Solving the MCM paradox by visualizing the scaffold of CMG helicase at active replisomes

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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.

OriginalsprogEngelsk
Artikelnummer6090
TidsskriftNature Communications
Vol/bind13
Antal sider11
ISSN2041-1723
DOI
StatusUdgivet - 2022

Bibliografisk note

Funding Information:
Research funding was provided by the Novo Nordisk Foundation (NNF14CC0001). H.P-S. was supported by the Czech Science Foundation Junior Star (22-20303M). T.C.R.M. was supported by the Danish National Research Foundation (DNRF115) and Carlsberg Foundation (CF21-0571). We thank Protein Imaging Platform at the Center for Protein Research for the technical assistance. Cell sorting was carried out at CPR and Danstem Flow Cytometry Platform. We thank D. Gilbert for sharing the CHO cell line ectopically expressing MCM4-mEmerald and PCNA-RFP. The pX335 plasmid was a gift from F. Zhang. We thank all members of the Lukas and Sedlackova lab for stimulating discussions and critical comments on the manuscript.

Publisher Copyright:
© 2022, The Author(s).

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