Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation.
Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation.
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DOI：
10.15252/embr.201641983
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发表时间：
2017-03
期刊：
影响因子：
7.7
通讯作者：
Donaldson AD
中科院分区：
文献类型：
--
作者：
Hiraga SI;Ly T;Garzón J;Hořejší Z;Ohkubo YN;Endo A;Obuse C;Boulton SJ;Lamond AI;Donaldson AD
人类RIF 1蛋白控制DNA复制，但其分子机制在很大程度上是未知的。在这里，我们证明了人RIF 1通过与蛋白磷酸酶1（PP 1）形成复合物来负调节DNA复制，该复合物限制磷酸化介导的MCM复制解旋酶的激活。我们确定了四个MCM解旋酶亚基上的特定残基，这些残基在RIF 1耗尽后显示出过度磷酸化，其中MCM 4的调节N末端结构域受到特别强烈的影响。除了限制起源激活的作用外，我们还发现了人类RIF 1 ‐ PP 1在介导有效起源许可方面的意想不到的新作用。具体而言，在细胞周期的G1期，RIF 1-PP 1保护结合origin的ORC 1蛋白免于过早磷酸化和随后的蛋白酶体降解。RIF 1的耗尽或PP 1的抑制使ORC 1不稳定，从而减少了来源许可。与减少的来源许可一致，RIF 1耗尽的细胞表现出活性来源之间的间隔增加。因此，人RIF 1作为PP 1靶向亚基，通过刺激起点许可步骤积极调节DNA复制，然后通过抵消复制起点激活来消极调节。
The human RIF1 protein controls DNA replication, but the molecular mechanism is largely unknown. Here, we demonstrate that human RIF1 negatively regulates DNA replication by forming a complex with protein phosphatase 1 (PP1) that limits phosphorylation‐mediated activation of the MCM replicative helicase. We identify specific residues on four MCM helicase subunits that show hyperphosphorylation upon RIF1 depletion, with the regulatory N‐terminal domain of MCM4 being particularly strongly affected. In addition to this role in limiting origin activation, we discover an unexpected new role for human RIF1‐PP1 in mediating efficient origin licensing. Specifically, during the G1 phase of the cell cycle, RIF1‐PP1 protects the origin‐binding ORC1 protein from untimely phosphorylation and consequent degradation by the proteasome. Depletion of RIF1 or inhibition of PP1 destabilizes ORC1, thereby reducing origin licensing. Consistent with reduced origin licensing, RIF1‐depleted cells exhibit increased spacing between active origins. Human RIF1 therefore acts as a PP1‐targeting subunit that regulates DNA replication positively by stimulating the origin licensing step, and then negatively by counteracting replication origin activation.