Lig4 and rad54 are required for repair of DNA double-strand breaks induced by P-element excision in Drosophila.
Site-specific double-strand breaks (DSBs) were generated in the white gene located on the X chromosome of Drosophila by excision of the w(hd) P-element. To investigate the role of nonhomologous end joining (NHEJ) and homologous recombination (HR) in the repair of these breaks, the w(hd) P-element was mobilized in flies carrying mutant alleles of either lig4 or rad54. The survival of both lig4- and rad54-deficient males was reduced to 25% in comparison to the wild type, indicating that both NHEJ and HR are involved in the repair P-induced gaps in males. Survival of lig4-deficient females was not affected at all, implying that HR using the homologous chromosome as a template can partially compensate for the impaired NHEJ pathway. In rad54 mutant females survival was reduced to 70% after w(hd) excision. PCR analysis indicated that the undamaged homologous chromosome may compensate for the potential loss of the broken chromosome in rad54 mutant females after excision. Molecular analysis of the repair junctions revealed microhomology (2-8 bp)-dependent DSB repair in most products. In the absence of Lig4, the 8-bp target site duplication is used more frequently for repair. Our data indicate the presence of efficient alternative end-joining mechanisms, which partly depend on the presence of microhomology but do not require Lig4.
Romeijn
Ron J
RJ
Department of Toxicogenetics, Leiden University Medical Center (LUMC), 2333 AL, Leiden, The Netherlands.
Gorski
Marcin M
MM
van Schie
Martijn A
MA
Noordermeer
Jasprina N
JN
Mullenders
Leon H
LH
Ferro
Wouter
W
Pastink
Albert
A
D000483:Alleles; D000818:Animals; D003433:Crosses, Genetic; D004249:DNA Damage; D004265:DNA Helicases; D011088:DNA Ligases / Q000502:physiology*; D004260:DNA Repair; D004251:DNA Transposable Elements / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology*; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins / Q000502:physiology*; D004527:Egg Proteins / Q000502:physiology*; D005260:Female; D017353:Gene Deletion; D017344:Genes, Insect; D019476:Insect Proteins / Q000502:physiology*; D008297:Male; D009154:Mutation; D015996:Survival Rate; D014960:X Chromosome
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D004527:Egg Proteins; D019476:Insect Proteins; C108443:okr protein, Drosophila; D004265:DNA Helicases; D011088:DNA Ligases