The Drosophila melanogaster RAD54 Homolog, DmRAD54, Is Involved in the Repair of Radiation Damage and Recombination

Abstract

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRAD54-deficient flies develop normally, but the females are sterile. Early development appears normal, but the eggs do not hatch, indicating an essential role for DmRAD54 in development. The larvae of mutant flies are highly sensitive to X rays and methyl methanesulfonate. Moreover, this mutant is defective in X-ray-induced mitotic recombination as measured by a somatic mutation and recombination test. These phenotypes are consistent with a defect in the repair of double-strand breaks and imply that the RAD54 gene is crucial in repair and recombination in a multicellular organism. The results also indicate that the recombinational repair pathway is functionally conserved in evolution.Double-strand breaks (DSBs) in DNA are induced by ionizing radiation and by various chemical compounds, such as free radicals and methyl methanesulfonate (MMS). In addition, DSBs arise as intermediates during V(D)J rearrangement in differentiating lymphocytes, transposition events, and meiotic recombination in germ cells. Unrepaired DSBs often lead to cell death or contribute to the formation of chromosomal aberrations such as deletions and translocations. In eukaryotes, DSBs can be repaired via two main pathways: (i) recombinational repair, which is dependent on the presence of an intact duplicate DNA sequence, and (ii) end-to-end rejoining, which is based on rejoining of the two DNA ends (46). The available evidence suggests that both mechanisms are conserved in eukaryotes from yeasts to humans. However, the relative contributions of both mechanisms in repair of DSBs differ considerably between lower and higher eukaryotes. Repair of DSBs in mammals has been investigated with radiation-sensitive rodent cell lines. Several of these cell lines have defects in repairing radiation-induced DSBs and are also strongly impaired in V(D)J rearrangement. Further studies have revealed that these mutants have defects in the end-to-end rejoining pathway (reviewed in references 26, 47, and 63). Repair of DSBs at defined sites in the genome of mammalian cells also occurs more frequently by end-to-end rejoining than by homologous recombination (21, 33). The same observations have been made in experiments studying the fate of linear plasmid molecules in mammalian cells and Xenopus laevis oocytes (27,29,31). Therefore, DSBs in higher eukaryotes appear to be repaired primarily by end-to-end rejoining mechanisms.In lower eukaryotes, however, repair of DSBs occurs almost exclusively by...

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.…”

Transcript profiling of Wilms tumors reveals connections to kidney morphogenesis and expression patterns associated with anaplasia

“…Increased expression of Huntingtin in anaplastic tumors might have led to inhibition of apoptosis and induction of BDNF and/or TrkB in the tumors. Reduced homologous recombination and ionizing radiation or X-ray resistance have been observed in RAD54 deficiency yeast, Drosophila, chick cells and mice (Bezzubova et al, 1997;Clever et al, 1997;Essers et al, 1997;Kooistra et al, 1997Kooistra et al, , 1999. Elevation of hRAD54 expression in anaplastic tumors may also contribute to their therapy resistant phenotype.…”

Promotion of Rad51-dependent D-loop formation by yeast recombination factor Rdh54/Tid1

“…In mouse embryonic stem cells, chicken DT40 cells, and Drosophila, deletion of the RAD54 gene engenders defects in recombination and repair of DNA damage induced by ionizing radiation, methyl methanesulfonate, and the DNA cross-linking agent mitomycin C (Esser et al 1997;Kooistra et al 1999;Takata et al 1999). Recently, another Rad54/Rdh54-like factor, Rad54B, has been identified in humans (Hiramoto et al 1999;Matsuda et al 1999).…”