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Molecular and Cellular Biology, September 1999, p. 6269-6275, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Rolf Kooistra, Albert Pastink,* José B. M. Zonneveld, Paul H. M. Lohman, and Jan C. J. Eeken

Department of Radiation Genetics and Chemical Mutagenesis, MGC, Leiden University Medical Center, Leiden, The Netherlands

Received 22 April 1999/Returned for modification 23 May 1999/Accepted 14 June 1999

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.

* Corresponding author. Mailing address: Department of Radiation Genetics and Chemical Mutagenesis, MGC, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. Phone: 31-71-5276152. Fax: 31-71-5221615. E-mail: Pastink{at}rullf2.medfac.leidenuniv.nl.

Molecular and Cellular Biology, September 1999, p. 6269-6275, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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