C. elegans Genomewide Synthetic Lethal Screens to uncover new DNA damage response genes

The Gartner group employs genetic approaches in the nematode worm C. elegans to better understand pathways needed for the maintenance of genome stability and apoptosis induction.

Homologous Recombination (HR) provides an error-free DNA repair pathway for DNA double strand breaks (DSBs). Holliday Junctions (HJs) are key late stage HR intermediates that must be processed to fully restore duplex DNA strands. However, very little is known about the mechanisms used by the alternative or redundant pathways that seem to resolve HJs. Very recently two HJ resolving nucleases, GEN1 and the SLX4/SLX1 complex, have been described in animals 35). Besides their resolution, a process called HJ dissolution can untangle HJs; alternatively, the need for HJ resolution can be bypassed by synthesis-dependent strand annealing (SSDA). Virtually nothing is known about how cells choose between these different HJ processing pathways and how the resolving enzymes are regulated. The Gartner group will train a Cancer Research UK funded student to undertake genome-wide synthetic lethal screens to identify and characterize key mediators of late-stage HR processes. C. elegans is a unique experimental system where RNAi can be applied at an organismal level at a scale amenable to 96-well liquid culture manipulation. We will use the RNAi by feeding procedure to screen a collection of E coli strains, where each strain expresses double-stranded RNA corresponding to a single worm gene. The dsRNA is ingested by worms and leads to specific gene inactivation, and by feeding worms with individual E. coli strains the majority of worm genes can be targeted for inactivation. We have already established liquid culture handling procedures to facilitate such large-scale RNAi screens for genes whose inactivation does not cause a phenotype in wild type worms, but is lethal for mutant worms. Initial experiments will include viable single and double mutant combinations of genes acting in the late stages of recombination repair. The PhD student will focus on identifying and characterizing genes that are synthetic lethal with the mus-81, gen-1, xpf-1, fan-1 and slx-4/slx-1 nucleases, all implicated in late stage recombinational repair. Synthetic lethal interactions involving mutations in genes involved in HR allow for the specific killing of cancer cells that have acquired mutations in DNA repair genes. We will thus validate our results obtained from the C. elegans model in appropriate vertebrate tissue culture models and will particularly focus on chicken DT40 cells as they allow for easy gene disruption procedures and as many DNA repair assays are set up in this system. We particularly encourage students with previous experimental experience to apply. Having worked on DNA damage response mechanisms, apoptosis and or C. elegans is a plus but we encourage all interested students to apply.