Mechanisms of monoubiquitination in the Fanconi Anemia tumour suppressor pathway.
Session type: Poster / e-Poster / Silent Theatre session
The Fanconi Anemia tumour suppressor pathway acts to repair DNA interstrand crosslinks (ICL) created during replication. Inefficient repair of these ICL can lead to leukaemia and bone marrow failure.
The vital step of the pathway is the monoubiquitination of FANCD2 and FANCI by the RING E3 ligase FANCL. Once FANCD2 and FANCI are modified they co-localise on the chromatin at the site of damage with other repair factors such as BRCA2, and the ICL is removed.
We are investigating the role of FANCL during the monoubiquitination of substrates FANCD2 and FANCI using and in vitro approach. Recombinant protein is overexpressed and purified from bacteria and insect cells, then used in a variety of biochemical and structural approaches to understand more about ubiquitination.
We initially uncovered that D. melanogaster FANCL non-covalently binds to ubiquitin in pull-downs and ITC (Isothermal Titration calorimetry). We then determined the ubiquitin binding site in the N-terminal E2-like domain of FANCL using NMR spectra. Via site-directed mutagenesis of FANCL we have highlighted the most important residues involved in the interaction. We are currently developing an in vitro assay to see how this interaction between FANCL and ubiquitin contributes to the monoubiquitination of FANCD2 and FANCI.
We have characterised a novel interaction between the E3 ligase FANCL and ubiquitin, which could contribute to substrate ubiquitination and therefore, DNA repair. Our future work shall couple an in vitro assay with structural and biochemical techniques to provide further insight into ubiquitination during DNA repair.