DNA-damage repair is implemented by proteins that are coordinated by specialized molecular signals. One such signal in the Fanconi anemia (FA) pathway for the repair of DNA interstrand crosslinks is the site-specific monoubiquitination of FANCD2 and FANCI. The signal is mediated by a multiprotein FA core complex (FA-CC) however, the mechanics for precise ubiquitination remain elusive. We show that FANCL, the RING-bearing module in FA-CC, allosterically activates its cognate ubiqutin-conjugating enzyme E2 UBE2T to drive site-specific FANCD2 ubiquitination. Unlike typical RING E3 ligases, FANCL catalyzes ubiquitination by rewiring the intraresidue network of UBE2T to influence the active site. Consequently, a basic triad unique to UBE2T engages a structured acidic patch near the target lysine on FANCD2. This three-dimensional complementarity, between the E2 active site and substrate surface, induced by FANCL is central to site-specific monoubiquitination in the FA pathway. Furthermore, the allosteric network of UBE2T can be engineered to enhance FANCL-catalyzed FANCD2–FANCI di-monoubiquitination without compromising site specificity.

DNA 损伤修复是由专门的分子信号协调的蛋白质实现的。范可尼贫血 (FA) 通路中用于修复 DNA 链间交联的一种信号是 FANCD2 和 FANCI 的位点特异性单泛素化。该信号由多蛋白 FA 核心复合物 (FA-CC) 介导，然而，精确泛素化的机制仍然难以捉摸。我们展示了 FANCL，FA-CC 中的环轴承模块，变构激活其同源泛素结合酶 E2 UBE2T 以驱动位点特异性 FANCD2 泛素化。与典型的 RING E3 连接酶不同，FANCL 通过重新连接 UBE2T 的残基内网络以影响活性位点来催化泛素化。因此，UBE2T 独有的基本三元组与 FANCD2 上目标赖氨酸附近的结构化酸性贴片接合。这种三维的互补性，在 E2 活性位点和底物表面之间，由 FANCL 诱导是 FA 通路中位点特异性单泛素化的中心。此外，可以设计 UBE2T 的变构网络以增强 FANCL 催化的 FANCD2-FANCI 二单泛素化而不影响位点特异性。