LEM-3 - A LEM domain containing nuclease involved in the DNA damage response in C. elegans

Abstract

The small nematode Caenorhabditis elegans displays a spectrum of DNA damage responses similar to humans. In order to identify new DNA damage response genes, we isolated in a forward genetic screen 14 new mutations conferring hypersensitivity to ionizing radiation. We present here our characterization of lem-3, one of the genes identified in this screen. LEM-3 contains a LEM domain and a GIY nuclease domain. We confirm that LEM-3 has DNase activity in vitro. lem-3(lf) mutants are hypersensitive to various types of DNA damage, including ionizing radiation, UV-C light and crosslinking agents. Embryos from irradiated lem-3 hermaphrodites displayed severe defects during cell division, including chromosome mis-segregation and anaphase bridges. The mitotic defects observed in irradiated lem-3 mutant embryos are similar to those found in baf-1 (barrier-to-autointegration factor) mutants. The baf-1 gene codes for an essential and highly conserved protein known to interact with the other two C. elegans LEM domain proteins, LEM-2 and EMR-1. We show that baf-1, lem-2, and emr-1 mutants are also hypersensitive to DNA damage and that loss of lem-3 sensitizes baf-1 mutants even in the absence of DNA damage. Our data suggest that BAF-1, together with the LEM domain proteins, plays an important role following DNA damage - possibly by promoting the reorganization of damaged chromatin.

Figure 1. lem-3(op444) phenotypes following DNA damage.

Wild-type animals and lem-3(op444) mutants were treated 24 h post L4/adult molt with (A) X-rays (B) UV-C, or (C) as L4 larvae with cisplatin. F1 embryonic lethality was assessed as described in Materials and Methods. Data shown represent the average of three independent experiments ± S.D. The progeny of 10 worms (A, B) or 5 worms (C) were analysed for each experiment. (D) Late radiation phenotypes. L1 animals were irradiated with 60 Gy. The uncoordinated phenotype (Unc) and protruding vulva phenotype (P-vul) were scored only for those animals that reached adulthood (as described in Materials and Methods). Data shown represent the average of three independent experiments ± S.D. (n for wild type, lem-3(op444) and cku-80(ok861) are 305, 236 and 373 respectively).

Figure 2. Characterization of the lem-3 locus.

(A) Gene structure of lem-3. Boxes represent exons, lines represent introns. The position and nature of the mutations are indicated. (B) LEM-3 is a 704 AA protein with 2 ankyrin repeats (ANK), a LEM domain (LEM) and a GIY-YIG domain (GIY) of the COG3680 type. (C) Expression of a wild-type copy of lem-3 rescues the radiation hypersensitivity of lem-3(op444) mutants. Data represent the average of three experiments ± S.D. (two experiments for opIs309). The progeny of 10 worms were analysed for each experiment. (D) Alignment of the C-terminus of LEM-3 of different species starting with the GIY domain. The number of the first amino acid is indicated.

Figure 3. The LEM-3 endonuclease acts in parallel to the NER pathway.

(A) Hermaphrodites were treated 24 h post L4/adult molt with 100 J/m² and F1 embryonic lethality scored as described in Materials and Methods. Data shown represent the average of three independent experiments ± S.D. The progeny of 10 worms were analysed for each experiment. (B) Non-specific endonuclease assay, in which supercoiled plasmid DNA was incubated with increasing amounts of Sf9-expressed MBP-LEM-3 fusion protein. Endonucleolytic activity of LEM-3 (WT) leads to nicks in the DNA strands and converts the supercoiled (sc) plasmid DNA first to open-circular (oc) and then into linear (lin) DNA species. Endonucleolytic activity of LEM-3 can be inhibited by the addition of EDTA. The figure is a negative image of a 0.8% agarose gel stained with ethidium bromide. M: 1 kb marker; (−): no protein; WT: LEM-3 wild type; L→F: mutant corresponding to lem-3(op444). (C) PhiX174 single-stranded DNA was incubated with increasing amounts of Sf9-expressed MBP-LEM-3 fusion protein. The single-stranded viral DNA of PhiX174 contains many secondary structures (hairpin loops, bulges etc.) that are cleaved by many structure-specific endonucleases. The figure is a negative image of a 0.8% agarose gel stained with SYBR Gold. (−): no protein; WT: LEM-3 wild type; L→F: mutant corresponding to lem-3(op444). EcExoIII, E. coli exonuclease III; HhaI, HinfI, MboI, restriction endonucleases.

Figure 4. lem-3(op444) mutants show normal cell cycle arrest and apoptotic responses.

(A) Schematic representation of a C. elegans gonad. The most distal part is capped by the somatic distal tip cell. The cells close to the DTC are in mitosis. As they travel down the gonad, they enter the pachytene stage of meiosis I. Near the gonadal bend, some cells die by apoptosis. Apoptotic bodies become visible as “refractive disks”. The mature oocytes are then pushed through the spermatheca and become fertilized. (B) Quantification of cell cycle arrest response following IR. The number of nuclei within 50 µm of the distant tip cell ± S.D was scored at various time points. n, number of germ lines analyzed. (C) Cell cycle arrest is normal in lem-3(op444) mutants. Representative DIC images of dissected gonads from wild-type and lem-3(op444) mutants are shown. Size bar: 10 µm. Black arrowheads indicate small (cycling) cell nuclei, white arrow heads indicate big (arrested) cells. (D) DNA damage-induced apoptosis is normal in lem-3(op444) mutants. Hermaphrodites were exposed to 60 Gy at L4/adult molt and apoptotic corpses were quantified at the indicated time points. Data shown represent the average of three independent experiments (10 germ lines per experiment) ± S.D.

Figure 5. RAD-54 foci formation and removal is normal in lem-3(op444) mutants.

DIC and fluorescence images of control (A) and irradiated (B) embryos expressing YFP::RAD-54(opIs257). RAD-54 foci (arrow heads for selected nuclei) are induced in the mitotic zone upon irradiation. (C) By 17 h post IR, foci are largely removed in both wild-type and lem-3(op444) animals. Size bar: 10 µm. (D) Quantification analysis of YFP::RAD-54 foci formation. n, number of germ lines analyzed.

Figure 6. Chromatin segregation defects in lem-3(op444) embryos after irradiation.

Embryos were dissected from irradiated hermaphrodites, DNA was stained with Dapi. Representative DIC and fluorescence pictures are shown. Arrows point to abnormally localized chromatin.

Figure 7. LEM-domain proteins and BAF-1 cooperate to protect embryos from DNA damage-induced lethality.

(A) Hermaphrodites were treated 36 h post L4/adult molt with 15 Gy and F1 embryonic lethality was scored 10 h later as described in Materials and Methods. All assays were done on 15°C, which is the permissive temperature for baf-1(t1639ts). All strains used had unc-32(e189) in the background. Data shown represent the average of three independent experiments ± S.D. The progeny of 10 worms were analysed for each experiment. (B) Animals were treated 24 h post L4/adult molt with 30 Gy. Data shown represent the average of three independent experiments ± S.D. The progeny of 10 worms were analysed for each experiment. (C) Model of lem-3 and baf-1 interaction. We propose that the requirements for BAF-1 activity are higher following DNA damage. In wild-type animals, LEM-domain proteins stimulate BAF-1 activity sufficiently to protect embryos from death. In the absence of LEM-3 this level is no longer met, and thus leads to embryonic lethality following DNA damage. In baf-1 ts mutants at permissive temperature, BAF-1 activity is compromised and does not meet the requirement following DNA damage, in the absence of LEM-3 it is further reduced so that it is even insufficient under normal conditions.