Fig. 4: Visualizing the heart of the replisome.

The complex biology of the MCMs has baffled researchers ever since their identification in the 1980s, through the discovery of their essential role in eukaryotic origin licensing in the mid-1990s, and up to the last decade marked by in vitro insights into MCMs as the structural core of the replicative helicase. The transfer of structural and biochemical knowledge of DNA replication to the in vivo settings has been hampered by the inability to detect MCMs at active replication sites in the cellular environment. In fact, most of the immunostaining data available in the literature reported rather the exclusion of MCMs from sites of active DNA replication^{49,171,172,173}. Although few potential explanations considering alternative replisome architecture and limitations of visualizing techniques to detect a minor fraction of active MCMs have been proposed^{49,172,173,174,175,176}, imaging of replisome dynamics in physiological settings has not been possible for several decades. In the era of CRISPR, a recent study provided an explanation of this paradox⁷⁴. a Upon full assembly of the replisomes, the MCM scaffold is shielded by various interacting partners, limiting the access of anti-MCM antibodies, which primarily detect the inactive form of MCM complexes. b CRISPR-Cas9 endogenous tagging of MCM subunits by flexible linkers fused with fluorescent tags enables to overcome the sterical hindrance and allows visualization of both inactive MCMs as well as a minor fraction of active MCMs that are part of the replisomes. Although simple, this work opens new avenues to validate and study replisome architecture and its dynamics in the cellular environment using super-resolution microscopy or nanoscopy techniques.