AP2M1 Supports TGF-ß Signals to Promote Collagen Expression by Inhibiting Caveolin Expression.

The extracellular matrix (ECM) is important for normal development and disease states, including inflammation and fibrosis. To understand the complex regulation of ECM, we performed a suppressor screening using Caenorhabditis elegans expressing the mutant ROL-6 collagen protein. One cuticle mutant has a mutation in dpy-23 that encodes the µ2 adaptin (AP2M1) of clathrin-associated protein complex II (AP-2). The subsequent suppressor screening for dpy-23 revealed the lon-2 mutation. LON-2 functions to regulate body size through negative regulation of the tumor growth factor-beta (TGF-ß) signaling pathway responsible for ECM production. RNA-seq analysis showed a dominant change in the expression of collagen genes and cuticle components. We noted an increase in the cav-1 gene encoding caveolin-1, which functions in clathrin-independent endocytosis. By knockdown of cav-1, the reduced TGF-ß signal was significantly restored in the dpy-23 mutant. In conclusion, the dpy-23 mutation upregulated cav-1 expression in the hypodermis, and increased CAV-1 resulted in a decrease of TßRI. Finally, the reduction of collagen expression including rol-6 by the reduced TGF-ß signal influenced the cuticle formation of the dpy-23 mutant. These findings could help us to understand the complex process of ECM regulation in organism development and disease conditions.

A novel functional cross-interaction between opioid and pheromone signaling may be involved in stress avoidance in Caenorhabditis elegans.

Upon sensing starvation stress, Caenorhabditis elegans larvae (L2d) elicit two seemingly opposing behaviors to escape from the stressful condition: food-seeking roaming mediated by the opioid peptide NLP-24 and dauer formation mediated by pheromones. Because opioid and pheromone signals both originate in ASI chemosensory neurons, we hypothesized that they might act sequentially or competitively to avoid starvation stress. Our data shows that NPR-17 opioid receptor signaling suppressed pheromone biosynthesis and the overexpression of opioid genes disturbed dauer formation. Likewise, DAF-37 pheromone receptor signaling negatively modulated nlp-24 expression in the ASI neurons. Under short-term starvation (STS, 3 h), both pheromone and opioid signaling were downregulated in gpa-3 mutants. Surprisingly, the gpa-3;nlp-24 double mutants exhibited much higher dauer formation than seen in either of the single mutants. Under long-term starvation (LTS, >24 h), the stress-activated SKN-1a downregulated opioid signaling and then enhanced dauer formation. Both insulin and serotonin stimulated opioid signaling, whereas NHR-69 suppressed opioid signaling. Thus, GPA-3 and SKN-1a are proposed to regulate cross-antagonistic interaction between opioids and pheromones in a cell-specific manner. These regulatory functions are suggested to be exerted via the selective interaction of GPA-3 with NPR-17 and site-specific SKN-1 binding to the promoter of nlp-24 to facilitate stress avoidance.

Hypersensitivity to DNA double-strand breaks associated with PARG deficiency is suppressed by exo-1 and polq-1 mutations in Caenorhabditis elegans.

Deficiency of either of the two homologs of poly(ADP-ribose) glycohydrolase (PARG), PARG-1 and PARG-2, in Caenorhabditis elegans leads to hypersensitivity to ionizing radiation (IR). In the germ cells of parg-2 mutant worms, the dissipation of recombinase RAD-51 foci was slower than in wild-type (WT) cells, suggesting defects in DNA double-strand break (DSB) repair via homologous recombination (HR). Nevertheless, RPA-1, the large subunit of replication protein A, accumulated faster in parg-2 worms and disappeared earlier than in WT worms. This accelerated RPA-1 accumulation may result from the enhanced expression of exonuclease-1 (EXO-1) after IR treatment. Accordingly, an exo-1 mutation reduced IR sensitivity and accumulation of RPA-1 in parg-2 worms. A mutation of polq-1, encoding for a key factor in the alternative end-joining (Alt-EJ) pathway, suppressed the IR hypersensitivity phenotype of parg-2 worms and normalized the kinetics of RAD-51 dissipation. This indicates that error-prone Alt-EJ may mediate DSB repair in parg-2 worms, causing hypersensitivity to IR. In summary, PARG-2 deficiency in C. elegans causes hyperactive DSB end resection likely through EXO-1 overproduction. DSBs with long single-stranded DNA ends in parg-2 worms are thought to be repaired by Alt-EJ instead of HR, causing genomic instability.

Single-strand annealing mediates the conservative repair of double-strand DNA breaks in homologous recombination-defective germ cells of Caenorhabditis elegans.

A missense mutation in C. elegans RAD-54, a homolog of RAD54 that operates in the homologous recombination (HR) pathway, was found to decrease ATPase activity in vitro. The hypomorphic mutation caused hypersensitivity of C. elegans germ cells to double-strand DNA breaks (DSBs). Although the formation of RAD-51 foci at DSBs was normal in both the mutant and knockdown worms, their subsequent dissipation was slow. The rad-54-deficient phenotypes were greatly aggravated when combined with an xpf-1 mutation, suggesting a conservative role of single-strand annealing (SSA) for DSB repair in HR-defective worms. The phenotypes of doubly-deficient rad-54;xpf-1 worms were partially suppressed by a mutation of lig-4, a nonhomologous end-joining (NHEJ) factor. In summary, RAD-54 is required for the dissociation of RAD-51 from DSB sites in C. elegans germ cells. Also, NHEJ and SSA exert negative and positive effects, respectively, on genome stability when HR is defective.

The Caenorhabditis elegans WRN helicase promotes double-strand DNA break repair by mediating end resection and checkpoint activation.

The protein associated with Werner syndrome (WRN), is involved in DNA repair, checkpoint activation, and telomere maintenance. To better understand the involvement of WRN in double-strand DNA break (DSB) repair, we analyzed the combinatorial role of WRN-1, the Caenorhabditis elegans WRN helicase, in conjunction with EXO-1 and DNA-2 nucleases. We found that WRN-1 cooperates with DNA-2 to resect DSB ends in a pathway acting in parallel to EXO-1. The wrn-1 mutants show an aberrant accumulation of replication protein A (RPA) and RAD-51, and the same pattern of accumulation is also observed in checkpoint-defective strains. We conclude that WRN-1 plays a conserved role in the resection of DSB ends and mediates checkpoint signaling, thereby influencing levels of RPA and RAD-51.

Roles of Caenorhabditis elegans WRN Helicase in DNA Damage Responses, and a Comparison with Its Mammalian Homolog: A Mini-Review.

Werner syndrome protein (WRN) is unusual among RecQ family DNA helicases in having an additional exonuclease activity. WRN is involved in the repair of double-strand DNA breaks via the homologous recombination and nonhomologous end joining pathways, and also in the base excision repair pathway. In addition, the protein promotes the recovery of stalled replication forks. The helicase activity is thought to unwind DNA duplexes, thereby moving replication forks or Holliday junctions. The targets of the exonuclease could be the nascent DNA strands at a replication fork or the ends of double-strand DNA breaks. However, it is not clear which enzyme activities are essential for repairing different types of DNA damage. Model organisms such as mice, flies, and worms deficient in WRN homologs have been investigated to understand the physiological results of defects in WRN activity. Premature aging, the most remarkable characteristic of Werner syndrome, is also seen in the mutant mice and worms, and hypersensitivity to DNA damage has been observed in WRN mutants of all three model organisms, pointing to conservation of the functions of WRN. In the nematode Caenorhabditis elegans, the WRN homolog contains a helicase domain but no exonuclease domain, so that this animal is very useful for studying the in vivo functions of the helicase without interference from the activity of the exonuclease. Here, we review the current status of investigations of C. elegans WRN-1 and discuss its functional differences from the mammalian homologs.

A PHF8 homolog in C. elegans promotes DNA repair via homologous recombination.

PHF8 is a JmjC domain-containing histone demethylase, defects in which are associated with X-linked mental retardation. In this study, we examined the roles of two PHF8 homologs, JMJD-1.1 and JMJD-1.2, in the model organism C. elegans in response to DNA damage. A deletion mutation in either of the genes led to hypersensitivity to interstrand DNA crosslinks (ICLs), while only mutation of jmjd-1.1 resulted in hypersensitivity to double-strand DNA breaks (DSBs). In response to ICLs, JMJD-1.1 did not affect the focus formation of FCD-2, a homolog of FANCD2, a key protein in the Fanconi anemia pathway. However, the dynamic behavior of RPA-1 and RAD-51 was affected by the mutation: the accumulations of both proteins at ICLs appeared normal, but their subsequent disappearance was retarded, suggesting that later steps of homologous recombination were defective. Similar changes in the dynamic behavior of RPA-1 and RAD-51 were seen in response to DSBs, supporting a role of JMJD-1.1 in homologous recombination. Such a role was also supported by our finding that the hypersensitivity of jmjd-1.1 worms to ICLs was rescued by knockdown of lig-4, a homolog of Ligase 4 active in nonhomologous end-joining. The hypersensitivity of jmjd-1.1 worms to ICLs was increased by rad-54 knockdown, suggesting that JMJD-1.1 acts in parallel with RAD-54 in modulating chromatin structure. Indeed, the level of histone H3 Lys9 tri-methylation, a marker of heterochromatin, was higher in jmjd-1.1 cells than in wild-type cells. We conclude that the histone demethylase JMJD-1.1 influences homologous recombination either by relaxing heterochromatin structure or by indirectly regulating the expression of multiple genes affecting DNA repair.

Transgene-mediated co-suppression of DNA topoisomerase-1 gene in Caenorhabditis elegans.

Ectopic expression of multi-transgenic copies can result in reduced expression of the transgene and can induce silence of endogenous gene; this process is called as co-suppression. Using a transgene-mediated co-suppression technique, we demonstrated the biological function of DNA topoisomerase-1 (top-1) in C. elegans development. Introduction of full-length top-1 transgene sufficiently induced the co-suppression of endogenous top-1 gene, causing embryonic lethality and abnormal germline development. We also found that the co-suppression of top-1 gene affected morphogenesis, lifespan and larval growth that were not observed in top-1 (RNAi) animals. Strikingly, co-suppression effects were significantly reduced by the elimination of top-1 introns, suggesting that efficient co-suppression may require intron(s) in C. elegans. Sequence analysis revealed that the introns 1 and 2 of top-1 gene possess consensus binding sites for several transcription factors, including MAB-3, LIN-14, TTX-3/CEH-10, CEH-1, and CEH-22. Among them, we examined a genetic link between ceh-22 and top-1. The ceh-22 is partially required for the specification of distal tip cells (DTC), which functions as a stem cell niche in the C. elegans gonad. Intriguingly, top-1 (RNAi) significantly enhanced DTC loss in ceh-22 mutant gonads, indicating that top-1 may play an important role in CEH-22-mediated DTC fate specification. Therefore, our findings suggest that transgene-mediated co-suppression facilitates the silencing of the specific genes and the study of gene function in vivo.

The 53BP1 homolog in C. elegans influences DNA repair and promotes apoptosis in response to ionizing radiation.

53BP1 contributes to activation of the G2/M checkpoint downstream of ATM and MDC1 in response to ionizing radiation and promotes nonhomologous end-joining (NHEJ) in mammalian cells. In order to determine whether the cellular activities of 53BP1 are conserved in the model organism C. elegans, we analyzed the function of its homolog, HSR-9 in response to DNA damage. Deletion or Mos1-insertion in hsr-9 did not affect the sensitivity of worms to double strand DNA breaks (DSBs), as reflected in embryonic survival and larval development. Nevertheless, the hsr-9 mutations, as well as a lig-4 deletion, reversed the hypersensitivity of rad-54-deficient worms to DSBs. In addition, oocyte chromosomal aberrations, which were increased by rad-54 knockdown in response to DSBs, were also reduced by the hsr-9 mutations. The hsr-9 mutations did not prevent the cell cycle arrest induced by DSBs in mitotically proliferating germ cells. However, they attenuated apoptosis induced by DSBs, but not when CEP-1 (a p53 ortholog) was absent, suggesting that HSR-9 functions in the same pathway as CEP-1. We concluded that the 53BP1 homolog in C. elegans is not directly involved in cell cycle arrest in response to DSBs, but that it promotes apoptosis and also a form of NHEJ that occurs only when rad-54 is deficient.

C. elegans ring finger protein RNF-113 is involved in interstrand DNA crosslink repair and interacts with a RAD51C homolog.

The Fanconi anemia (FA) pathway recognizes interstrand DNA crosslinks (ICLs) and contributes to their conversion into double-strand DNA breaks, which can be repaired by homologous recombination. Seven orthologs of the 15 proteins associated with Fanconi anemia are functionally conserved in the model organism C. elegans. Here we report that RNF-113, a ubiquitin ligase, is required for RAD-51 focus formation after inducing ICLs in C. elegans. However, the formation of foci of RPA-1 or FCD-2/FANCD2 in the FA pathway was not affected by depletion of RNF-113. Nevertheless, the RPA-1 foci formed did not disappear with time in the depleted worms, implying serious defects in ICL repair. As a result, RNF-113 depletion increased embryonic lethality after ICL treatment in wild-type worms, but it did not increase the ICL-induced lethality of rfs-1/rad51C mutants. In addition, the persistence of RPA-1 foci was suppressed in doubly-deficient rnf-113;rfs-1 worms, suggesting that there is an epistatic interaction between the two genes. These results lead us to suggest that RNF-113 and RFS-1 interact to promote the displacement of RPA-1 by RAD-51 on single-stranded DNA derived from ICLs.

Physical and functional interactions of Caenorhabditis elegans WRN-1 helicase with RPA-1.

The Caenorhabditis elegans Werner syndrome protein, WRN-1, a member of the RecQ helicase family, has a 3'-5' DNA helicase activity. Worms with defective wrn-1 exhibit premature aging phenotypes and an increased level of genome instability. In response to DNA damage, WRN-1 participates in the initial stages of checkpoint activation in concert with C. elegans replication protein A (RPA-1). WRN-1 helicase is stimulated by RPA-1 on long DNA duplex substrates. However, the mechanism by which RPA-1 stimulates DNA unwinding and the function of the WRN-1-RPA-1 interaction are not clearly understood. We have found that WRN-1 physically interacts with two RPA-1 subunits, CeRPA73 and CeRPA32; however, full-length WRN-1 helicase activity is stimulated by only the CeRPA73 subunit, while the WRN-1(162-1056) fragment that harbors the helicase activity requires both the CeRPA73 and CeRPA32 subunits for the stimulation. We also found that the CeRPA73(1-464) fragment can stimulate WRN-1 helicase activity and that residues 335-464 of CeRPA73 are important for physical interaction with WRN-1. Because CeRPA73 and the CeRPA73(1-464) fragment are able to bind single-stranded DNA (ssDNA), the stimulation of WRN-1 helicase by RPA-1 is most likely due to the ssDNA binding activity of CeRPA73 and the direct interaction of WRN-1 and CeRPA73.

STR-33, a novel G protein-coupled receptor that regulates locomotion and egg laying in Caenorhabditis elegans.

Despite their predicted functional importance, most G protein-coupled receptors (GPCRs) in Caenorhabditis elegans have remained largely uncharacterized. Here, we focused on one GPCR, STR-33, encoded by the str-33 gene, which was discovered through a ligand-based screening procedure. To characterize STR-33 function, we performed UV-trimethylpsolaren mutagenesis and isolated an str-33-null mutant. The resulting mutant showed hypersinusoidal movement and a hyperactive egg-laying phenotype. Two types of egg laying-related mutations have been characterized: egg laying-deficient (Egl-d) and hyperactive egg laying (Egl-c). The defect responsible for the egg laying-deficient Egl-d phenotype is related to Gα(q) signaling, whereas that responsible for the opposite, hyperactive egg-laying Egl-c phenotype is related to Gα(o) signaling. We found that the hyperactive egg-laying defect of the str-33(ykp001) mutant is dependent on the G protein GOA-1/Gα(o). Endogenous acetylcholine suppressed egg laying in C. elegans via a Gα(o)-signaling pathway by inhibiting serotonin biosynthesis or release from the hermaphrodite-specific neuron. Consistent with this, in vivo expression of the serotonin biosynthetic enzyme, TPH-1, was up-regulated in the str-33(ykp001) mutant. Taken together, these results suggest that the GPCR, STR-33, may be one of the neurotransmitter receptors that regulates locomotion and egg laying in C. elegans.

Gene expression profiling of oxidative stress response of C. elegans aging defective AMPK mutants using massively parallel transcriptome sequencing.

BACKGROUND: A strong association between stress resistance and longevity in multicellular organisms has been established as many mutations that extend lifespan also show increased resistance to stress. AAK-2, the C. elegans homolog of an alpha subunit of AMP-activated protein kinase (AMPK) is an intracellular fuel sensor that regulates cellular energy homeostasis and functions in stress resistance and lifespan extension. FINDINGS: Here, we investigated global transcriptional responses of aak-2 mutants to oxidative stress and in turn identified potential downstream targets of AAK-2 involved in stress resistance in C. elegans. We employed massively parallel Illumina sequencing technology and performed comprehensive comparative transcriptome analysis. Specifically, we compared the transcriptomes of aak-2 and wild type animals under normal conditions and conditions of induced oxidative stress. This research has presented a snapshot of genome-wide transcriptional activities that take place in C. elegans in response to oxidative stress both in the presence and absence of AAK-2. CONCLUSIONS: The analysis presented in this study has enabled us to identify potential genes involved in stress resistance that may be either directly or indirectly under the control of AAK-2. Furthermore, we have extended our current knowledge of general defense responses of C. elegans against oxidative stress supporting the function for AAK-2 in inhibition of biosynthetic processes, especially lipid synthesis, under oxidative stress and transcriptional regulation of genes involved in reproductive processes.

Caenorhabditis elegans mitofilin homologs control the morphology of mitochondrial cristae and influence reproduction and physiology.

Human mitofilin is a mitochondrial protein that controls cristae formation. Here, we investigated the role of the Caenorhabditis elegans mitofilin homologs, IMMT-1 and -2, in reproduction, physiology, and mitochondrial cristae formation. Mutation of either immt-1 or immt-2 produced defects in germline development and egg-laying. These defects were exacerbated by the double mutation, which greatly reduced motility, increased levels of reactive oxygen species, decreased mitochondrial mass, and imparted resistance to oxidative stress. Cryo-electron microscopy and electron tomography revealed that each of the single mutations resulted in curved and stacked mitochondrial crista tubules as well as a reduced number of crista junctions. The immt-2 mutation was also associated with the presence of outer mitochondrial membrane pores, which were larger in the double mutant. IMMT-1 and IMMT-2 proteins were localized to the inner mitochondrial membrane, as seen by immunoelectron microscopy, and they behaved as oligomers or large complexes with F(1)F(0) ATP synthase in native polyacrylamide gel electrophoresis. These findings suggest that the two C. elegans mitofilin isoforms have non-overlapping functions in controlling mitochondrial cristae formation.

The involvement of FANCM, FANCI, and checkpoint proteins in the interstrand DNA crosslink repair pathway is conserved in C. elegans.

Fanconi anemia (FA) patients are specifically defective in the repair of interstrand DNA crosslinks (ICLs), a complex process involving at least 13 FA proteins and other repair/checkpoint proteins. Of the 13 FA proteins, FANCD1/BRCA2, FANCD2, and FANCJ were previously found to be functionally conserved in C. elegans. We have also identified C. elegans homologs of FANCM and FANCI, and determined their epistatic relationships with homologs of FANCD2, checkpoint proteins, and RAD51 upon DNA crosslinking. The counterparts of FANCM, FANCI, and three checkpoint proteins (RPA, ATR and CHK1) are required for focus formation and ubiquitination associated with FANCD2 in C. elegans. However, C. elegans FANCM affects neither RPA focus formation nor CHK1 phosphorylation induced by ICLs, unlike the reported role of human FANCM, which influences ATR-CHK1 signaling at stalled replication forks. Although focus formation by both FANCD2 and RAD51 requires ATR-CHK1 signaling, FANCD2 and RAD51 acted independently in the formation of their respective foci. Thus, the FANCD2 activation pathway involving FANCM, FANCI, and the checkpoint proteins is conserved in C. elegans but with distinct differences.

The Caenorhabditis elegans Werner syndrome protein functions upstream of ATR and ATM in response to DNA replication inhibition and double-strand DNA breaks.

WRN-1 is the Caenorhabditis elegans homolog of the human Werner syndrome protein, a RecQ helicase, mutations of which are associated with premature aging and increased genome instability. Relatively little is known as to how WRN-1 functions in DNA repair and DNA damage signaling. Here, we take advantage of the genetic and cytological approaches in C. elegans to dissect the epistatic relationship of WRN-1 in various DNA damage checkpoint pathways. We found that WRN-1 is required for CHK1 phosphorylation induced by DNA replication inhibition, but not by UV radiation. Furthermore, WRN-1 influences the RPA-1 focus formation, suggesting that WRN-1 functions in the same step or upstream of RPA-1 in the DNA replication checkpoint pathway. In response to ionizing radiation, RPA-1 focus formation and nuclear localization of ATM depend on WRN-1 and MRE-11. We conclude that C. elegans WRN-1 participates in the initial stages of checkpoint activation induced by DNA replication inhibition and ionizing radiation. These functions of WRN-1 in upstream DNA damage signaling are likely to be conserved, but might be cryptic in human systems due to functional redundancy.

DIC-1 over-expression enhances respiratory activity in Caenorhabditis elegans by promoting mitochondrial cristae formation.

Deficiency of the Caenorhabditis elegans protein, DIC-1, located in the inner membrane of mitochondria produces an abnormal mitochondrial morphology. The mechanism by which DIC-1 controls the topology of the inner membrane was investigated by transiently over-expressing DIC-1 in C. elegans. Cryo-electron microscopy showed that DIC-1 over-expression greatly increased the number and fractional area of mitochondrial cristae, suggesting that DIC-1 actively participates in cristae formation. These morphological changes were accompanied by increases in the oxygen consumption rate and ATP content of C. elegans worms, and decreases in reactive oxygen species (ROS) and sensitivity to paraquat. DIC-1 knockdown induced the opposite changes in ATP, ROS and paraquat-sensitivity. The ability of DIC-1 to increase cristae formation and secondarily, oxidative phosphorylation, suggests a potential use of this factor to control mitochondrial activity.

The efficiency of RNA interference in Bursaphelenchus xylophilus.

RNA interference (RNAi) was performed on several essential genes in the pinewood nematode Bursaphelenchus xylophilus, which causes pine wilt disease. Double-stranded RNA (dsRNA) was delivered to larvae or adult worms by soaking, electroporation, or microinjection. Soaking and electroporation of L2-L3 stage worms in solutions containing dsRNA for essential genes induced over 25% lethality after 5 days, and gene-specific phenotypes were observed. This lethality agreed with significant reductions of the targeted transcripts, as assayed by reverse-transcription coupled with real time PCR. Microinjection was the most efficient route as measured by the hatching rate of F1 embryos, which was reduced by 46%. When adult worms were soaked in dsRNA, lethality was induced in the F1 larvae, revealing the persistence of knockdown phenotypes. The penetrance of the RNAi phenotypes for essential genes was relatively low but consistent, indicating that RNAi should be useful for studying the in vivo functions of B. xylophilus gene products.

The Caenorhabditis elegans AMP-activated protein kinase AAK-2 is phosphorylated by LKB1 and is required for resistance to oxidative stress and for normal motility and foraging behavior.

AAK-2 is one of two alpha isoforms of the AMP-activated protein kinase in Caenorhabditis elegans and is involved in life span maintenance, stress responses, and germ cell cycle arrest upon dauer entry. We found that AAK-2 was phosphorylated at threonine 243 in response to paraquat treatment and that this phosphorylation depends on PAR-4, the C. elegans LKB1 homologue. Both aak-2 mutation and par-4 knockdown increased the sensitivity of C. elegans worms to paraquat, and the double deficiency did not further increase sensitivity, indicating that aak-2 and par-4 act in a linear pathway. Both mutations also slowed body bending during locomotion and failed to reduce head oscillation in response to anterior touch. Consistent with this abnormal motility and behavioral response, expression of the AAK-2::green fluorescent protein fusion protein was observed in the ventral cord, some neurons, body wall muscle, pharynx, vulva, somatic gonad, and excretory cell. Our study suggests that AMPK can influence the behavior of C. elegans worms in addition to its well known function in metabolic control.

Developmental stage- and DNA damage-specific functions of C. elegans FANCD2.

In this study, we set out to investigate the role of Fanconi anemia complementation group D2 protein (FANCD2) in developmental stage-specific DNA damage responses in Caenorhabditis elegans. A mutant C. elegans strain containing a deletion in the gene encoding the FANCD2 homolog, FCD-2, exhibited egg-laying defects, precocious oogenesis, and partial defects in fertilization. The mutant strain also had a lower hatching rate than the wild-type after gamma-irradiation of embryos, but not after the irradiation of pachytene stage germ cells. This mutation sensitized pachytene stage germ cells to the genotoxic effects of photoactivated psoralen, as seen by a greatly reduced hatching rate and increased chromosomal aberrations. This mutation also enhanced physiological M-phase arrest and apoptosis. Taken together, our data reveal that the C. elegans FANCD2 homolog participates in the repair of spontaneous DNA damage and DNA crosslinks, not only in proliferating cells but also in pachytene stage cells, and it may have an additional role in double-stranded DNA break repair during embryogenesis.