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

Autor: Polasek-Sedlackova, Hana, Miller, Thomas C. R., Krejci, Jana, Rask, Maj-Britt, Lukas, Jiri
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Zdroj: Nature Communications; 10/14/2022, Vol. 13, p1-11, 11p
Abstrakt: 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. For several decades the MCM2-7 proteins, the core of the DNA replicative helicase, eluded detection at DNA replication sites. Here, the authors solve this conundrum by gene editing, which enables visualization of replication dynamics in living cells. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index
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