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Genome duplication is safeguarded by constantly adjusting the activity of the replicative CMG (CDC45-MCM2-7-GINS) helicase. However, minichromosome maintenance proteins (MCMs)-the structural core of the CMG helicase-have never been visualized at sites of DNA synthesis inside a cell (the so-called MCM paradox). Here, we solve this conundrum by showing that anti-MCM antibodies primarily detect inactive MCMs. Upon conversion of inactive MCMs to CMGs, factors that are required for replisome activity bind to the MCM scaffold and block MCM antibody binding sites. Tagging of endogenous MCMs by CRISPR-Cas9 bypasses this steric hindrance and enables MCM visualization at active replisomes. Thus, by defining conditions for detecting the structural core of the replicative CMG helicase, our results explain the MCM paradox, provide visual proof that MCMs are an integral part of active replisomes in vivo, and enable the investigation of replication dynamics in living cells exposed to a constantly changing environment., (© 2022. The Author(s).)

Minichromosome maintenance proteins (MCMs) are DNA-dependent ATPases that bind to replication origins and license them to support a single round of DNA replication. A large excess of MCM2-7 assembles on chromatin in G1 phase as pre-replication complexes (pre-RCs), of which only a fraction become the productive CDC45-MCM-GINS (CMG) helicases that are required for genome duplication 1-4 . It remains unclear why cells generate this surplus of MCMs, how they manage to sustain it across multiple generations, and why even a mild reduction in the MCM pool compromises the integrity of replicating genomes 5,6 . Here we show that, for daughter cells to sustain error-free DNA replication, their mother cells build up a nuclear pool of MCMs both by recycling chromatin-bound (parental) MCMs and by synthesizing new (nascent) MCMs. Although all MCMs can form pre-RCs, it is the parental pool that is inherently stable and preferentially matures into CMGs. By contrast, nascent MCM3-7 (but not MCM2) undergo rapid proteolysis in the cytoplasm, and their stabilization and nuclear translocation require interaction with minichromosome-maintenance complex-binding protein (MCMBP), a distant MCM paralogue 7,8 . By chaperoning nascent MCMs, MCMBP safeguards replicating genomes by increasing chromatin coverage with pre-RCs that do not participate on replication origins but adjust the pace of replisome movement to minimize errors during DNA replication. Consequently, although the paucity of pre-RCs in MCMBP-deficient cells does not alter DNA synthesis overall, it increases the speed and asymmetry of individual replisomes, which leads to DNA damage. The surplus of MCMs therefore increases the robustness of genome duplication by restraining the speed at which eukaryotic cells replicate their DNA. Alterations in physiological fork speed might thus explain why even a minor reduction in MCM levels destabilizes the genome and predisposes to increased incidence of tumour formation.