Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, College of Medical and Dental Sciences, University of Birmingham, Vincent Drive, Birmingham United Kingdom.
Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, College of Medical and Dental Sciences, University of Birmingham, Vincent Drive, Birmingham United Kingdom.
J Biol Chem. 2022 Aug;298(8):102234. doi: 10.1016/j.jbc.2022.102234. Epub 2022 Jul 4.
Complex cellular processes are driven by the regulated assembly and disassembly of large multiprotein complexes. While we are beginning to understand the molecular mechanism for assembly of the eukaryotic DNA replication machinery (replisome), we still know relatively little about the regulation of its disassembly at replication termination. Recently, the first elements of this process have emerged, revealing that the replicative helicase, at the heart of the replisome, is polyubiquitylated prior to unloading and that this unloading requires p97 segregase activity. Two different E3 ubiquitin ligases have now been shown to ubiquitylate the helicase under different conditions: Cul2 and TRAIP. Here, using Xenopus laevis egg extract cell-free system and biochemical approaches, we have found two p97 cofactors, Ubxn7 and Faf1, which can interact with p97 during replisome disassembly during S-phase. We show only Ubxn7, however, facilitates efficient replisome disassembly. Ubxn7 delivers this role through its interaction via independent domains with both Cul2 and p97 to allow coupling between Mcm7 ubiquitylation and its removal from chromatin. Our data therefore characterize Ubxn7 as the first substrate-specific p97 cofactor regulating replisome disassembly in vertebrates and a rationale for the efficacy of the Cul2 replisome unloading pathway in unperturbed S-phase.
复杂的细胞过程是由大型多蛋白复合物的调节组装和拆卸驱动的。虽然我们开始了解真核 DNA 复制机器（复制体）的组装分子机制，但我们仍然相对较少了解其在复制终止时的拆卸调节。最近，这个过程的第一个要素已经出现，揭示了复制体核心的复制性螺旋酶在卸载之前被多聚泛素化，并且这种卸载需要 p97 分体型酶的活性。现在已经显示两种不同的 E3 泛素连接酶可以在不同条件下泛素化螺旋酶：Cul2 和 TRAIP。在这里，我们使用非洲爪蟾卵提取物无细胞系统和生化方法，发现了两个 p97 共因子 Ubxn7 和 Faf1，它们可以在 S 期复制体拆卸过程中与 p97 相互作用。然而，只有 Ubxn7 可以促进有效的复制体拆卸。Ubxn7 通过其与 Cul2 和 p97 的独立结构域相互作用来发挥此作用，从而允许 Mcm7 泛素化与其从染色质上的去除之间的偶联。因此，我们的数据将 Ubxn7 确定为第一个底物特异性 p97 共因子，用于调节脊椎动物复制体的拆卸，并且为 Cul2 复制体卸载途径在未受干扰的 S 期的功效提供了依据。
