mus304 encodes a novel DNA damage checkpoint protein required during Drosophila development

Abstract

Checkpoints block cell cycle progression in eukaryotic cells exposed to DNA damaging agents. We show that several Drosophila homologs of checkpoint genes, mei-41, grapes, and 14-3-3epsilon, regulate a DNA damage checkpoint in the developing eye. We have used this assay to show that the mutagen-sensitive gene mus304 is also required for this checkpoint. mus304 encodes a novel coiled-coil domain protein, which is targeted to the cytoplasm. Similar to mei-41, mus304 is required for chromosome break repair and for genomic stability. mus304 animals also exhibit three developmental defects, abnormal bristle morphology, decreased meiotic recombination, and arrested embryonic development. We suggest that these phenotypes reflect distinct developmental consequences of a single underlying checkpoint defect. Similar mechanisms may account for the puzzling array of symptoms observed in humans with mutations in the ATM tumor suppressor gene.

Figure 1

A DNA damage checkpoint in wild-type and mutant imaginal discs. Larvae were either unirradiated (A,G,J) or irradiated with 4000 rads of X-rays (B–F, H–I, K–L). Discs were dissected and fixed 1 hr following irradiation. Histochemical staining with a mitotic-specific antibody reveals the number and distribution of mitotic cells. The genotype of each disc is indicated. Alleles are listed in Materials and methods. (A–I) Eye-antennal imaginal discs with anterior left and dorsal on top. In each disc, the round tissue on the left is the antennal disc and the oval tissue to the right is the eye disc. (J–L) Wing imaginal discs with the future notum to the left and the wing pouch to the right. (M) A time course of mitotic cells in irradiated wild-type and mus304¹/Df(3L)WR4 eye discs. Irradiation with 4000 rads was performed at time zero. Error bars, s.e.m..

Figure 2

Rough eye phenotypes in checkpoint mutants. Scanning electron micrographs of adult male eyes are shown with anterior to the left and dorsal on top. (A) Wild type. (B) mus304¹/mus304². (C) mus304¹, +/mus304², E(mus304). (D) mei-41^29D/Y; +/E(mus304)¹. (E) 41^29D/Y; mus304¹, +/mus304², E(mus304). (F) Wild type following irradiation with 2000 rads during third instar.

Figure 3

High rates of LOH in mus304 animals. (A) The genetic basis of the LOH assay. The fertilized egg is heterozygous for a mutation in the mwh gene. If the wild-type copy of the gene is lost during the proliferation of a wing cell precursor, that cell's progeny will exhibit the recessive mwh phenotype. Loss of the wild-type gene can occur because of a chromosome break (shown here), point mutation, chromosome loss, or nondisjunction. (B) Individual wing hairs in an animal heterozygous for mwh. Each hair cell has one hair. There are ∼20,000 hair cells per wing. (C) In an animal homozygous for mwh, each hair cell has two or more hairs. (D) In an animal heterozygous for mwh and homozygous for a mutation that increases genomic instability, individual cells that lost the wild-type mwh gene (arrow) can be seen within a field of heterozygous cells. (E) The frequency of mwh clones per wing in wild-type and mus304 animals. Error bars, s.e.m..

Figure 4

Macrochaete defects in mus304 adults. (A) The genetic basis of bristle defects due to genomic instability. Minute loci (M) are present throughout the genome and are haploinsufficient for macrochaete (bristle) development. If one of the two wild-type copies of a Minute locus is lost in a macrochaete precursor, the resulting macrochaete will exhibit a Minute phenotype. (B) A wild-type notum. Two specific macrochaetae are indicated (arrows). (C) A mus304 notum. The indicated macrochaetae exhibit a Minute phenotype (i.e., shorter and thinner). (D) The frequency of Minute bristles in different genetic backgrounds. Error bars, s.e.m..

Figure 5

Cell cycle defects in mus304 embryos. (A) Nuclear morphologies are visualized in wild-type and mus304 embryos using a maternally contributed Histone–GFP fusion protein. Following interphase 14, the mus304 embryo enters an additional, asynchronous mitosis. (B) The length of interphase (white bars) and M phase (black bars) is indicated for the wild-type and mus304 embryos shown in A.

Figure 6

Disrupted zygotic gene expression in irradiated or mus304 embryos. Embryos from wild-type mothers (A,D,E,F,I,J,K) or mus304 mothers (B,C,G,H,L,M) were processed for either runt RNA expression (A–E) and DAPI staining (F–I), or cyclin B RNA expression (J–M) and DAPI staining (not shown). The nuclear division cycle for each embryo, as determined by timed collection and DAPI staining, is indicated in parentheses. X-ray indicates wild-type embryos irradiated 1 hr prior to fixation (D,E,I). Note that the stripes of staining by the RNA in situ procedure interferes with the DAPI signal, causing the apparent stripes in F.

Figure 7

mus304 encodes a novel protein targeted to the cytoplasm. (A) mus304 gene structure. (Top) A map of the mus304 genomic sequence with the following restriction enzyme sites indicated: EcoRI (Ec); SacII (Sa); XbaI (Xb); XhoI (Xh). The broken lines indicate genomic DNA deleted in two deficiencies that contain mus304. The mus304 cDNA (GenBank accession no. AF224715) is shown to scale with the restriction map. Untranslated regions and introns are indicated as a thin line and coding regions are indicated as boxes. The position of each mutant allele is shown; the allele number is followed by the wild-type amino acid, the amino acid number, and the mutant amino acid or stop codon. The position of a potential dimeric coiled-coil region within the coding region is highlighted in gray. (B) mus304 RNA expression in a wild-type ovary. (C) mus304 RNA expression in a wild-type interphase 13 embryo. (D) mus304 RNA expression in a wild-type interphase 14 embryo. (E) Anti-Flag staining (red) of an interphase S2 cell transfected with a Flag–MUS304 transgene. (F) DAPI staining (blue) of the cell in E. (G) Anti-Flag staining (red) of a mitotic S2 cell transfected with a Flag–MUS304 transgene. (H) DAPI staining (blue) of the cell in G.