Distinct roles of MCM2-7 subunits in replication licensing in human cells

Eukaryotic DNA replication requires the precise assembly of MCM2-7 single hexamers (SHs) into head-to-head double hexamers (DHs) at replication origins. While DH formation is well-characterized in budding yeast, the underlying mechanisms in human cells remain poorly understood. Here, we report cryo-electron microscopy structures of endogenous human MCM2-7 SH isolated from G1-phase cells. In these structures, human MCM2-7 adopts a latched spiral conformation in an autoinhibited state where the carboxyl-terminal extension (CTE) of MCM5 occupies the central channel, and MCM3-CTE is capable of locking the MCM2-5 gate to occlude DNA entry. Systematic functional analysis demonstrates that the six CTEs of MCM2-7 play distinct roles in SH stability, MCM loading, and DH formation on chromatin. Surprisingly, unlike in yeast, the human MCM3-CTE is dispensable for cell viability but ensures efficient genome-wide replication initiation. Our findings establish how human MCM2-7 enables flexible yet precise MCM loading via its CTEs, providing a framework for understanding the regulation of DNA replication initiation in higher eukaryotes.