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

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.

Regulation of MCM2-7 function

“…We observed that human MCM10 mainly binds to MCM4, 6 and 7 (unpublished results), which may directly bind to single-stranded DNA during DNA unwinding, and we also showed that MCM10 binds to the ATP-binding domain of MCM6 (Hosoi et al, 2016). A recent study showed that MCM10 activity to stimulate DNA replication in human cells requires its two MCM2-7-interacting domains, located in the central domain (amino acids 200 -482) and carboxyl-terminal region (amino acids 530 -655) (Izumi et al, 2017). It was also suggested that an interaction of Mcm10 and double-hexameric Mcm2-7 is required for helicase splitting and activation during S phase (Quan et al, 2015).…”

Distinct MCM10 Proteasomal Degradation Profiles by Primate Lentiviruses Vpr Proteins

“…The region encompassing amino acids 530-655, also known as the MCM2-7 interaction region, was mapped as a determinant domain of MCM10 degradation under induction by Vpr [20,43]. Previously, little was known about the 530-633 region of MCM10, identified as a newly identified functional domain, flanked by an ID and involved in parts of CTD.…”

Human NK cell deficiency as a result of biallelic mutations in MCM10

“…While quantitative measurement of protein levels did not identify differences in expression of full-length protein between the patient and healthy donors, the cell-cycle dependent changes in expression of MCM10 also make it difficult to draw quantitative conclusions based on this measurement. With relative expression of MCM10 highest in S phase (28), it is likely that cell cycle-dependent protein regulation affects the overall expression of MCM10 in patient cells, which we have shown are more frequently detected in S phase than healthy donor control cells.…”

“…Furthermore, direct interaction of di-ubiquitinated MCM10 with PCNA is required for DNA elongation (42). Human MCM10 interacts directly with the CMG helicase through MCM2 and CDC45, supporting chromatin association of MCM10 and efficient firing and elongation during replication (28,30). To further probe the effect of the R426C mutation on replisome formation, we immunoprecipitated WT MCM10 or MCM10 R426C using turboGFP and probed for POLA and MCM2, CDC45 and PCNA.…”

“…MCM10 is a highly conserved and non-redundant component of the eukaryotic replisome that binds directly to the MCM2-7 complex, CDC45, and both double-and single-stranded DNA (27). MCM10 is thought to play a critical role in CMG complex assembly and activation and replication fork processivity (28)(29)(30). Here, we sought to define the molecular nature of the MCM10 mutations discovered in a patient with NKD.…”