MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
Institute of Molecular Cell and Systems Biology, University of Glasgow, Glasgow G12 8QQ, U.K.
Biosci Rep. 2020 Jun 26;40(6). doi: 10.1042/BSR20191304.
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 cross-links. 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 recognizing 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 cross-linking agent. We find that the FANCL mutations I136V, L154S, W212A and L214A, R221W, R221C, and V287G are destabilizing, with N270K and E289Q destabilizing 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.
范可尼贫血症（FA）是一种罕见的遗传性疾病，其特征为发育缺陷、骨髓衰竭和癌症高发。FA 的 DNA 修复途径在人类中是必需的，用于协调 DNA 链间交联的修复。该途径的激活的中心事件是 E2-E3 对 Ube2T-FANCL 对 FANCD2 和 FANCI 的单泛素化，其中 FANCL 的中央 UBC-RWD（URD）结构域识别底物。对来自患者的癌细胞的全基因组测序研究发现 FANCL URD 结构域的点突变。我们分析了包括已知底物结合突变体（W212A、W214A 和 L248A、F252A、L254A、I265A）、FA 突变（R221C）和 14 个癌症相关突变体（F110S、I136V、L149V、L154S、A192G、E215Q、E217K、R221W、T224K、M247V、F252L、N270K、V287G、E289Q）在内的 17 种 FANCL 变体，通过重组表达分析、热位移测定、与 FANCD2 的相互作用、体外泛素化活性以及细胞对链间交联剂的敏感性进行分析。我们发现 FANCL 突变体 I136V、L154S、W212A 和 L214A、R221W、R221C 和 V287G 是不稳定的，而 N270K 和 E289Q 则使 URD 结构域的 C 末端螺旋不稳定。疏水性斑突变体（L248A、F252A、L254A、I265A）以及突变体 E217K、T224K 和 M247V 导致 FANCL 的催化功能缺陷。这突出了 FANCL URD 结构域的 C 末端叶对 FANCL 的活性和功能很重要。这些影响 FANCL 折叠和活性的突变可能导致这些非 FA 癌症患者的肿瘤发生，这表明 FA 基因一般与癌症进展有关。
