Raices M et al. (2008) Cell "C. elegans telomeres contain G-strand and C-strand overhangs that are bound by distinct proteins."

Haggblom CI, Raices M, Verdun RE, Karlseder J, Dillin A, Compton SA, Griffith JD

[Cell, 2008]

Single-strand extensions of the G strand of telomeres are known to be critical for chromosome-end protection and length regulation. Here, we report that in C. elegans, chromosome termini possess 3'' G-strand overhangs as well as 5'' C-strand overhangs. C tails are as abundant as G tails and are generated by a well-regulated process. These two classes of overhangs are bound by two single-stranded DNA binding proteins, CeOB1 and CeOB2, which exhibit specificity for G-rich or C-rich telomeric DNA. Strains of worms deleted for CeOB1 have elongated telomeres as well as extended G tails, whereas CeOB2 deficiency leads to telomere-length heterogeneity. Both CeOB1 and CeOB2 contain OB (oligo-saccharide/oligo-nucleotide binding) folds, which exhibit structural similarity to the second and first OB folds of the mammalian telomere binding protein hPOT1, respectively. Our results suggest that C. elegans telomere homeostasis relies on a novel mechanism that involves 5'' and 3'' single-stranded termini.

Jessica B Bessler et al. (2005) International Worm Meeting "Chromatin Modification and Double-Strand Breaks in C. elegans Meiosis"

Jessica B Bessler, Anne M Villeneuve

[International Worm Meeting, 2005]

Meiosis is the specialized cell division program that allows diploid organisms to generate haploid gametes. Successful meiotic cell divisions require that programmed DNA double-strand breaks (DSBs) are formed and repaired utilizing the homologous chromosome as the repair partner, thereby generating chiasmata and crossover recombination products. While the highly conserved protein SPO-11 is universally required to catalyze meiotic DSB formation, relatively little is known about how chromosomal competence for DSB formation is regulated. Several lines of evidence have suggested a connection between chromosome structure and DSB formation. For example, the C. elegans protein HIM-17 has been identified as a chromatin-associated factor that is required for both the formation of SPO-11-dependent DSBs and for the proper accumulation of histone H3 lysine 9 methylation (H3MeK9) on chromosomes during meiotic prophase progression. The precise nature of the link between HIM-17, DSBs and H3MeK9 is not well understood. Previous analyses have ruled out the possibility that H3MeK9 is a consequence of DSB formation but left open the question of whether acquisition of the H3MeK9 mark might be a prerequisite for either onset or cessation of DSB formation. One way to investigate these links is to determine what proteins interact with HIM-17 in vivo. In experiments currently underway, we are utilizing a HIM-17::GFP fusion protein to immunoprecipitate HIM-17 interacting factors from C. elegans extract. The identification of HIM-17 interacting proteins will allow us to narrow down the potential functions of HIM-17 in DSB formation and chromatin modification. In addition, we are working on localizing C. elegans SPO-11 using both microscopy and extract fractionation coupled with SDS-PAGE and Western analysis in order to determine if the presence or absence of HIM-17 affects SPO-11 localization.

Iuval Clejan et al. (2005) International Worm Meeting "NON-HOMOLOGOUS END-JOINING OF RADIATION-INDUCED DNA DOUBLE STRAND BREAKS IN C. ELEGANS"

Iuval Clejan, Shawn Ahmed

[International Worm Meeting, 2005]

Non-homologous end-joining (NHEJ) of double-strand DNA breaks is an important repair pathway in many organisms, but the particular circumstances under which this pathway is used and the way NHEJ is coordinated with other repair pathways are still unclear. We have observed that C. elegans strains harboring mutations in any of three NHEJ genes, cku-80, cku-70 and lig-4, are defective for repairing DNA double-strand breaks in non-cycling somatic cells. Several phenotypes are observed upon gamma irradiation of these mutants, including Gro, Unc, Pvl, Vul, Rup, and Egl. One possible upstream molecular event for all these phenotypes might be mis-segregation of improperly repaired chromosomes during mitosis following radiation induced DNA breaks. In support of this hypothesis, we observed failure of intestinal nuclei to undergo karyokinesis upon irradiation of NHEJ mutants. Further, the somatic radiation-induced phenotypes of NHEJ mutants synergized with him-10(e1511), a mutation in a known kinetochore component that results in unstable mitotic chromosome segregation. Although NHEJ is important for non-cycling somatic cells, the preferred DNA double-strand break repair pathway in both cycling and non-cycling germline cells and in cycling somatic cells is homologous recombination (HR). Worms depleted for the HR proteins RAD-51, MRE-11 and RAD-54 show sensitivity to gamma radiation in their germlines, and somatic sensitivity to gamma irradiation if irradiated during early embryogenesis. However, we observed no role for HR genes in dsDNA break repair in non cycling somatic cells. This is interesting because it implies MRE-11 does not participate in NHEJ in C. elegans (and possibly higher eukaryotes), as it does in S. cerevisiae. There was no apparent role for NHEJ genes in dsDNA break repair in the germline, even in non-cycling germ cells. In conclusion, we have shown that NHEJ repair of dsDNA breaks occurs preferentially in non-cycling somatic cells, whereas HR repair of dsDNA breaks occurs in cycling somatic cells and in the germline.

Marcel Tijsterman et al. (2005) International Worm Meeting "Double-strand break responses in C. elegans"

Joris Pothof, Marcel Tijsterman, Evelien Kruisselbrink, Gijs van Haaften, Ronald Plasterk

[International Worm Meeting, 2005]

The DNA within our cells is constantly being damaged by both environmental and endogenous agents; of the many forms of DNA damage, the DNA double strand break (DSB) is considered to be most dangerous. Many genes that play a role in the processing of DSBs have been identified over the past decades, however, it is clear that many components required in higher eukaryotes are not yet known and the identification of those will be a major challenge for future research.To identify the complete set of genes that are crucial for a proper response to DNA DSBs we performed a genome-wide screen in which we fed animals the Ahringer RNAi library and monitored the brood size after exposure to sub-lethal doses of ionizing radiation (see abstract van Haaften). This screen revealed 45 genes, that when knocked down result in ionizing radiation sensitivity; some of these also have defects in the apoptotic and/or cell cycle response.One of the genes we previously found to be required for a proper response to DNA DSBs is C. elegans lin-61, the worm homolog of human L3MBTL2 and MBTD1. lin-61 is essential for ionizing radiation induced cell cycle arrest and apoptosis, has an impaired intra-S-phase checkpoint and is not able to activate the apoptotic program upon E2F deregulation in a lin-35(n745)/Rb background or after cep-1/p53 upregulation. Furthermore we show that LIN-61 is upregulated after DNA damage in an ATM-1 dependent manner. LIN-61 interacts with a SET domain containing histone methyltransferase, a Sharp like protein and to EFL-2, one of the two worm homologs of the transcription factor E2F, and subsequent analyses revealed that both the chromatin remodeling genes and efl-2 are also essential for the DNA damage checkpoint.In a complementing approach, we recently developed a transgenic system that allows the visualisation of repair of a single localized DSB. This system is based on pie-1 driven (germ line) expression of the rare-cutting yeast endonuclease I-SceI that is combined with transgenes containing LacZ reporters that are interrupted with I-SceI restriction sites; homology driven repair of the I-SceI-induced DSB can restore B-galactosidase expression, which is seen as blue staining of somatic sub-lineages.

Ketting RF et al. (2006) CSH Protoc "Introduction of Double-Stranded RNA in C. elegans by Feeding."

Plasterk RH, Ketting RF, Tijsterman M

[CSH Protoc, 2006]

Ketting RF et al. (2006) CSH Protoc "Introduction of Double-Stranded RNA in C. elegans by Injection."

Tijsterman M, Ketting RF, Plasterk RH

[CSH Protoc, 2006]

Ketting RF et al. (2006) CSH Protoc "Introduction of Double-Stranded RNA in C. elegans by Soaking."

Tijsterman M, Plasterk RH, Ketting RF

[CSH Protoc, 2006]

Kotagama K et al. (2024) Nucleic Acids Res "Catalytic residues of microRNA Argonautes play a modest role in microRNA star strand destabilization in C. elegans."

McJunkin K, Yu G, Kotagama K, Braviner L, Grimme AL, Joshua-Tor L, Sakhawala RM, Benner LK, Yang B

[Nucleic Acids Res, 2024]

Many microRNA (miRNA)-guided Argonaute proteins can cleave RNA ('slicing'), even though miRNA-mediated target repression is generally cleavage-independent. Here we use Caenorhabditis elegans to examine the role of catalytic residues of miRNA Argonautes in organismal development. In contrast to previous work, mutations in presumed catalytic residues did not interfere with development when introduced by CRISPR. We find that unwinding and decay of miRNA star strands is weakly defective in the catalytic residue mutants, with the largest effect observed in embryos. Argonaute-Like Gene 2 (ALG-2) is more dependent on catalytic residues for unwinding than ALG-1. The miRNAs that displayed the greatest (albeit minor) dependence on catalytic residues for unwinding tend to form stable duplexes with their star strand, and in some cases, lowering duplex stability alleviates dependence on catalytic residues. While a few miRNA guide strands are reduced in the mutant background, the basis of this is unclear since changes were not dependent on EBAX-1, an effector of Target-Directed miRNA Degradation (TDMD). Overall, this work defines a role for the catalytic residues of miRNA Argonautes in star strand decay; future work should examine whether this role contributes to the selection pressure to conserve catalytic activity of miRNA Argonautes across the metazoan phylogeny.

van Luenen HGAM et al. (1996) Worm Breeder's Gazette "Double strand DNA repair in C. elegans"

Plasterk RHA, van Luenen HGAM, Hartman PS

[Worm Breeder's Gazette, 1996]

Transposition of Tc1 and Tc3 is initiated by binding of the respective transposases to the terminal inverted repeats of each transposon (1,2). Subsequently, the element is excised by double strand breaks near each transposon end (3). The cut at the 3' end of the element is between the last nucleotide of the transposon and the flanking genomic sequence. The cut at the 5' end is not at the end of the transposon, but two nucleotides within the transposon. After excision the donor site contains a double strand break with a two nucleotide extension at each 3' end. The cellular DNA repair processes have to seal the break to ensure cell viability. We have set up an in vivo assay to monitor the repair of excision induced double strand breaks in real time. A Tc3 allele of unc-22 (r750::Tc3) was crossed into a strain containing an inducible Tc3 transposase gene. Upon heat shock induction the transposase gene is expressed, resulting in transposition of Tc3. Digested genomic DNA of this strain was analysed on a Southern blot using a fragment of the unc-22 gene flanking the donor site as a probe. After a two hour induction at an elevated temperature and a subsequent recovery of the worms at normal temperature a band appears of the size of the band expected from the wild type unc-22 gene. This fragment was cloned and sequenced. It contains the unc-22 sequence with characteristic Tc3 footprints (3). Twenty hours after the induction of transposase expression approximately 10 % of the unc-22(r750::Tc3) alleles have reverted. Hybridisation of the blot with the unc-22 probe lead to the detection of an additional band. Based on the size and the specific hybridization of the band we conclude that this band corresponds to the broken unc-22 fragment created during excision of Tc3. Approximately two hours after induction this band represent 1-2 % of all unc-22::Tc3 sites. During the remainder of the experiment (up to twenty hours after induction) the intensity of this band stays constant, whereas the intensity of the band representing the repaired chromosome continues to increase. This indicates that the Tc3 elements are continuously excised and that the broken chromosome is continuously repaired. The structure of the left end and the right end of the broken chromosome was determined. The 5' ends and the 3' ends are in agreement with the structure predicted from the excised transposon (the 3' ends on both sides of the double strand break contain the two nucleotides which are not co-excised with the transposon, the 5' ends contain the target sequence and no transposon sequence).

Stamper, Ericca et al. (2011) International Worm Meeting "Regulation of Meiotic Double-Strand Break Formation in C. elegans."

Stamper, Ericca, Dernburg, Abby

[International Worm Meeting, 2011]

Meiotic recombination is essential for the successful execution of meiosis, and therefore for sexual reproduction. Recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs), catalyzed by the conserved endonuclease Spo11. If not properly repaired, DSBs pose a threat to genomic integrity. The activity of Spo11 is thought to be tightly regulated to control both the number and location of DSBs. In budding yeast, where the regulation of DSB formation is best understood, at least 9 proteins in addition to Spo11 are required for meiotic DSBs. However, little is understood about how recombination initiation and DSB formation is regulated in other organisms, including C. elegans, which has emerged as an important system for studying meiosis. To gain new insights into the regulation of SPO-11 and DSB formation in C. elegans, I have been characterizing a novel gene that we have named dsb-1. Animals lacking dsb-1 function are viable and fertile, but do not make crossovers during meiosis and therefore produce many inviable progeny and a high incidence of males (Him). This defect in recombination can be rescued by irradiation, indicating that they are specifically defective in DSB formation and not repair. DSB-1 shares homology with another C. elegans gene implicated in DSB formation (Rosu et al., 2009), but lacks obvious homology with proteins outside of Caenorhabditis. I have found that epitope-tagged DSB-1, expressed from single-copy transgenes integrated by MosSCI, localizes as foci on meiotic chromosomes during early meiotic prophase, corresponding to the timing of DSB formation. One hypothesis is that DSB-1 may recruit SPO-11 to meiotic chromosomes. However, analysis by yeast two-hybrid (Y2H) assays failed to detect an interaction between DSB-1 and SPO-11. Y2H analysis did detect a weak interaction between DSB-1 and HTP-3, complementing previous studies that suggested a role for HTP-3 in DSB formation through the recruitment of recombination proteins to meiotic chromosomes (Goodyer et al., 2008). I plan to investigate DSB-1 function through co-immunoprecipitation and ChIP experiments. Future goals of my project aim to address some of the many remaining questions regarding the role of DSB-1 in promoting DSBs and the regulation of meiotic recombination initiation in C. elegans. References: Goodyer W, Kaitna S, Couteau F, Ward JD, Boulton SJ, Zetka M. 2008. HTP-3 links DSB formation with homolog pairing and crossing over during C. elegans meiosis. Dev Cell 14:263-74. Rosu S, Tam A, Villeneuve A. 2009. Identification of novel components of the C. elegans meiotic machinery. International Worm Meeting.