Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
Cell. 2019 Jan 10;176(1-2):167-181.e21. doi: 10.1016/j.cell.2018.10.053. Epub 2018 Dec 27.
Covalent DNA-protein cross-links (DPCs) impede replication fork progression and threaten genome integrity. Using Xenopus egg extracts, we previously showed that replication fork collision with DPCs causes their proteolysis, followed by translesion DNA synthesis. We show here that when DPC proteolysis is blocked, the replicative DNA helicase CMG (CDC45, MCM2-7, GINS), which travels on the leading strand template, bypasses an intact leading strand DPC. Single-molecule imaging reveals that GINS does not dissociate from CMG during bypass and that CMG slows dramatically after bypass, likely due to uncoupling from the stalled leading strand. The DNA helicase RTEL1 facilitates bypass, apparently by generating single-stranded DNA beyond the DPC. The absence of RTEL1 impairs DPC proteolysis, suggesting that CMG must bypass the DPC to enable proteolysis. Our results suggest a mechanism that prevents inadvertent CMG destruction by DPC proteases, and they reveal CMG's remarkable capacity to overcome obstacles on its translocation strand.
共价 DNA-蛋白质交联物 (DPCs) 会阻碍复制叉的前进，并威胁基因组的完整性。我们之前使用非洲爪蟾卵提取物表明，复制叉与 DPC 碰撞会导致其蛋白水解，随后进行跨损伤 DNA 合成。在这里，我们发现当 DPC 蛋白水解被阻断时，在领头链模板上移动的复制性 DNA 解旋酶 CMG（CDC45、MCM2-7、GINS）会绕过完整的领头链 DPC。单分子成像显示，在绕过过程中 GINS 不会从 CMG 上解离，并且 CMG 在绕过后会急剧减速，可能是由于与停滞的领头链解耦。DNA 解旋酶 RTEL1 促进了绕过，显然是通过在 DPC 之外产生单链 DNA 来实现的。RTEL1 的缺失会损害 DPC 的蛋白水解，表明 CMG 必须绕过 DPC 才能进行蛋白水解。我们的结果表明了一种防止 DPC 蛋白酶意外破坏 CMG 的机制，并且它们揭示了 CMG 在其转位链上克服障碍的非凡能力。
