The Comet Assay in Drosophila: A Tool to Study Interactions between DNA Repair Systems in DNA Damage Responses In Vivo and Ex Vivo

Rubén Rodríguez; Isabel Gaivão; Leticia Aguado; Marta Espina; Jorge García; Pablo Martínez-Camblor; L María Sierra

doi:10.3390/cells12151979

The Comet Assay in Drosophila: A Tool to Study Interactions between DNA Repair Systems in DNA Damage Responses In Vivo and Ex Vivo

Cells. 2023 Jul 31;12(15):1979. doi: 10.3390/cells12151979.

Authors

Rubén Rodríguez^{1

2}, Isabel Gaivão³, Leticia Aguado^{1

2}, Marta Espina^{1

2}, Jorge García¹, Pablo Martínez-Camblor^{4

5}, L María Sierra^{1

2

6}

Affiliations

¹ Department of Functional Biology (Genetic Area), University of Oviedo, 33006 Oviedo, Spain.
² Oncology University Institute from Asturias (IUOPA), University of Oviedo, 33006 Oviedo, Spain.
³ Department of Genetics and Biotechnology and CECAV, University of Trás-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal.
⁴ Department of Biomedical Data Science, Geisel Medical School at Dartmouth, Hanover, NH 03755, USA.
⁵ Faculty of Health Sciences, Universidad Autónoma de Chile, Provedencia 7500912, Chile.
⁶ Institute of Sanitary Research of the Principality of Asturias, Av. del Hospital Universitario, s/n, 33011 Oviedo, Spain.

Abstract

The comet assay in Drosophila has been used in the last few years to study DNA damage responses (DDR) in different repair-mutant strains and to compare them to analyze DNA repair. We have used this approach to study interactions between DNA repair pathways in vivo. Additionally, we have implemented an ex vivo comet assay, in which nucleoids from treated and untreated cells were incubated ex vivo with cell-free protein extracts from individuals with distinct repair capacities. Four strains were used: wild-type OregonK (OK), nucleotide excision repair mutant mus201, dmPolQ protein mutant mus308, and the double mutant mus201;mus308. Methyl methanesulfonate (MMS) was used as a genotoxic agent. Both approaches were performed with neuroblasts from third-instar larvae; they detected the effects of the NER and dmPolQ pathways on the DDR to MMS and that they act additively in this response. Additionally, the ex vivo approach quantified that mus201, mus308, and the double mutant mus201;mus308 strains presented, respectively, 21.5%, 52.9%, and 14.8% of OK strain activity over MMS-induced damage. Considering the homology between mammals and Drosophila in repair pathways, the detected additive effect might be extrapolated even to humans, demonstrating that Drosophila might be an excellent model to study interactions between repair pathways.

Keywords: DNA damage response; Drosophila melanogaster; MMS; NER; dmPolQ; ex vivo comet assay; in vivo comet assay; mus308 and OK strains; mus308, mus201, mus201.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Comet Assay
DNA Damage
DNA Repair
Drosophila melanogaster* / genetics
Drosophila* / genetics
Humans
Mammals / genetics
Methyl Methanesulfonate / pharmacology

Substances

Methyl Methanesulfonate

Grants and funding

This research was funded by MEC Spain (project CT2004-03005), FICYT (PCTI Asturias, project PC07-018), and the Instituto Universitario de Oncología del Principado de Asturias, Obra Social Cajastur to LMS. By Instituto Universitario de Oncología del Principado de Asturias, Obra Social Cajastur to LA. And by the Portuguese Science and Technology Foundation (FCT) under Projects UIDP/00772/2020 and LA/P/0059/2020 to IG.