Rapid Detection of γ-H2Av Foci in Ex Vivo MMS-Treated Drosophila Imaginal Discs

Khodaverdian, Varandt Y.; McVey, Mitch

doi:10.1007/978-1-4939-7187-9_19

Cited by 3 publications

(2 citation statements)

References 16 publications

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“…This is thought to result from massive cell death due to DNA double-strand breaks in rapidly dividing imaginal disc tissues, which are precursors for adult structures including wings, eyes, and other appendages. To test whether this could be responsible for the MMS hypersensitivity observed in rev1Δ mutants, we dissected wing imaginal discs from homozygous rev1Δ third instar larvae and treated them ex vivo with MMS for 5 hours, during which time all cells should replicate their DNA at least once (method per [59]) (Figure 2A). We then quantified the number of γ-H2Av foci, which are indicative of a checkpoint response to double-strand breaks.…”

Section: Resultsmentioning

confidence: 99%

“…Discs were washed 4X for 5 minutes with PBST and incubated for 2 hours at room temperature in 1:1000 goat anti-Rabbit IgG Rhodamine Red conjugated antibody and 500 μg/mL DAPI in 1xPBS + 5% BSA. Discs were washed and mounted in VECTASHEILD on microscope slides [59]. γ-H2Av foci were imaged at 10-20x magnification using a Zeiss Z-stacking microscope and with filter sets compatible with DAPI and Rhodamine.…”

Section: Imaginal Disc Culture and Immunofluorescencementioning

confidence: 99%

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REV1 Coordinates a Multi-Faceted Tolerance Response to DNA Alkylation Damage and Prevents Chromosome Shattering inDrosophila melanogaster

Khodaverdian,

Sano,

Maggs

et al. 2024

Preprint

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When replication forks encounter damaged DNA, cells utilize DNA damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent to which these different damage tolerance mechanisms are utilized depends on cell, tissue, and developmental context-specific cues, the last two of which are poorly understood. To address this gap, we have investigated damage tolerance responses following alkylation damage inDrosophila melanogaster. We report that translesion synthesis, rather than template switching, is the preferred response to alkylation-induced damage in diploid larval tissues. Furthermore, we show that the REV1 protein plays a multi-faceted role in damage tolerance in Drosophila. Drosophila larvae lacking REV1 are hypersensitive to methyl methanesulfonate (MMS) and have highly elevated levels of γ-H2Av foci and chromosome aberrations in MMS-treated tissues. Loss of the REV1 C-terminal domain (CTD), which recruits multiple translesion polymerases to damage sites, sensitizes flies to MMS. In the absence of the REV1 CTD, DNA polymerases eta and zeta become critical for MMS tolerance. In addition, flies lacking REV3, the catalytic subunit of polymerase zeta, require the deoxycytidyl transferase activity of REV1 to tolerate MMS. Together, our results demonstrate that Drosophila prioritize the use of multiple translesion polymerases to tolerate alkylation damage and highlight the critical role of REV1 in the coordination of this response to prevent genome instability.

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Section: Resultsmentioning

confidence: 99%

Section: Imaginal Disc Culture and Immunofluorescencementioning

confidence: 99%

REV1 Coordinates a Multi-Faceted Tolerance Response to DNA Alkylation Damage and Prevents Chromosome Shattering inDrosophila melanogaster

Khodaverdian,

Sano,

Maggs

et al. 2024

Preprint

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Sertraline induces DNA damage and cellular toxicity in Drosophila that can be ameliorated by antioxidants

Jajoo

Donlon

Shnayder

et al. 2020

Sci Rep

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Sertraline hydrochloride is a commonly prescribed antidepressant medication that acts by amplifying serotonin signaling. Numerous studies have suggested that children of women taking sertraline during pregnancy have an increased risk of developmental defects. Resolving the degree of risk for human fetuses requires comprehensive knowledge of the pathways affected by this drug. We utilized a Drosophila melanogaster model system to assess the effects of sertraline throughout development. Ingestion of sertraline by females did not affect their fecundity or embryogenesis in their progeny. However, larvae that consumed sertraline experienced delayed developmental progression and reduced survival at all stages of development. Genetic experiments showed that these effects were mostly independent of aberrant extracellular serotonin levels. Using an ex vivo imaginal disc culture system, we showed that mitotically active sertraline-treated tissues accumulate DNA double-strand breaks and undergo apoptosis at increased frequencies. Remarkably, the sertraline-induced genotoxicity was partially rescued by co-incubation with ascorbic acid, suggesting that sertraline induces oxidative DNA damage. These findings may have implications for the biomedicine of sertraline-induced birth defects.

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REV1 coordinates a multi-faceted tolerance response to DNA alkylation damage and prevents chromosome shattering in Drosophila melanogaster

Khodaverdian,

Sano,

Maggs

et al. 2024

PLoS Genet

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When replication forks encounter damaged DNA, cells utilize damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent to which these different damage tolerance mechanisms are utilized depends on cell, tissue, and developmental context-specific cues, the last two of which are poorly understood. To address this gap, we have investigated damage tolerance responses in Drosophila melanogaster. We report that tolerance of DNA alkylation damage in rapidly dividing larval tissues depends heavily on translesion synthesis. Furthermore, we show that the REV1 protein plays a multi-faceted role in damage tolerance in Drosophila. Larvae lacking REV1 are hypersensitive to methyl methanesulfonate (MMS) and have highly elevated levels of γ-H2Av (Drosophila γ-H2AX) foci and chromosome aberrations in MMS-treated tissues. Loss of the REV1 C-terminal domain (CTD), which recruits multiple translesion polymerases to damage sites, sensitizes flies to MMS. In the absence of the REV1 CTD, DNA polymerases eta and zeta become critical for MMS tolerance. In addition, flies lacking REV3, the catalytic subunit of polymerase zeta, require the deoxycytidyl transferase activity of REV1 to tolerate MMS. Together, our results demonstrate that Drosophila prioritize the use of multiple translesion polymerases to tolerate alkylation damage and highlight the critical role of REV1 in the coordination of this response to prevent genome instability.

show abstract

Rapid Detection of γ-H2Av Foci in Ex Vivo MMS-Treated Drosophila Imaginal Discs

Cited by 3 publications

References 16 publications

REV1 Coordinates a Multi-Faceted Tolerance Response to DNA Alkylation Damage and Prevents Chromosome Shattering inDrosophila melanogaster

REV1 Coordinates a Multi-Faceted Tolerance Response to DNA Alkylation Damage and Prevents Chromosome Shattering inDrosophila melanogaster

Sertraline induces DNA damage and cellular toxicity in Drosophila that can be ameliorated by antioxidants

REV1 coordinates a multi-faceted tolerance response to DNA alkylation damage and prevents chromosome shattering in Drosophila melanogaster

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