C. elegans mre-11 is required for meiotic recombination and DNA repair but is dispensable for the meiotic G(2) DNA damage checkpoint

Abstract

We investigated the roles of Caenorhabditis elegans MRE-11 in multiple cellular processes required to maintain genome integrity. Although yeast Mre11 is known to promote genome stability through several diverse pathways, inviability of vertebrate cells that lack Mre11 has hindered elucidation of the in vivo roles of this conserved protein in metazoan biology. Worms homozygous for an mre-11 null mutation are viable, allowing us to demonstrate in vivo requirements for MRE-11 in meiotic recombination and DNA repair. In mre-11 mutants, meiotic crossovers are not detected, and oocyte chromosomes lack chiasmata but appear otherwise intact. gamma-irradiation of mre-11 mutant germ cells during meiotic prophase eliminates progeny survivorship and induces chromosome fragmentation and other cytologically visible abnormalities, indicating a defect in repair of radiation-induced chromosome damage. Whereas mre-11 mutant germ cells are repair-deficient, they retain function of the meiotic G(2) DNA damage checkpoint that triggers germ cell apoptosis in response to ionizing radiation. Although mre-11/mre-11 animals derived from heterozygous parents are viable and produce many embryos, there is a marked drop both in the number and survivorship of embryos produced by succeeding generations. This progressive loss of fecundity and viability indicates that MRE-11 performs a function essential for maintaining reproductive capacity in the species.

Figure 1

C. elegans MRE-11 predicted protein sequence aligned with orthologs from human, mouse, Drosophila, fission yeast, and budding yeast. The solid underline corresponds to the conserved phosphoesterase domain, with the thicker segments indicating motifs conserved between Mre11 and E. coli SbcD nuclease. The asterisk marks the alternative initiator methionine predicted by cDNAs obtained by 5′ RACE. The position of the glutamate to lysine change caused by the mre-11(me41) missense mutation is indicated above the sequence alignment. The in-frame deletion in mre-11(ok179) removes the segment of sequence encoding the amino acids indicated by the dashed line below the alignment.

Figure 2

Chiasmata are absent in mre-11 mutants but pachytene morphology and homolog pairing are normal. (a–c) Single DAPI-stained oocyte nuclei at diakinesis, the final stage of meiotic prophase. Each panel shows a projection of a three-dimensional data stack through an entire nucleus. (a) Wild-type nucleus with six DAPI-stained bodies, corresponding to six pairs of homologous chromosomes attached by chiasmata. (b,c) mre-11 mutant oocytes with 12 univalents, indicating an absence of chiasmata. (d–f) Fields of nuclei at the pachytene stage earlier in meiotic prophase. Images shown are projections halfway through the nuclei to highlight nuclear organization. (d) Pachytene nuclei in wild-type. Discrete tracks of DAPI-stained chromatin corresponding to pairs of side-by-side aligned and synapsed chromosomes are arranged at the periphery of each nucleus. (e,f) Pachytene nuclei in the mre-11 mutants. DAPI-stained chromatin tracks are similar in thickness, number, and arrangement to those seen in wild-type pachytene nuclei. (g–i) Homolog pairing assayed by FISH in wild-type (g) and mre-11 mutant (h,i) nuclei in the pachytene region of the germ-line. DAPI-stained chromosomes are shown in blue, and a probe from the left end of chromosome I is shown in orange. A single hybridization signal indicative of intimate pairing is visible in each nucleus for all three genotypes. Images shown are projections of three-dimensional data stacks through entire nuclei. Scale bars, 2 μm.

Figure 3

IR-induced chromosome defects in mre-11 mutant oocytes. Wild-type and mre-11 worms at the late L4 larval stage were exposed to 5 krad of γ-irradiation, and their germ lines were fixed with formaldehyde 18 h later and stained with DAPI. Oocyte nuclei examined at the diakinesis stage at this time were likely at the pachytene stage at the time of irradiation. (a) Wild-type oocyte nucleus, which contains six pairs of homologs attached by chiasmata; the chromosomes appear morphologically normal and intact. (b–f) mre-11 mutant oocytes exhibiting multiple chromosomal abnormalities. Chromosomes tend to be clumped or aggregated with a frayed appearance. Chromosome fragments are indicated by arrows in c and f. Images are projections of three-dimensional data stacks through entire nuclei. Scale bars, 2 μm.

Figure 4

A functional meiotic G₂ DNA damage checkpoint in mre-11 mutants. Late-stage L4 hermaphrodites were exposed to 0, 5, or 10 krad of γ-irradiation, and induction of germ cell apoptosis (a readout of the meiotic G₂ DNA damage checkpoint) was assessed 24–28 h later using Nomarski microscopy. The y-axis indicates the number of apoptotic nuclei observed per gonad arm. Fifty-two to seventy gonad arms were scored for each data point; error bars indicate standard error of the mean.