Multiple genetic pathways involving the Caenorhabditis elegans Bloom's syndrome genes him-6, rad-51, and top-3 are needed to maintain genome stability in the germ line

Abstract

Bloom's syndrome (BS) is an autosomal-recessive human disorder caused by mutations in the BS RecQ helicase and is associated with loss of genomic integrity and an increased incidence of cancer. We analyzed the mitotic and the meiotic roles of Caenorhabditis elegans him-6, which we show to encode the ortholog of the human BS gene. Mutations in him-6 result in an enhanced irradiation sensitivity, a partially defective S-phase checkpoint, and in reduced levels of DNA-damage induced apoptosis. Furthermore, him-6 mutants exhibit a decreased frequency of meiotic recombination that is probably due to a defect in the progression of crossover recombination. In mitotically proliferating germ cells, our genetic interaction studies, as well as the assessment of the number of double-strand breaks via RAD-51 foci, reveal a complex regulatory network that is different from the situation in yeast. Although the number of double-strand breaks in him-6 and top-3 single mutants is elevated, the combined depletion of him-6 and top-3 leads to mitotic catastrophe concomitant with a massive increase in the level of double-strand breaks, a phenotype that is completely suppressed by rad-51. him-6 and top-3 are thus needed to maintain low levels of double-strand breaks in normally proliferating germ cells, and both act in partial redundant pathways downstream of rad-51 to prevent mitotic catastrophy. Finally, we show that topoisomerase IIIalpha acts independently during a late stage of meiotic recombination.

FIG. 1.

him-6 encodes a RecQ-like helicase. (A) him-6 gene structure. The mutation him-6(ok412) is a 1,687-bp deletion of five exons. The numbers indicate the nucleotide positions in the cosmid T04A11. (B) Schematic representation of RecQ-like DNA helicase; the name of the gene product and its length in amino acids is shown on the left, and the name of the organism is shown on the right. Helicase domains are shown as gray boxes; acidic domains are shown as black boxes. The proteins were arranged by aligning the helicase domains. The percent values indicate the level of conservation throughout the helicase domain between HIM-6 and its counterparts in other organisms. Arrowheads indicate residues mutated in him-6 alleles: K479Stop in e1423 and G561Q in e1104. The HIM-6 GenBank accession number is AY095296.

FIG. 2.

Meiotic phenotypes of him-6. (A) Wild-type gonad (left panel) and him-6(e1423) gonad (right panel) stained with DAPI, which show a normal progression of the germ cells through meiotic prophase. (B) Wild-type oocyte at diakinesis stained with DAPI. Six stained bodies can be observed corresponding to the six sets of homologous chromosomes attached by chiasmata, him-6(e1423) oocyte and him-6(ok412) oocyte: more than six structures are present, which correspond to a mix of bivalents and univalents. (C) Meiotic prophase nuclei stained with anti-RAD-51 antibody (red) and DAPI (blue). The RAD-51 immunostaining reveals similar foci pattern in wild-type and him-6(e1423) transition zone and early-mid-pachytene nuclei. Foci are absent from late pachytene nuclei in wild type but persist in the him-6(e1423) mutant. (D) Meiotic prophase nuclei stained with anti-HIM-3 antibody (green) and DAPI (red). The staining revealed no pairing defects in the transition zone and pachytene nuclei of the him-6 mutant. MZ, mitotic zone; TZ, transition zone; EMP, early-mid pachytene; LP, late pachytene; P, pachytene; DK, diakinesis. Scale bars, 10 μm.

FIG. 3.

him-6 mutants are defective in responding to DNA damage. (A) him-6 germ cells are more sensitive to gamma irradiation. To compare the irradiation sensitivity (IR-sens) curve of the mutant, the survival rates of N2, him-6(e1423), and him-6(ok412) are set to 100 at 0 Gy, although the survival rate of the him-6(e1423) and him-6(ok412) progenies is only 21 and 36%, respectively. (B) him-6(e1423) mutant show decreased germ cell apoptosis. Late-stage L4 hermaphrodites were exposed to 0, 60, and 120 Gy of gamma irradiation and apoptosis was scored after 2, 4 and 6 h later as described previously (19). The y axis indicates the number of dead cells (cell corpses) per gonad arm. (C) him-6 germ lines are sensitive to HU. him-6 and wild-type worms were grown on plates containing 0, 1, 5, 10, or 25 mM HU. Cell cycle arrest was measured by counting the average number of cells in a defined volume of the germ line. The him-6 mutants show a higher number of germ cells upon HU treatment compared to the wild type. (D) Nomarski pictures of wild-type and him-6(e1423) mitotic zones exposed to 0 and 5 mM HU. him-6(e1423) has more mitotic nuclei when exposed to 5 mM HU due to a defect in cell cycle arrest.

FIG. 4.

top-3(RNAi); him-6(e1423) worms show mitotic catastrophy and massive RAD-51 recruitment in the germ line. The defects are suppressed by mutation in rad-51. (A) top-3(RNAi); him-6(e1423) gonad stained with DAPI, which shows germ line nuclei with severe chromosomal abnormalities; (B) top-3(RNAi); him-6(e1423) gonad stained with DAPI (blue) and anti-RAD-51 antibody (red); (C) mitotic germ nuclei of wild type, him-6(e1423) and top-3(RNAi) worms stained with DAPI (blue) and anti-RAD-51 antibody (red), which revealed a higher number of RAD-51 foci compared to wild type; (D) top-3(RNAi); rad-51(lg08701)him-6(e1423) gonad stained with DAPI, which shows a normal proliferation of the germ line nuclei and a normal progression through meiotic prophase. Scale bars, 10 μm.

FIG. 5.

top-3(RNAi) worms show defects, which depend on meiotic recombination. (A) top-3(RNAi) gonad stained with DAPI, which revealed an extended transition zone. (B) Transition zone nuclei stained with an anti-RAD-51 antibody (red) and DAPI (blue). top-3(RNAi) nuclei show an increased number of RAD-51 foci compared to wild type. (C) top-3(RNAi); spo-11(ok79) gonad stained with DAPI, which shows a normal progression of the germ nuclei through meiotic prophase. Arrows point toward nuclei in the transition zone (TZ) and at diakinesis (DK). (D) RAD-51 foci are absent from top-3(RNAi); spo-11(ok79) nuclei in the transition zone and in early pachytene. (E) top-3(RNAi); rad-51(lg08701) gonad stained with DAPI, which shows a normal proliferation of the germ line nuclei and a normal progression through meiotic prophase. Scale bars, 10 μm.

FIG. 6.

him-6 functions during meiosis and mitosis. During meiotic recombination him-6 functions downstream of spo-11 and rad-51. We cannot address the epistatic relationship between top-3 and him-6 during meiotic recombination as top-3; him-6 worms do not enter meiosis. During mitotic proliferation of the germ line, him-6 acts partially redundantly with top-3 on DNA lesions that have been processed by rad-51.