Functions and regulations of Mcm10 protein in eukaryotic chromosomal replication

Mcm10首先在酿酒酵母中被鉴定为染色体DNA复制所必需的蛋白质。最近的研究表明，Mcm10在DNA复制的起始和复制的延伸中都具有多种功能，但其确切的作用尚不清楚。最近我们发现GFP标记的人Mcm10在细胞核内出现离散的病灶，并且GFP-Mcm10病灶的分布模式先于模式复制的变化30~60分钟。这些结果表明，人Mcm10在复制前30-60分钟被招募到复制站点。在这项研究中，我们通过使用填充长度为874个氨基酸的人Mcm10的截断导数来定位其焦点形成所需的结构域。Mcm10在染色质上的保留需要包含473-693氨基酸的区域，该区域与与Mcm2-7相互作用的区域重叠。尽管CDC45和DNA聚合酶被负载到染色质上，但siRNA耗尽Mcm10减缓了S期的进展，并干扰了RPA和增殖细胞核抗原的染色质负载。S时相进展的延迟是由于抑制了新起源的放电，而不是抑制了已建立的复制分叉。这些结果表明，人Mcm10通过与Mcm2-7的相互作用被负载到染色质上，并参与了CDC45和DNA聚合酶被负载到染色质上后复制前复合体的激活。

Mcm10 was first identified in S. cerevisiae as an essential protein for chromosomal DNA replication. Recent studies have shown that Mcm10 has multiple functions both in the initiation of DNA replication and the replication elongation, although the precise roles of Mcm10 are still unknown. Recently we have hind that GFP-tagged human Mcm10 fanned discrete foci in the nuclei, and the distribution patterns of GFP-Mcm10 foci preceded changes in replication of patterns by 30〜60 min. These results suggest that human Mcm10 is recruited to the replication sites 30-60 min before they replicate. In this study, we have mapped a domain required for its foci formation by using the truncated derivatives of the fill-length 874-amino acid human Mcm10. The retention of Mcm10 onto chromatin required the domain containing amino acids 473-693, which was overlapped with the domain interacting with Mcm2-7. Depletion of Mcm10 by siRNA slowed down the S phase progression and interfered the chromatin loading of RPA and PCNA, although Cdc45 and DNA polymerases were loaded onto chromatin. The delay of S phase progression was caused by the inhibition of new origin firing, not by the inhibition of established replication fork. These results suggest that human Mcm10 is loaded onto the chromatin through the interaction with Mcm2-7 and involved in the activation of pre-replication complex after Cdc45 and DNA polymerases are loaded onto chromatin