The DNA Pol E stimulatory activity of Mrc1 is modulated by phosphorylation
The DNA Pol E stimulatory activity of Mrc1 is modulated by phosphorylation
复制标题
Mrc1 的 DNA Pol E 刺激活性受磷酸化调节
DOI：
10.1080/15384101.2017.1403680
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发表时间：
2018
期刊：
影响因子：
4.3
通讯作者：
Lou Huiqiang
中科院分区：
文献类型：
--
作者：
Zhang Zhong-Xin;Zhang Jingjing;Cao Qinhong;Campbell Judith L.;Lou Huiqiang
DNA 复制检查点（芽殖酵母中的 Mec1-Mrc1-Rad53）是一种进化保守的监视系统，可确保所有真核生物中正确的 DNA 复制和基因组稳定性。与众所周知的复制检查点中介功能相比，Mrc1 作为正常复制叉组成部分的确切作用仍然相对不清楚。在这项研究中，我们提供了体外生化证据来支持酵母 Mrc1 能够增强主要前导链复制酶 DNA 聚合酶 ϵ (Pol ϵ) 的活性。 Mrc1 可以选择性地与带有单链区域的引物/模板 DNA 强烈结合，但不能与双链 DNA (dsDNA) 结合。基本补丁 1 (BP1) 内赖氨酸残基的突变会损害 DNA 结合和聚合酶刺激活性。有趣的是，Mrc1-3D 是一种模仿 Hog1/MAPK 激酶在渗透应激反应过程中磷酸化的突变体，保留了 DNA 结合但不保留聚合酶刺激。从用羟基脲 (HU) 处理的细胞纯化的 Mrc1 中，刺激作用也被消除，从而引发复制检查点激活。与之前的发现结合起来，这些结果表明，在不受干扰的条件下，Mrc1 具有 DNA 合成刺激活性，可以通过响应复制和/或渗透应激的 Mrc1 磷酸化来消除这种活性。
DNA replication checkpoint (Mec1-Mrc1-Rad53 in budding yeast) is an evolutionarily conserved surveillance system to ensure proper DNA replication and genome stability in all eukaryotes. Compared to its well-known function as amediator ofreplicationcheckpoint, the exact role of Mrc1 as a component of normal replication forks remains relatively unclear. In this study, we providein vitrobiochemical evidence to support that yeast Mrc1 is able to enhance the activity of DNA polymerase ϵ (Pol ϵ), the major leading strand replicase. Mrc1 can selectively bind avidly to primer/template DNA bearing a single-stranded region, but not to double-stranded DNA (dsDNA). Mutations of the lysine residues within basic patch 1 (BP1) compromise both DNA binding and polymerase stimulatory activities. Interestingly, Mrc1-3D, a mutant mimicking phosphorylation by the Hog1/MAPK kinase during the osmotic stress response, retains DNA binding but not polymerase stimulation. The stimulatory effect is also abrogated in Mrc1 purified from cells treated with hydroxyurea (HU), which elicits replication checkpoint activation. Taken together with previous findings, these results imply that under unperturbed condition, Mrc1 has a DNA synthesis stimulatory activity, which can be eliminated via Mrc1 phosphorylation in response to replication and/or osmotic stresses.