Alver Robert C, Chadha Gaganmeet Singh, Gillespie Peter J, Blow J Julian
Centre for Gene Regulation & Expression, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
Centre for Gene Regulation & Expression, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
Cell Rep. 2017 Mar 7;18(10):2508-2520. doi: 10.1016/j.celrep.2017.02.042.
Dbf4-dependent kinases (DDKs) are required for the initiation of DNA replication, their essential targets being the MCM2-7 proteins. We show that, in Xenopus laevis egg extracts and human cells, hyper-phosphorylation of DNA-bound Mcm4, but not phosphorylation of Mcm2, correlates with DNA replication. These phosphorylations are differentially affected by the DDK inhibitors PHA-767491 and XL413. We show that DDK-dependent MCM phosphorylation is reversed by protein phosphatase 1 (PP1) targeted to chromatin by Rif1. Loss of Rif1 increased MCM phosphorylation and the rate of replication initiation and also compromised the ability of cells to block initiation when challenged with replication inhibitors. We also provide evidence that Rif1 can mediate MCM dephosphorylation at replication forks and that the stability of dephosphorylated replisomes strongly depends on Chk1 activity. We propose that both replication initiation and replisome stability depend on MCM phosphorylation, which is maintained by a balance of DDK-dependent phosphorylation and Rif1-mediated dephosphorylation.
Dbf4 依赖性激酶（DDK）是 DNA 复制起始所必需的，其主要作用靶点是 MCM2-7 蛋白。我们发现，在非洲爪蟾卵提取物和人类细胞中，与 DNA 结合的 Mcm4 的过度磷酸化而非 Mcm2 的磷酸化与 DNA 复制相关。这些磷酸化受到 DDK 抑制剂 PHA-767491 和 XL413 的不同影响。我们表明，靶向染色质的 Rif1 所招募的蛋白磷酸酶 1（PP1）可逆转 DDK 依赖性的 MCM 磷酸化。Rif1 的缺失增加了 MCM 磷酸化和复制起始速率，并且在细胞受到复制抑制剂挑战时，也损害了细胞阻止起始的能力。我们还提供证据表明，Rif1 可在复制叉处介导 MCM 去磷酸化，并且去磷酸化的复制体的稳定性强烈依赖于 Chk1 活性。我们提出，复制起始和复制体稳定性均依赖于 MCM 磷酸化，而这种磷酸化通过 DDK 依赖性磷酸化和 Rif1 介导的去磷酸化之间的平衡得以维持。
