the amount of DNA damage within a cell can be increased, leading to cell death. In this report, we develop a mathematical model that describes how molecular processes at individual sites of DNA damage give rise to quantifiable foci. Equations that describe stochastic mean behaviours at individual DSB sites are derived and parametrized using population-scale, time-series measurements from two different cancer cell lines. The model is used to examine two case studies in which the introduction of an antibody (anti- γ H2AX-TAT) that targets a key component in the DSB repair pathway influences system behaviour. We investigate: (i) how the interaction between anti- γ H2AX-TAT and γ H2AX effects the kinetics of H2AX phosphorylation and DSB repair and (ii) model behaviour when the anti- γ H2AX antibody is labelled with Auger electron-emitting 111 In and can thus instigate additional DNA damage. This work supports the conclusion that DSB kinetics are largely unaffected by the introduction of the anti- γ H2AX antibody, a result that has been validated experimentally, and hence the hypothesis that the use of anti- γ H2AX antibody to quantify DSBs does not violate the image tracer principle. Moreover, it provides a novel model of DNA damage accumulation in the presence of Auger electron-emitting 111 In that is supported qualitatively by the available experimental data.

scholarly journals A Simulated Shift Work Schedule Does Not Increase DNA Double-Strand Break Repair by NHEJ in the Drosophila Rr3 System

Genes ◽  
2022 ◽  
Vol 13 (1) ◽  
pp. 150
Author(s):  
Lydia Bergerson ◽  
Caleb Fitzmaurice ◽  
Tyler Knudtson ◽  
Halle McCormick ◽  
Alder M. Yu
Keyword(s):  
Shift Work ◽  
Strand Break ◽  
Double Strand Break ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Significant Difference ◽  
Nhej Repair ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Shift Work Schedule

Long-term shift work is widely believed to increase the risk of certain cancers, but conflicting findings between studies render this association unclear. Evidence of interplay between the circadian clock, cell cycle regulation, and DNA damage detection machinery suggests the possibility that circadian rhythm disruption consequent to shift work could alter the DNA double-strand break (DSB) repair pathway usage to favor mutagenic non-homologous end-joining (NHEJ) repair. To test this hypothesis, we compared relative usage of NHEJ and single-strand annealing (SSA) repair of a complementary ended chromosomal double-stranded break using the Repair Reporter 3 (Rr3) system in Drosophila between flies reared on 12:12 and 8:8 (simulated shift work) light:dark schedules. Actimetric analysis showed that the 8:8 light:dark schedule effectively disrupted the rhythms in locomotor output. Inaccurate NHEJ repair was not a frequent outcome in this system overall, and no significant difference was seen in the usage of NHEJ or SSA repair between the control and simulated shift work schedules. We conclude that this circadian disruption regimen does not alter the usage of mutagenic NHEJ DSB repair in the Drosophila male pre-meiotic germline, in the context of the Rr3 system.

Quantification of DNA double-strand break induced by radiation in cervix cancer cells: in vitro study

2018 ◽  
Vol 18 (1) ◽  
pp. 52-54
Author(s):  
Sothing Vashum ◽  
Rabi Raja Singh I ◽  
Saikat Das ◽  
Mohammed Azharuddin KO ◽  
Prabhakaran Vasudevan
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Ionising Radiation ◽  
Dependent Manner ◽  
Survival Fraction ◽  
Dna Double Strand Break ◽  
Cervical Cancer Cells

AbstractAimDNA double-strand break (DSB) results in the phosphorylation of the protein, H.2AX histone. In this study, the effect of radiotherapy and chemotherapy on DNA DSB in cervical cancer cells is analysed by the phosphorylation of the protein.MethodsThe cervical cancer cells (HeLa cells) were cultured and exposed to ionising radiation. Radiation sensitivity was measured by clonogenic survival fraction after exposing to ionising radiation. Since the phosphorylation of H.2AX declines with time, the DNA damage was quantified at different time points: 1 hour, 3 hours and 1 week after exposed to the radiation. The analysis of γ-H.2AX was done by Western-blot technique. The protein expression was observed at different dose of radiation and combination of both radiation and paclitaxel.ResultsLow-dose hypersensitivity was observed. By 1 week after radiation at 0·5, 0·8 and 2 Gy, there was no expression of phosphorylated H.2AX. Previous experiments on the expression of phosphorylated H.2AX (γ-H.2AX) in terms of foci analysis was found to peak at 1 hour and subsequently decline with time. In cells treated with the DNA damaging agents, the expression of phosphorylated H.2AX decreases in a dose-dependent manner when treated with radiation alone. However, when combined with paclitaxel, at 0·5 Gy, the expression peaked and reduces at 0·8 Gy and slightly elevated at 2 Gy.FindingsIn this study, the peak phosphorylation was observed at 3 hour post irradiation indicating that DSBs are still left unrepaired.

scholarly journals The Roles of Bacterial DNA Double-Strand Break Repair Proteins in Chromosomal DNA Replication

2020 ◽  
Vol 44 (3) ◽  
pp. 351-368 ◽  
Author(s):  
Anurag Kumar Sinha ◽  
Christophe Possoz ◽  
David R F Leach
Keyword(s):  
Dna Replication ◽  
Cell Viability ◽  
Strand Break ◽  
Double Strand Break ◽  
Replication Initiation ◽  
Genome Replication ◽  
Chromosomal Dna ◽  
Dsb Repair ◽  
Bacterial Dna ◽  
Dna Double Strand Break

ABSTRACT It is well established that DNA double-strand break (DSB) repair is required to underpin chromosomal DNA replication. Because DNA replication forks are prone to breakage, faithful DSB repair and correct replication fork restart are critically important. Cells, where the proteins required for DSB repair are absent or altered, display characteristic disturbances to genome replication. In this review, we analyze how bacterial DNA replication is perturbed in DSB repair mutant strains and explore the consequences of these perturbations for bacterial chromosome segregation and cell viability. Importantly, we look at how DNA replication and DSB repair processes are implicated in the striking recent observations of DNA amplification and DNA loss in the chromosome terminus of various mutant Escherichia coli strains. We also address the mutant conditions required for the remarkable ability to copy the entire E. coli genome, and to maintain cell viability, even in the absence of replication initiation from oriC, the unique origin of DNA replication in wild type cells. Furthermore, we discuss the models that have been proposed to explain these phenomena and assess how these models fit with the observed data, provide new insights and enhance our understanding of chromosomal replication and termination in bacteria.

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