AutoComet: A fully automated algorithm to quickly and accurately analyze comet assays

Barbé, Lise; Lam, Stephanie; Holub, Austin; Faghihmonzavi, Zohreh; Deng, Minnie; Iyer, Rajshri; finkbeiner, steven

doi:10.1016/j.redox.2023.102680

Cited by 8 publications

(3 citation statements)

References 28 publications

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“…Then, 100 μL of either UNG buffer with DNA glycosylase enzyme (2 U/mL, Roche, Basel, Switzerland) or UNG buffer alone were added, and the slides were covered with a glass coverslip. The slides were incubated in a moist atmosphere at 37°C for 1 h. After enzyme treatment, the slides were placed in a tank with cold running buffer (300 mM NaOH, 1 mM Na 2 EDTA, pH was adjusted to 13 using HCl) for 40 min before electrophoresis for 90 min at 25 V. Comet Analysis System OpenComet [ 73 ] was used to quantify tail DNA moments in DT40 cells. 150 cells were scored per sample.…”

Section: Methodsmentioning

confidence: 99%

REV3 promotes cellular tolerance to 5-fluorodeoxyuridine by activating translesion DNA synthesis and intra-S checkpoint

Washif,

Kawasumi,

Hirota

2024

PLoS Genet

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The drug floxuridine (5-fluorodeoxyuridine, FUdR) is an active metabolite of 5-Fluorouracil (5-FU). It converts to 5-fluorodeoxyuridine monophosphate (FdUMP) and 5-fluorodeoxyuridine triphosphate (FdUTP), which on incorporation into the genome inhibits DNA replication. Additionally, it inhibits thymidylate synthase, causing dTMP shortage while increasing dUMP availability, which induces uracil incorporation into the genome. However, the mechanisms underlying cellular tolerance to FUdR are yet to be fully elucidated. In this study, we explored the mechanisms underlying cellular resistance to FUdR by screening for FUdR hypersensitive mutants from a collection of DT40 mutants deficient in each genomic maintenance system. We identified REV3, which is involved in translesion DNA synthesis (TLS), to be a critical factor in FUdR tolerance. Replication using a FUdR-damaged template was attenuated in REV3-/- cells, indicating that the TLS function of REV3 is required to maintain replication on the FUdR-damaged template. Notably, FUdR-exposed REV3-/- cells exhibited defective cell cycle arrest in the early S phase, suggesting that REV3 is involved in intra-S checkpoint activation. Furthermore, REV3-/- cells showed defects in Chk1 phosphorylation, which is required for checkpoint activation, but the survival of FUdR-exposed REV3-/- cells was further reduced by the inhibition of Chk1 or ATR. These data indicate that REV3 mediates DNA checkpoint activation at least through Chk1 phosphorylation, but this signal acts in parallel with ATR-Chk1 DNA damage checkpoint pathway. Collectively, we reveal a previously unappreciated role of REV3 in FUdR tolerance.

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Section: Methodsmentioning

confidence: 99%

REV3 promotes cellular tolerance to 5-fluorodeoxyuridine by activating translesion DNA synthesis and intra-S checkpoint

Washif,

Kawasumi,

Hirota

2024

PLoS Genet

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“…Images were captured using a BZ-X810 fluorescence microscope (Keyence, Tokyo, Japan). Tail DNA percentage, reflecting the number of DSBs, was measured for cells exposed to 0 or 20 nM CPT for 1 h. OpenComet ( 66 ) was used to quantify the tail DNA percentage;150 cells were scored per sample.…”

Section: Methodsmentioning

confidence: 99%

The proofreading exonuclease of leading-strand DNA polymerase epsilon prevents replication fork collapse at broken template strands

Ahmad,

Kawasumi,

Taniguchi

et al. 2023

Nucleic Acids Research

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Leading-strand DNA replication by polymerase epsilon (Polϵ) across single-strand breaks (SSBs) causes single-ended double-strand breaks (seDSBs), which are repaired via homology-directed repair (HDR) and suppressed by fork reversal (FR). Although previous studies identified many molecules required for hydroxyurea-induced FR, FR at seDSBs is poorly understood. Here, we identified molecules that specifically mediate FR at seDSBs. Because FR at seDSBs requires poly(ADP ribose)polymerase 1 (PARP1), we hypothesized that seDSB/FR-associated molecules would increase tolerance to camptothecin (CPT) but not the PARP inhibitor olaparib, even though both anti-cancer agents generate seDSBs. Indeed, we uncovered that Polϵ exonuclease and CTF18, a Polϵ cofactor, increased tolerance to CPT but not olaparib. To explore potential functional interactions between Polϵ exonuclease, CTF18, and PARP1, we created exonuclease-deficient POLE1exo−/−, CTF18−/−, PARP1−/−, CTF18−/−/POLE1exo−/−, PARP1−/−/POLE1exo−/−, and CTF18−/−/PARP1−/− cells. Epistasis analysis indicated that Polϵ exonuclease and CTF18 were interdependent and required PARP1 for CPT tolerance. Remarkably, POLE1exo−/− and HDR-deficient BRCA1−/− cells exhibited similar CPT sensitivity. Moreover, combining POLE1exo−/− with BRCA1−/− mutations synergistically increased CPT sensitivity. In conclusion, the newly identified PARP1-CTF18-Polϵ exonuclease axis and HDR act independently to prevent fork collapse at seDSBs. Olaparib inhibits this axis, explaining the pronounced cytotoxic effects of olaparib on HDR-deficient cells.

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“…While tail moment and Olive moment are expressed in arbitrary units, rendering them potentially variable across image analysis systems, the DNA percentage in the tail offers a readily comparable metric across platforms. OpenComet (Barbé et al, 2023) in its analytical output, computes and presents these three pivotal damage measures alongside additional comet-related parameters. This comprehensive approach empowers users to select the most relevant metric for subsequent in-depth analysis.…”

Section: Comet Measurementsmentioning

confidence: 99%

Exploring the Impact of Simulated Microgravity on Cellular DNA: A Comparative Analysis of Cancer and Normal Cell Lines

Altaie,

Alrawi,

Duan

et al. 2024

Microgravity Sci. Technol.

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The examination of the impact of microgravity on biological systems has gained considerable attention owing to its potential implications for health and disease. Simulated microgravity serves as a valuable methodology for elucidating the intricate cellular responses to altered gravitational conditions. This study investigates the effects of simulated microgravity on cellular DNA, employing four distinct cell lines-breast, brain, and esophageal cancer cells, in conjunction with normal cells for comparative analysis. The experiment utilized the comet assay test to quantitatively assess DNA damage. The results revealed a discernible disparity in the response to simulated microgravity, notably with cancer cells exhibiting a signi cant increase in DNA damage compared to the relatively minimal effects observed in both control and normal cells. Furthermore, within the cancer cell lines, signi cant variations in the extent of DNA damage were evident, implying a cell type-dependent response to simulated microgravity. These ndings illuminate the potential differential susceptibility of cancerous and normal cells to microgravity-induced DNA damage. Consequently, this research substantially contributes to our comprehension of microgravity-induced cellular responses and unveils promising avenues for targeted interventions in cancer therapy.

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AutoComet: A fully automated algorithm to quickly and accurately analyze comet assays

Cited by 8 publications

References 28 publications

REV3 promotes cellular tolerance to 5-fluorodeoxyuridine by activating translesion DNA synthesis and intra-S checkpoint

REV3 promotes cellular tolerance to 5-fluorodeoxyuridine by activating translesion DNA synthesis and intra-S checkpoint

The proofreading exonuclease of leading-strand DNA polymerase epsilon prevents replication fork collapse at broken template strands

Exploring the Impact of Simulated Microgravity on Cellular DNA: A Comparative Analysis of Cancer and Normal Cell Lines

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