Simon Boulton et al. (2001) International C. elegans Meeting "Identification of C. elegans genes required for the DNA damage response using a combination of functional genomic approaches"

Jerome Reboul, Simon Boulton, David Hill, Marc Vidal, Anton Gartner, Philippe Vaglio

[International C. elegans Meeting, 2001]

To address novel questions about metazoan DNA damage checkpoints and DNA repair (referred to as the DNA damage response or "DDR"), we are using functional genomic approaches in C. elegans . Our first goal was to identify novel genes involved in DDR by generating a protein-protein interaction map. Database searches using known DDR proteins from other organisms identified 75 C. elegans ORFs that encode putative orthologs (dORFs). These dORFs were cloned using the Gateway recombinational cloning technology and transferred to both RNAi-feeding and two-hybrid vectors. High-throughput RNAi was used to establish that many dORFs are indeed required for DDR in the worm. A DDR protein interaction map was generated by screening each of the 75 dORFs for two-hybrid interactors. This map contains 165 potential interactions. Finally RNAi was used for many of the novel potential interactors. In each case we scored for defects in DNA damage-induced cell cycle arrest and apoptosis to ascertain checkpoint integrity. Using this combination of large-scale two-hybrid and RNAi approaches we have identified novel DDR worm genes and many of those have potential orthologs in mammals. These findings suggest that protein interaction mapping and RNAi can be complementary and can be used in combination to uncover novel genes involved in other biological processes.

Jolanta Polanowska et al. (2006) European Worm Meeting "Regulation of BRC-1 - Dependent Ubiquitylation at DNA Damage Sites"

Jolanta Polanowska, Simon Boulton, Tatiana Garcia-Muse, Julie Martin

[European Worm Meeting, 2006]

Jolanta Polanowska1,2, Julie Martin1, Tatiana Garcia-Muse1 and Simon Boulton1. The BRCA1 tumour suppressor and its heterodimeric partner BARD1 constitute an E3-ubiquitin (Ub) ligase and function in DNA repair by unknown mechanisms. We have previously described C. elegans BRCA1 and BARD1 orthologues (brc-1 and brd-1, respectively) that possess many of the functional domains present in their human counterparts, including RING, ankyrin, and BRCT domains (Boulton et al., 2004). Consistent with conserved roles in DNA repair, BRC-1 and BRD-1 interact to form a heterodimer via their respective RING domains. To explore the mechanistic role of BRC-1 and BRD-1 in DNA repair processes we have characterized a C. elegans BRC-1/BRD-1 complex (CeBCD) purified by tandem immunoaffinity before and at different time points after IR-treatment. This approach is a first for C. elegans and demonstrates that protein complexes purified in this manner are amenable to biochemical analysis and can be used in combination with genetics and cell biology to accelerate functional discoveries. We present evidence that the CeBCD complex possesses an E3-Ub ligase that is activated on chromatin in response to IR-treatment and further demonstrate that the DNA damage checkpoint promotes association of the CeBCD complex with E2-Ub conjugating enzyme, Ubc5(LET-70), to form an active E3-Ub ligase in response to DNA damage. We also show that ubiquitylation events at DNA damage sites require brc-1, brd-1, ubc5(let-70), mre-11 and atl-1, thus providing in vivo evidence to support our biochemical analysis.. Boulton, S.J., Martin, J.S., Polanowska, J., Hill, D.E., Gartner, A. and Vidal, M. (2004) Curr Biol, 14, 33-39.

Saito, Takamune T. et al. (2009) International Worm Meeting "C. elegans HIM-18/SLX-4 interacts with SLX-1 and XPF-1 and maintains genomic integrity in the germline by processing recombination intermediates."

Boulton, Simon J., Saito, Takamune T., Youds, Jillian L., Colaiacovo, Monica P.

[International Worm Meeting, 2009]

DNA double-strand breaks (DSBs) can arise in various ways, including as a result of the collapse of stalled replication forks, exposure to DNA damaging agents and the formation of programmed meiotic DSBs. The importance of DSB repair is therefore highlighted by its critical roles in replication fork restart, the maintenance of genomic integrity and promoting faithful meiotic chromosome segregation. Homologous recombination (HR) provides an efficient and accurate repair of DSBs, in part through the use of an intact donor template for repair. Through the process of HR, two four-way DNA junction intermediates referred to as double Holliday junctions (dHJ) are formed. dHJ resolution is essential for the repair of blocked or collapsed replication forks and for the production of crossovers between homologs that promote accurate meiotic chromosome segregation. In D. melanogaster, MUS312 is thought to be required for dHJ resolution along with XPF-ERCC1 known as a structure specific endonuclease complex. In S. cerevisiae, the Slx4-Slx1 complex cleaves branched DNA substrates in vitro. Through a functional genomics approach, we identified HIM-18, an ortholog of MUS312/Slx4, as a critical player required in vivo for dHJ resolution in C. elegans. HIM-18 is localized to mitotic nuclei at the premeiotic tip and to meiotic nuclei from late pachytene through diakinesis in wild type germlines. DNA damage sensitivity and an accumulation of recombination intermediates (RAD-51 foci) during premeiotic entry in him-18 mutants suggest that HIM-18 is required for DNA duplex resolution at stalled replication forks. A reduction in crossover recombination frequencies, accompanied by an increase in recombination intermediates during meiosis, germ cell apoptosis, unstable bivalent attachments and subsequent chromosome nondisjunction, support a role for HIM-18 in meiotic HJ resolution. The evidence of a physical interaction of HIM-18 with SLX-1 and XPF-1, accompanied by the synthetic lethality of him-18 with him-6, the C. elegans BLM homolog, suggest that the proposed function of MUS312/Slx4 in DNA repair is conserved in C. elegans. We propose that HIM-18 facilitates resolution of recombination intermediates resulting from replication fork collapse and programmed meiotic DSBs in the C. elegans germline.

Iuval Clejan et al. (2003) International Worm Meeting "cku-80 and lifespan in C. elegans"

Shawn Ahmed, Bettina Meier, Iuval Clejan

[International Worm Meeting, 2003]

RNAi of cku-80, which functions in non-homologous end-joining, silencing, and telomere maintenance in other organisms, results in a 15%; increase in mean lifespan of wildtype worms. RNAi knockdown of several other non-homologous end-joining genes produced no lifespan extension, suggesting that the mechanism by which cKU-80 affects lifespan is unrelated to non-homologous end-joining. Consistent with this hypothesis, non-homologous end-joining does not appear to play a major role in repair of double-strand DNA breaks, either in the germline or soma of C. elegans, as cku-80 RNAi worms are only weakly sensitive to ionizing radiation. Since Ku is known to interact with telomerase in other organisms, we are also testing telomerase mutants for lifespan extension. So far no lifespan extension has been observed, suggesting that the mechanism of life extension in cKU-80 knockdown is unrelated to telomere maintenance. Since Ku interacts with the DNA damage response, we performed epistasis between cku-80 deficient worms and the long-lived checkpoint mutant clk-2, but the effects were additive, suggesting that clk-2 and cku-80 affect lifespan through distinct pathways. Based on the physical interaction observed between DAF-21 and cKU-80 in a yeast two-hybrid (Boulton et al. 2002), we are measuring lifespan of daf-21 mutants with cku-80 RNAi, to see if we can observe an epistatic interaction between these genes. We are also checking for epistatic interactions between cku-80 and members of the insulin-signaling pathway. Boulton, S. J. et al. (2002). Combined functional genomic maps of the C. elegans DNA damage response. Science 295: 127-131.

Jillian L. Youds et al. (2006) European Worm Meeting "The DOG-1 Helicase, Genomic Instability and Fanconi Anemia"

Ann M. Rose, Nigel J. O'Neil, Simon J. Boulton, Louise J. Barber, Jillian L. Youds

[European Worm Meeting, 2006]

Jillian L. Youds1, Nigel J. O''Neil1, Louise J. Barber2, Simon J. Boulton2 & Ann M. Rose1. In Caenorhabditis elegans, DOG-1 is required for the maintenance of polyG/polyC-tracts (G-tracts). In the absence of DOG-1, it is thought that G-tracts form secondary structures that block replication, leading to deletions that initiate in the G-tracts. Using our assay for deletions forming in the absence of DOG-1, we have assayed the in vivo contribution of various repair genes to the maintenance of these tracts. We show that DOG-1 and the BLM ortholog, HIM-6, act synergistically during replication; simultaneous loss of function of both genes results in replicative stress and an increase in the formation of small deletions that initiate in G-tracts. Similarly, we show that genes implicated in homologous recombinational repair and trans-lesion synthesis are required to prevent G-tract deletions in the dog-1 background. However, genes essential to the non-homologous end-joining and nucleotide excision repair pathways do not appear to be involved in deletion prevention or formation. By investigating the mechanisms that maintain genomic stability in dog-1 mutants, we might better understand the genomic instability associated with Fanconi anemia, as the human gene most similar to dog-1, BRIP1/FANCJ, was recently shown to be mutated in a subset of patients with Fanconi anemia, implicating it in interstrand cross-link (ICL) repair. In collaboration with the Boulton lab, we are currently investigating the possibility that DOG-1 and BRIP1 have functionally conserved roles in DNA repair. Our preliminary data indicate that dog-1 mutants are sensitive ICL-inducing agents and we are assessing the relationship to fcd-2 and other genes involved in ICL repair. . This research is funded by the Natural Sciences and Engineering Council and the Michael Smith Foundation for Health Research.

Judith Yanowitz (2007) International Worm Meeting "Genome stability and telomere maintenance are regulated by him-15/rfs-1."

Judith Yanowitz

[International Worm Meeting, 2007]

I have identified him-15 as the rad-51 paralogue gene, rfs-1. rfs-1 has a 2% HIM phenotype and is sensitive to ionizing radiation. rfs-1 displays a classic MRT phenotype with populations of worms going sterile after 20 passages from starved plates, with evidence of telomere shortening, and with end-to-end fusions in the generation preceding sterility. However, rfs-1 has several characteristics that make it a unique MRT gene and have important implications for telomere maintenance. Specifically, although shorter telomeres can be seen in rfs-1 mutants, lengthening events are also observed. Similarly, rfs-1 can enhance the MRT phenotype of telomerase mutant, trt-1; but it also partially suppresses the phenotype. Consistent with these results, rfs-1 trt-1 double mutants show a slower rate of telomere attrition than the trt-1 signle mutants. The rfs-1 trt-1 "survivors" have stabilized karyotypes with fewer chromosomes- in one case, with only 2 chromosomes observed in all diakinetic nuclei. These results suggest that in rfs-1 mutants, an alternative pathway for telomere regulation has been uncovered. Simon Boulton''s lab has shown that rfs-1 regulates replication fork progression through stalled forks. My results are consistent with a model in which replication fork progression is the sole function of rfs-1. Defects in replicating telomeric repeats lead to stalled forks that need to be repaired by recombination, leading to both lengthening and shortening events. Rescue in the rfs-1 trt-1 double mutant can result from interchromosomal recombination. Additional genomic instability arises when telomeres recombine with internal telomeric repeats leading to inversions, deletions, and traslocations or when replication of other repetitive elements leads to stalling and subsequent recombination repair.

Chung, George et al. (2015) International Worm Meeting "The molecular identification of a gene which controls the distribution of meiotic recombination events in Caenorhabditis elegans."

Sanchez-Pulido, Luis, Petalcorin, Mark, Chu, Jeffrey, Chung, George, Boulton, Simon, Rose, Ann, Ponting, Chris, Yanowitz, Judith, Kessler, Zebulin, O'Neil, Nigel, Martin, Julie

[International Worm Meeting, 2015]

The placement of meiotic crossover events is regulated such that crossover events are not distributed randomly along the chromosome. In Caenorhabditis elegans, meiotic crossover placement favours chromosome arms over chromosome centres, a bias abolished by mutations in the rec-1 gene. This mutant phenotype has precluded positional cloning efforts for 30 years. Here, we describe the identity of rec-1, which was uncovered by whole-genome sequencing and confirmed by new alleles engineered with MosSCI and CRISPR-Cas9. The rec-1 gene encodes a previously uncharacterized protein, which possesses putative phosphorylation motifs as well as weak homology to the meiotic protein HIM-5. We demonstrate that the REC-1 protein is a substrate for CDK phosphorylation in vitro and in C. elegans extracts. This phosphorylation was abolished in a REC-1(8A) mutant lacking the 8 CDK phosphorylation sites. Moreover, the rec-1(8A) and rec-1(8E) (phosphor-mimetic) mutant transgenes did not rescue the rec-1 defect. Given the homology shared between REC-1 and HIM-5, we also examined their genetic relationship. Strikingly, we found that in contrast to the single mutants, the rec-1; him-5 double mutant was defective for the formation of meiotic double-strand breaks (DSBs), which was rescued by artificially introducing DSBs using ionizing radiation. Thus, we establish that REC-1 plays an indispensable role in the distribution of meiotic crossovers, which is controlled by CDK phosphorylation. Furthermore, we uncover an unappreciated redundancy in the mechanisms required to generate meiotic DSBs involving REC-1 and HIM-5.

Dengg M. et al. (2006) European Worm Meeting "Using Caenorhabditis elegans to Study Cellular Responses to Endogenous DNA Damage"

Boulton S., Nilsen H., Garcia-Muse T., Dengg M.

[European Worm Meeting, 2006]

Dengg, M. 1, Garcia-Muse, T.2, Boulton, S.2, and Nilsen, H1 DNA repair pathways and intracellular signalling cascades contribute to DNA damage response (DDR) mechanisms that determine cellular responses to DNA damage. C.elegans has been used successfully to study DDR in response to DNA damage induced by physical or chemical agents. Whether DDR is activated in response to the most abundant forms of DNA damage, arising spontaneously through the reaction of water and reactive oxygen species with DNA, is poorly understood. Compared with treatment with physical or chemical DNA damaging agents, the study of DDR in response to endogenous lesions must overcome technical problems with respect to inducing sufficiently high levels of DNA lesions to allow studies of downstream responses. We have exploited RNA interference (RNAi) technology in C.elegans to modulate dUTP pools in order to achieve incorporation of uracil above background levels. Here, we present data describing the phenotypical consequences of increased incorporation of uracil into DNA. The role of DNA repair and DNA damage checkpoints in activating DNA damage response pathways in response to uracil will be presented.

Lars Nilsson et al. (2006) European Worm Meeting "C. elegans NUM-1 Modulates Endocytosis by Targeting the TAT-1 Phospholipid Flippase"

Lars Nilsson, Simon Tuck

[European Worm Meeting, 2006]

Lars Nilsson and Simon Tuck. We have previously reported that C. elegans NUM-1 modulates both endocytosis and the activity of LIN-12, a receptor that mediates a number of cell fate specification events in the worm. In order to understand better how NUM-1 functions, we carried out a yeast 2-hybrid screen for interacting proteins. One clone we isolated encodes TAT-1, a transmembrane P-type ATPase. Work in S. cerevisiae has shown that these proteins function as so-called inward phospholipid flippases to concentrate specific phospholipids to the cytosolic leaflet of biological membranes. They are known to be associated with endosomes and to be required for correct vesicle trafficking. We have found that TAT-1 is expressed in tissues in which NUM-1 functions. Mapping of the regions required for binding revealed that the PTB domain of NUM-1 associates with an NXXF motif at the C-terminus of TAT-1. RNAi experiments show that tat-1 affects membrane trafficking in C. elegans. Our results suggest that NUM-1 functions in the worm by modulating TAT-1 activity.

JJ Yochem (1999) International C. elegans Meeting "Mutations that confer aspects of the molting defects seen with lrp-1 mutations or sterol starvation"

JJ Yochem

[International C. elegans Meeting, 1999]

Mutations that produce larvae that are completely stuck in old cuticle, that have rings of unshed cuticle that constrict regions of the body, or that have attached to the tail a train of old cuticle will be described. Such larvae can be detected with a dissecting microscope, permitting a semi-clonal screen. The search for such mutations was begun because a failure to complete molting is an aspect of the phenotype conferred by loss-of-function mutations in lrp-1 , the product of which resembles mammalian gp330/megalin, a large member of the LDL receptor family of proteins [Yochem et al., Development 126, 597 (1999)]. It is hoped that new mutations will reveal genes whose products are required for the same process as LRP-1. The mutations also have the potential for increasing our understanding of molting, a process that has not been much studied genetically in nematodes. To date, mutations have defined eight candidate genes. Of particular note are mlt-3(sv8) and mlt-7(sv16) , both of which closely resemble the lrp-1 mutant phenotype. Thanks to Simon Tuck, Min Han, and Bob Herman.