The Drosophila melanogaster DmRAD54 gene plays a crucial role in double-strand break repair after P-element excision and acts synergistically with Ku70 in the repair of X-ray damage

Abstract

The RAD54 gene has an essential role in the repair of double-strand breaks (DSBs) via homologous recombination in yeast as well as in higher eukaryotes. A Drosophila melanogaster strain deficient in the RAD54 homolog DmRAD54 is characterized by increased X-ray and methyl methanesulfonate (MMS) sensitivity. In addition, DmRAD54 is involved in the repair of DNA interstrand cross-links, as is shown here. However, whereas X-ray-induced loss-of-heterozygosity (LOH) events were completely absent in DmRAD54(-/-) flies, treatment with cross-linking agents or MMS resulted in only a slight reduction in LOH events in comparison with those in wild-type flies. To investigate the relative contributions of recombinational repair and nonhomologous end joining in DSB repair, a DmRad54(-/-)/DmKu70(-/-) double mutant was generated. Compared with both single mutants, a strong synergistic increase in X-ray sensitivity was observed in the double mutant. No similar increase in sensitivity was seen after treatment with MMS. Apparently, the two DSB repair pathways overlap much less in the repair of MMS-induced lesions than in that of X-ray-induced lesions. Excision of P transposable elements in Drosophila involves the formation of site-specific DSBs. In the absence of the DmRAD54 gene product, no male flies could be recovered after the excision of a single P element and the survival of females was reduced to 10% compared to that of wild-type flies. P-element excision involves the formation of two DSBs which have identical 3' overhangs of 17 nucleotides. The crucial role of homologous recombination in the repair of these DSBs may be related to the very specific nature of the breaks.

FIG. 1

(A) Mechanism of induction and repair of a single DSB after P-element excision. The w^hd element has an internal deletion and does not code for a functional P-element transposase. It is excised by the transposase encoded by the Δ2-3 element on the third chromosome, leaving behind a DSB. In males this DSB can be repaired via homologous recombination using the w^alter template, located at position 25F on the second chromosome, resulting in a functional white gene. Repair via NHEJ will in most cases result in a nonfunctional white gene. Females can also use the w^a allele on the other X chromosome as a template. (B) Cross to combine the w^hd transposition system with a DmRAD54-deficient background. Cy and Ubx are dominant marker mutations on the second and third balancer chromosomes, respectively. The third chromosome, carrying the Δ2-3 element, is marked by the dominant Sb mutation. The males used in this cross carry the w^a mutation on the X chromosome to ensure a white-eye background in the female offspring. In the control cross, DmRAD54-proficient females carrying a second chromosome containing only the w^alter construct were used.

FIG. 2

Somatic recombination induced by DNA-damaging agents in a DmRAD54-deficient background. DmRAD54-proficient control larvae (▵) and DmRAD54-deficient larvae (▴) were treated with increasing doses of ionizing radiation (A), the cross-linking agent MMC (B) or cisDDP (C), or the alkylating agent MMS (D). Twelve to 18 days after treatment, the eyes of the adult flies were scored for colorless spots. Each spot is caused by one LOH event. Results are presented as spots per eye; for each point, at least 200 eyes were scored.