Davo Martinez, Carlota et al. (2021) International Worm Meeting "Persistent DNA repair complex binding in the absence of DNA damage excision impairs neuron functionality"

Ribeiro-Silva, Cristina, Muniesa Vargas, Alba, Theil, Arjan, Davo Martinez, Carlota, Vermeulen, Wim, Lans, Hannes

[International Worm Meeting, 2021]

Nucleotide excision repair (NER) is a major DNA repair pathway that removes a large variety of helix-distorting DNA damage. Hereditary mutations in NER genes that encode proteins that make up the core NER machinery, such in endonucleases XPF and XPG, can give rise to the cancer prone disorder xeroderma pigmentosum (XP) or to more severe disorders in which XP is combined with progressive neurodegeneration or progeroid Cockayne syndrome (CS) features, called XP-CS. Intriguingly, mutations in other factors of the core NER complex give rise to a milder phenotype. It is not properly understood why mutations in genes that act in the same DNA repair pathway cause different types of disease. Here, we use C. elegans as a model to study the phenotypic impact of different mutations in the evolutionary conserved NER pathway. We found that XPF-1 or XPG-1 deletion mutants show severe developmental arrest and neuronal dysfunction upon induction of UV damage. In the absence of XPF-1 or XPG-1, core NER-intermediates accumulate at DNA damage and stay stably bound to DNA. We hypothesized that this could shield the lesion from other DNA repair pathways and block transcription, which could cause this severe phenotype. Intriguingly, we found that inhibiting the binding of the core NER-intermediates to DNA in XPF-1 or XPG-1 worms alleviated the severe phenotype, confirming that the accumulation of repair intermediates can indeed be more toxic for cells than the damage itself. Together, these results strongly suggest that the persistence of NER intermediates adversely affects cell functionality, which may be a plausible explanation for why mutations in XPF or XPG can lead to more severe disease features than mutations in other core subunits.

Touroutine, D. et al. (2019) International Worm Meeting "Superselective primers - a powerful molecular tool with broad application to detect SNPs."

Touroutine, D., Tanis, J.

[International Worm Meeting, 2019]

With wide spread use of single nucleotide variants generated through mutagenesis screens, the million mutation project, and genome editing technologies there is pressing need for an efficient and low-cost strategy to detect SNPs. Current SNP-discriminating methods involve PCR amplification of the variant containing region followed by either restriction digest if there is a SNP induced change in a restriction site or direct sequencing. These methods are labor intensive, requiring extra steps beyond PCR, and expensive, demanding purchase of unique restriction enzymes or sequencing reactions. Other strategies, including simple allele-discriminating PCR and ARMS-PCR require extensive troubleshooting to identify primers and conditions that can accurately distinguish variants. We have developed a rapid and cheap method for detection of point mutants through optimization of superselective (SS) primers [1] for end PCR. A SS primer consists of three sections: an anchor, bridge and foot. The anchor region, which anneals to the template, is followed by a bridge containing non-specific sequence and then a short foot which anneals to the template to distinguish mismatches. We explored how length, stability and sequence composition of each segment affected primer selectivity in order to develop simple rules for primer design that allow for discrimination between any mismatches in various genetic contexts over a broad range of annealing temperatures. Based on our design, SS primers can easily detect even (A/T-G/C) transitions, which are primarily generated by the EMS mutagen and greatly tolerated by Taq polymerase. We have demonstrated that SS primers are capable of identifying a desired mutation in the presence of a thousand fold excess wild-type template, allowing for pooling screening strategies for detection of genome editing events. Further, SS primers can be utilized for colony PCR to identify successful site-directed mutagenesis constructs. In summary, we adapted and optimized SS primers to enable greater flexibility in design and demonstrated their utility for various laboratory applications. Vargas DY, Kramar FR, Tyagi S, Marras S (2016) Multiplex real-time PCR Assays that measure the abundance of extremely rare mutations associated with cancer. PLoS One, 11(5):e0156546

Valansi, C. et al. (2015) International Worm Meeting "Understanding the mechanism of EFF-1-mediated fusion."

Valansi, C., Oren-Suissa, M., Avinoam, O., Podbilewicz, B., Fridman, K.

[International Worm Meeting, 2015]

Cell fusion is an important developmental process in many organisms. The first identified eukaryotic fusogen, the C. elegans epithelial fusion failure-1 (EFF-1), mediates fusion of cells in the hypodermis, pharynx, and vulva. Ectopic expression of EFF-1 is sufficient to fuse cells in C. elegans and in heterologous cells. Here, we use mammalian cells and vesicular stomatitis pseudoviruses as model systems to study how eff-1 affects membrane dynamics. We expressed wild-type and mutant forms of EFF-1 and compared their ability to fuse mammalian cells in culture. We found that EFF-1 fusion failure mutations identified in worms, had a similar phenotype in baby hamster kidney (BHK) cells. We also found that expression of EFF-1 in BHK cells stabilizes actin-rich membrane protrusions that arise through retraction of the plasma membrane, resulting in a 'hairy' phenotype that is observable by immunofluorescence and EM. Mutations in EFF-1 had a measurable effect both on cell-cell fusion and on localization to these protrusions. hy21ts allele (P183L) shows strong fusion failure at the restrictive temperature in L4 stage, as well as dumpy and protruded vulva phenotypes, whereas in BHK cells it is retained in the ER blocking both multinucleation and localization to protrusions. oj55 (S441L) hypomorphic mutation results in failure in most epidermal fusion events in the embryo. We found that in BHK cells EFF-1(S441L) partially reduces both cell fusion and localization to EFF-1. In contrast to the other mutations, EFF-1(G260A) (Perez-Vargas et al., 2014) inhibits cell fusion but does not affect the localization to protrusions.To elucidate the structure of EFF-1 molecules at the membrane we used a combination of molecular genetics, biochemistry, electron and light microscopy and found that EFF-1 localizes to micron-long tubes that had a diameter of 30 nm. We determined that EFF-1 wild-type protein and the different mutants studied have 70% monomers, 20% dimers and 10% trimers on the surface of BHK cells.Based on our findings in worms and mammalian cells, together with the atomic structure of EFF-1, we propose that EFF-1 has two self-organizing activities: First, it maintains long nano-tubes via monomer-monomer interactions in cis. Second, it drives bilateral zippering and cell-to-cell fusion via transient homo-dimerization and -trimerization in trans..