Cullin ubiquitin ligases drive replisome disassembly during DNA replication termination. In worm, frog and mouse cells, CUL2LRR1 is required to ubiquitylate the MCM7 subunit of the CMG helicase. Here, we show that cullin ligases also drive CMG-MCM7 ubiquitylation in human cells, thereby making the helicase into a substrate for the p97 unfoldase. Using purified human proteins, including a panel of E2 ubiquitin-conjugating enzymes, we have reconstituted CMG helicase ubiquitylation, dependent upon neddylated CUL2LRR1. The reaction is highly specific to CMG-MCM7 and requires the LRR1 substrate targeting subunit, since replacement of LRR1 with the alternative CUL2 adaptor VHL switches ubiquitylation from CMG-MCM7 to HIF1. CUL2LRR1 firstly drives monoubiquitylation of CMG-MCM7 by the UBE2D class of E2 enzymes. Subsequently, CUL2LRR1 activates UBE2R1/R2 or UBE2G1/G2 to extend a single K48-linked ubiquitin chain on CMG-MCM7. Thereby, CUL2LRR1 converts CMG into a substrate for p97, which disassembles the ubiquitylated helicase during DNA replication termination.

中文翻译：

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通过 CUL2LRR1 和多种 E2 酶重建人 CMG 解旋酶泛素化

Cullin 泛素连接酶在 DNA 复制终止过程中驱动复制体分解。在蠕虫、青蛙和小鼠细胞中，CUL2LRR1 需要泛素化 CMG 解旋酶的 MCM7 亚基。在这里，我们表明 cullin 连接酶还驱动人类细胞中的 CMG-MCM7 泛素化，从而使解旋酶成为 p97 去折叠酶的底物。使用纯化的人类蛋白质，包括一组 E2 泛素结合酶，我们重建了 CMG 解旋酶泛素化，依赖于 neddylated CUL2LRR1。该反应对 CMG-MCM7 具有高度特异性，需要 LRR1 底物靶向亚基，因为用替代 CUL2 适配器 VHL 替换 LRR1 会将泛素化从 CMG-MCM7 切换到 HIF1。CUL2LRR1 首先通过 UBE2D 类 E2 酶驱动 CMG-MCM7 的单泛素化。随后，CUL2LRR1 激活 UBE2R1/R2 或 UBE2G1/G2 以在 CMG-MCM7 上扩展单个 K48 连接的泛素链。因此，CUL2LRR1 将 CMG 转化为 p97 的底物，在 DNA 复制终止期间分解泛素化解旋酶。