Characterisation of FANCL variants observed in patient cancer cells

Frost, M. G., Mazloumi Aboukheili, A. M., Toth, R. and Walden, H. (2020) Characterisation of FANCL variants observed in patient cancer cells. Bioscience Reports, 40(6), 20191304. (doi: 10.1042/BSR20191304) (PMID:32420600) (PMCID:PMC7273913)

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Fanconi Anemia (FA) is a rare genetic disorder characterized by developmental defects, bone marrow failure and high predisposition to cancer. The FA DNA repair pathway is required in humans to coordinate repair of DNA interstrand crosslinks. The central event in the activation of the pathway is the monoubiquitination of FANCD2 and FANCI by the E2-E3 pair, Ube2T-FANCL, with the central UBC-RWD (URD) domain of FANCL recognising the substrates. Whole genome sequencing studies of cancer cells from patients identified point mutations in the FANCL URD domain. We analysed 17 such variants of FANCL, including known substrate binding mutants (W212A, W214A and L248A, F252A, L254A, I265A), a FA mutation (R221C) and 14 cancer-associated mutations (F110S, I136V, L149V, L154S, A192G, E215Q, E217K, R221W, T224K, M247V, F252L, N270K, V287G, E289Q) through recombinant expression analysis, thermal shift assay, interaction with FANCD2, in vitro ubiquitination activity, and cellular sensitivity to an interstrand crosslinking agent. We find that the FANCL mutations I136V, L154S, W212A and L214A, R221W, R221C, and V287G are destabilising, with N270K and E289Q destabilising the C-terminal helices of the URD domain. The hydrophobic patch mutant (L248A, F252A, L254A, I265A), along with mutations E217K, T224K, and M247V, cause defects in the catalytic function of FANCL. This highlights the C-terminal lobe of the FANCL URD domain as important for the activity and function of FANCL. These mutations which affect the fold and activity of FANCL may contribute to tumorigenesis in these non-FA cancer patients, and this implicates FA genes in general cancer progression.

Item Type:Articles
Additional Information:This study was supported by a Cancer Research UK studentship 17739 (to M.G.F.) and the 427 MRC [grant number MC_UU_12016/12 (to R.T and H. W.)]; and ERC-2015-CoG-681582 ICLUb (to H.W.).
Glasgow Author(s) Enlighten ID:Walden, Professor Helen
Authors: Frost, M. G., Mazloumi Aboukheili, A. M., Toth, R., and Walden, H.
College/School:College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Journal Name:Bioscience Reports
Publisher:Portland Press
ISSN (Online):1573-4935
Published Online:18 May 2020
Copyright Holders:Copyright © 2020 The Author(s)
First Published:First published in Bioscience Reports
Publisher Policy:Reproduced under a Creative Commons license

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