BPS2026 – Visualization of double-hexameric MCM in cellulo using minflux nanoscopy

DNA replication in eukaryotes is thought to initiate with the loading of the minichromosome maintenance (MCM) helicase complex as a double hexamer at replication origins. Our understanding of MCM assembly and function has largely been derived from cryo-EM studies of reconstituted proteins and DNA replication-associated in situ HiC. However, currently there is no direct visualization of double-hexameric MCM in cellulo. According to the mantra “seeing is believing,” we utilize MINFLUX nanoscopy to image individual MCM inside the nucleus. MINFLUX nanoscopy is a powerful localization technique providing localization precisions down to 1 nm. In G1 phase, we observe pairs of MCM with a 20 nm distance, resembling loaded double-hexameric MCM. During S phase, MCM helicases are responsible for unwinding the DNA at replication origins. Accordingly, we observe additional to the double-hexameric MCM, pairs of MCM with an increased distance up to 35–60 nm. This suggests the formation of a diverging replication fork as observed in cryo-EM studies. Additionally, we observe that pairs of MCM organize into cluster, reminiscent to the coupling of sister forks. All in all, this work provides the first direct visualization of double-hexameric MCM in their native cellular environment, revealing dynamic structural transitions associated with replication initiation and progression. Our findings offer new insights into the spatial organization and functional behavior of MCM helicases during the cell cycle.