Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Blavatnik Institute, Boston, Massachusetts 02115, USA.
Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA.
Genes Dev. 2020 Nov 1;34(21-22):1534-1545. doi: 10.1101/gad.339739.120. Epub 2020 Sep 17.
When converging replication forks meet during replication termination, the CMG (Cdc45-MCM2-7-GINS) helicase is polyubiquitylated by CRL2 and unloaded from chromatin by the p97 ATPase. Here, we investigate the signal that triggers CMG unloading in 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.
当复制叉在复制终止过程中相遇时，CMG（Cdc45-MCM2-7-GINS）解旋酶被 CRL2 多泛素化，并被 p97 ATP 酶从染色质上卸载。在这里，我们使用单分子和整体方法研究了在卵提取物中触发 CMG 卸载的信号。我们表明，汇聚的 CMG 相互通过并以与汇聚前相同的速度继续转运，之后它们被快速且独立地卸载。当 CMG 卸载被阻断时，发散的 CMG 不支持 DNA 合成，这表明在绕过 CMG 后，它们遇到了汇聚叉的新生滞后链，然后沿着双链 DNA（dsDNA）转运。然而，CMG 从染色质上的去除不需要 dsDNA 上的转运，因为在没有新生链合成的情况下，汇聚的 CMG 仍然被卸载。此外，在没有任何 DNA 的情况下，重组 CMG 加入核提取物中会经历泛素化和组装，并且 DNA 消化会在停滞的复制叉上触发 CMG 的泛素化。我们的发现表明，DNA 在延伸过程中抑制 CMG 的泛素化，并且当 CMG 汇聚时，这种抑制被解除，导致 CMG 卸载。
