Inlay

During eukaryotic DNA replication initiation, inactive MCM2-7 double-hexamers assembled at replication origins must be converted into two active CMG helicases, yet how this transition is coupled to origin DNA unwinding in vivo remains unclear. Here, we identify a DNA-bound intermediate with an extended genomic footprint that forms during helicase activation. Genome-wide mapping of initial strand separation reveals that DNA unwinding initiates near the N-terminal interface of opposing MCM2-7 hexamers. At these sites, the origin DNA exhibits a conserved AT-rich/GC-rich/AT-rich sequence architecture on which the helicase complex is centred, consistent with a role in promoting DNA extrusion following double-hexamer separation. We further show that restricting hexamer rotation and splitting delays release of the Cdc45-loading factor Sld3, demonstrating that mechanical transitions during helicase activation are tightly coupled to complex disassembly. Finally, we provide in vivo evidence that single-stranded DNA is ejected through a specialised DNA exit gate at the Mcm2/5 interface during helicase activation, which is dispensable for ongoing DNA synthesis. Together, these findings establish a mechanistic framework for how replication origins are remodelled to initiate DNA replication and reveal key intermediates and DNA transactions during helicase activation. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, ZA 153/28-1, 393547839 - SFB1361, 505087959 Engineering and Physical Sciences Research Council, EP/N509486/1 European Commission, 101109916 Biotechnology and Biological Sciences Research Council, BB/N000323/1, BB/S001387/1 Medical Research Council, MC_U120085811 Wellcome Trust, 107903/Z/15/Z