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Site-specific double-strand breaks (DSBs) were generated in the white gene located on the X chromosome of Drosophila by excision of the whd P-element. To investigate the role of nonhomologous end joining (NHEJ) and homologous recombination (HR) in the repair of these breaks, the whd 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 whd 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.

genetics

Lig4 and Rad54 Are Required for Repair of DNA Double-Strand Breaks Induced by P-Element Excision in DrosophilaThis article is dedicated to the memory of our colleague and friend Dr. Jan C. J. Eeken, who died unexpectedly on May 24, 2002.