TheDrosophila melanogasterDNALigase IVGene Plays a Crucial Role in the Repair of Radiation-Induced DNA Double-Strand Breaks and Acts Synergistically WithRad54

Abstract

DNA Ligase IV has a crucial role in double-strand break (DSB) repair through nonhomologous end joining (NHEJ). Most notably, its inactivation leads to embryonic lethality in mammals. To elucidate the role of DNA Ligase IV (Lig4) in DSB repair in a multicellular lower eukaryote, we generated viable Lig4-deficient Drosophila strains by P-element-mediated mutagenesis. Embryos and larvae of mutant lines are hypersensitive to ionizing radiation but hardly so to methyl methanesulfonate (MMS) or the crosslinking agent cis-diamminedichloroplatinum (cisDDP). To determine the relative contribution of NHEJ and homologous recombination (HR) in Drosophila, Lig4; Rad54 double-mutant flies were generated. Survival studies demonstrated that both HR and NHEJ have a major role in DSB repair. The synergistic increase in sensitivity seen in the double mutant, in comparison with both single mutants, indicates that both pathways partially overlap. However, during the very first hours after fertilization NHEJ has a minor role in DSB repair after exposure to ionizing radiation. Throughout the first stages of embryogenesis of the fly, HR is the predominant pathway in DSB repair. At late stages of development NHEJ also becomes less important. The residual survival of double mutants after irradiation strongly suggests the existence of a third pathway for the repair of DSBs in Drosophila.

DNA ligase IV mutations confer shorter lifespan and increased sensitivity to nutrient stress in Drosophila melanogaster

“…We obtained three strains of D. melanogaster from the Bloomington Drosophila Stock Center: w 1118 (wild-type DNAlig4 ) and two DNAlig4 mutants: DNAlig4 5 and DNAlig4 57 ( Gorski 2003 ). The D. melanogaster stocks were maintained at 25 °C on normal yeast-cornmeal food (ingredients: 337.5 g yeast, 195 g soy flour, 1425 g cornmeal, 95 g Drosophila food-grade Agar type II, 900 g malt extract, 1.5 g molasses, 100 mL propionic acid, 250 mL 10% Tegosept, and 25 L tap water) unless mentioned otherwise.…”

“…melanogaster has been extensively used as a model to study DNA repair. The DSB repair in D. melanogaster , like in mammals, is via the two primary pathways: the template-dependent homologous recombination and template-independent end-joining (EJ) pathway as well as the alternative end-joining (altEJ) pathway (Gorski 2003 ; Mota 2019 ). These pathways and corresponding components in D. melanogaster are similar to the mammalian system, yet there are some key differences.…”

“…Lig4 hypomorphic mutations in humans cause the Lig4 syndrome (recently reviewed (Altmann and Gennery 2016 )), and Lig4 −/− mice are inviable, as mutations cause p53-mediated neuronal apoptosis resulting in embryonic lethality (Frank 2000 ). In contrast, D. melanogaster males and females lacking DNAlig4 function are viable and fertile; however, these mutants are hypersensitive to ionizing radiation (IR)–induced DNA damage during early development (Gorski 2003 ; McVey et al 2004 ). An accumulation of DNA damage is commonly observed as a result of aging, and metabolic alterations resulting from nutrient deprivation can induce oxidative stress which in turn induces DNA damage (Filomeni et al 2015 ).…”

Disruption of the Schizosaccharomyces japonicus lig4 Disturbs Several Cellular Processes and Leads to a Pleiotropic Phenotype

“…DNA ligases catalyze the joining of two DNA molecules. One of them is LIG4, which can be found in various species, from yeasts to humans [1][2][3][4][5]. Several studies have proved that LIG4 proteins are essential components of the non-homologous end joining (NHEJ) mechanism, which repairs the double-strand breaks of DNA [1,[5][6][7].…”

“…In contrast, the Drosophila and fungal mutants were viable. The Drosophila mutants had hypersensitivity to radiation [3], while the budding yeast mutants showed impaired hyphal growth and cell division [2,22,23], unlike the Magnaporthe grisea, Monascus ruber, or Mortierella alpina mutant cells, which showed no defect in sexual or asexual growth [5,24,25].…”