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.

Abstract

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.

Drosophila mus301/spindle-C Encodes a Helicase With an Essential Role in Double-Strand DNA Break Repair and Meiotic Progression

“…In support of our model, budding yeast mer3 encodes an ATP-dependent DNA helicase that unwinds dsDNA with a 39-59 polarity and that stimulates 39-59 heteroduplex extension by Rad51 in crossover recombination (Mazina et al 2004). Finally, okra, a gene required for the repair of DSB after P-element excision and for DNA repair during oogenesis (Kooistra et al 1997;Ghabrial et al 1998;Kooistra et al 1999;Romeijn et al 2005), is the Drosophila homolog of yeast Rad54, a SWI2/SNF2 chromatin-remodeling dsDNAdependent ATPase that binds Rad51 directly and that stimulates DSB repair in both meiotic and mitotic cells (Mazin et al 2000;Krogh and Symington 2004). Since Okra, Mus301, Spn-A, and, to a lesser extent, Spn-B are also involved in the repair of DNA damage caused by MMS treatment and ionizing radiation in mitotic cells (this work; Oliveri et al 1990;Staeva-Vieira et al 2003), it is likely that the Mus301-Rad51-Okra interaction is also maintained in DSB repair in the soma.…”

“…okra encodes the Drosophila homolog of the yeast DNA-repair protein Rad54, a chromatin-remodeling dsDNA-dependent ATPase with a known function in DSB metabolism; Spn-A is homologous to yeast Rad51, a protein with an essential role in DNA repair and meiotic checkpoint control; spn-B is another Drosophila homolog of Rad51 and has been shown to participate in the repair of meiotic DSBs; finally, spn-D encodes a Rad51C-like protein required exclusively during meiosis. Furthermore, since the patterning defects of okra, spn-A, spn-B, and spn-D mutants can be suppressed by mutations in mei-W68 and in mei-41, the primary defect in these spn mutations seems to be a failure to repair DSBs (Ghabrial et al 1998;Abdu et al 2003;Staeva-Vieira et al 2003;Romeijn et al 2005).…”

Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases

“…This might be expected when the major NHEJ process is disabled. It has been observed, however, that removing DNA ligase IV does not always reduce end joining to this extent (39)(40)(41)(42). Since lig4 Ϫ flies are viable and fertile, we see no major drawbacks to using such strains as recipients for RNA and DNA injections when gene replacements are desired.…”

Differential Usage of Alternative Pathways of Double-Strand Break Repair in Drosophila

“…Yet the overall conclusions are similar for both types of ends: (1) SDSA is used much more frequently than DHJ as evidenced by low crossover rates (Engels et al 1990;Johnson-Schlitz and Engels 1993;Nassif and Engels 1993); (2) SSA can be very efficient (Preston et al 2002;Rong and Golic 2003); and (3) the majority of NHEJ products have simple deletions, insertions, or a combination of deletion and insertion (Rong and Golic 2003;McVey et al 2004b;Weinert et al 2005). With both P-and I-SceI-induced breaks, many of the simple deletion products appear to exploit microhomology to align the ends (Staveley et al 1995;Romeijn et al 2005).…”