When converging replication forks meet during replication termination, the CMG (Cdc45–MCM2–7–GINS) helicase is polyubiquitylated by CRL2^Lrr1 and unloaded from chromatin by the p97 ATPase. Here, we investigate the signal that triggers CMG unloading in Xenopus egg extracts using single-molecule and ensemble approaches. We show that converging CMGs pass each other and keep translocating at the same speed as before convergence, whereafter they are rapidly and independently unloaded. When CMG unloading is blocked, diverging CMGs do not support DNA synthesis, indicating that after bypass CMGs encounter the nascent lagging strands of the converging fork and then translocate along double-stranded DNA (dsDNA). However, translocation on dsDNA is not required for CMG's removal from chromatin because in the absence of nascent strand synthesis, converging CMGs are still unloaded. Moreover, recombinant CMG added to nuclear extract undergoes ubiquitylation and disassembly in the absence of any DNA, and DNA digestion triggers CMG ubiquitylation at stalled replication forks. Our findings suggest that DNA suppresses CMG ubiquitylation during elongation and that this suppression is relieved when CMGs converge, leading to CMG unloading.

中文翻译：

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DNA复制叉可在终止前抑制CMG从染色质中卸载。

当聚合复制叉在复制终止期间相遇时，CMG（Cdc45–MCM2–7–GINS）解旋酶被CRL2 ^Lrr1泛泛化，^并被p97 ATPase从染色质中卸载。在这里，我们调查了触发非洲爪蟾CMG卸载的信号蛋提取物采用单分子和整体方法。我们表明，收敛的CMG彼此通过并保持与收敛之前相同的速度移位，此后它们被快速独立地卸载。当CMG卸载受阻时，分散的CMG不支持DNA合成，这表明旁路CMG在遇到汇聚叉的新生滞后链后，会沿着双链DNA（dsDNA）转移。但是，从染色质中去除CMG不需要dsDNA上的易位，因为在没有新生链合成的情况下，会聚的CMG仍然没有负载。此外，添加到核提取物中的重组CMG在不存在任何DNA的情况下会发生泛素化和分解，并且DNA消化会在停滞的复制叉处触发CMG泛素化。