The Mcm2-7-interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing
The Mcm2-7-interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing
复制标题
DOI：
10.1074/jbc.m117.779371
复制
发表时间：
2017-08-04
影响因子：
4.8
通讯作者：
Hanaoka, Fumio
中科院分区：
文献类型：
--
作者：
Izumi, Masako;Mizuno, Takeshi;Hanaoka, Fumio
蛋白质微型染色体维护 10 (Mcm10) 最初被鉴定为维持微型染色体质粒的必需酵母蛋白。随后，Mcm10 被证明是染色体 DNA 复制过程中起始和延伸所必需的。然而，目前尚不完全清楚Mcm10的多种功能是如何协调的，或者Mcm10如何与复制叉上的其他因子相互作用。在这里，我们鉴定并表征了人类 Mcm10 中的 Mcm2-7 相互作用结构域。与 Mcm2-7 的相互作用需要包含氨基酸 530-655 的 Mcm10 结构域，该结构域与 Mcm10 在染色质上稳定保留所需的结构域重叠。通过 siRNA 耗尽内源性 Mcm10 的 HeLa 细胞中截短的 Mcm10 的表达表明，Mcm10 保守结构域（氨基酸 200-482）对于 DNA 复制至关重要，而保守结构域和 Mcm2-7 结合结构域都是其完整活性所必需的。 Mcm10 缺失降低了 DNA 复制的起始频率，并干扰复制蛋白 A、DNA 聚合酶 (Pol) 和增殖细胞核抗原的染色质负载，而 Cdc45 和 Pol E 的染色质负载不受影响。这些结果表明，人 Mcm10 通过与 Mcm2-7 相互作用与染色质结合，并且主要参与加载 Cdc45 和 Pol E 后 DNA 复制的启动。
The protein mini-chromosome maintenance 10 (Mcm10) was originally identified as an essential yeast protein in the maintenance of mini-chromosome plasmids. Subsequently, Mcm10 has been shown to be required for both initiation and elongation during chromosomal DNA replication. However, it is not fully understood how the multiple functions of Mcm10 are coordinated or how Mcm10 interacts with other factors at replication forks. Here, we identified and characterized the Mcm2-7-interacting domain in human Mcm10. The interaction with Mcm2-7 required the Mcm10 domain that contained amino acids 530-655, which overlapped with the domain required for the stable retention of Mcm10 on chromatin. Expression of truncated Mcm10 in HeLa cells depleted of endogenous Mcm10 via siRNA revealed that the Mcm10 conserved domain (amino acids 200-482) is essential for DNA replication, whereas both the conserved and the Mcm2-7-binding domains were required for its full activity. Mcm10 depletion reduced the initiation frequency of DNA replication and interfered with chromatin loading of replication protein A, DNA polymerase (Pol) , and proliferating cell nuclear antigen, whereas the chromatin loading of Cdc45 and Pol E was unaffected. These results suggest that human Mcm10 is bound to chromatin through the interaction with Mcm2-7 and is primarily involved in the initiation of DNA replication after loading of Cdc45 and Pol E.