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J Biol Chem. 2017 Aug 4;292(31):13008-13021. doi: 10.1074/jbc.M117.779371. Epub 2017 Jun 22.

The Mcm2-7-interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing.

Author information

1
From the Accelerator Applications Research Group, Nishina Center for Accelerator-Based Science and mizumi@riken.jp.
2
Cellular Dynamics Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.
3
Department of Life Science, Graduate School of Bioresources, Mie University, Tsu, Mie 514-8507, Japan.
4
From the Accelerator Applications Research Group, Nishina Center for Accelerator-Based Science and.
5
Department of Life Science, Faculty of Science, Gakushuin University, Tokyo 171-8588, Japan.
6
Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki 305-8577, Japan.

Abstract

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) α, and proliferating cell nuclear antigen, whereas the chromatin loading of Cdc45 and Pol ϵ 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 ϵ.

KEYWORDS:

DNA binding protein; DNA helicase; DNA replication; DNA-protein interaction; Mcm10; Mcm2–7; cell cycle

PMID:
28646110
PMCID:
PMC5546039
DOI:
10.1074/jbc.M117.779371
[Indexed for MEDLINE]
Free PMC Article

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