DNA replication origins retain mobile licensing proteins

Autor: Humberto Sanchez, Edo van Veen, Nynke H. Dekker, Kaley McCluskey, John F.X. Diffley, Belen Solano, Theo van Laar, Filip M. Asscher
Rok vydání: 2021
Předmět:
Zdroj: Nature Communications, Vol 12, Iss 1, Pp 1-12 (2021)
Nature Communications, 12(1)
Nature Communications
ISSN: 2041-1723
DOI: 10.1038/s41467-021-22216-x
Popis: DNA replication in eukaryotes initiates at many origins distributed across each chromosome. Origins are bound by the origin recognition complex (ORC), which, with Cdc6 and Cdt1, recruits and loads the Mcm2-7 (MCM) helicase as an inactive double hexamer during G1 phase. The replisome assembles at the activated helicase in S phase. Although the outline of replisome assembly is understood, little is known about the dynamics of individual proteins on DNA and how these contribute to proper complex formation. Here we show, using single-molecule optical trapping and confocal microscopy, that yeast ORC is a mobile protein that diffuses rapidly along DNA. Origin recognition halts this search process. Recruitment of MCM molecules in an ORC- and Cdc6-dependent fashion results in slow-moving ORC-MCM intermediates and MCMs that rapidly scan the DNA. Following ATP hydrolysis, salt-stable loading of MCM single and double hexamers was seen, both of which exhibit salt-dependent mobility. Our results demonstrate that effective helicase loading relies on an interplay between protein diffusion and origin recognition, and suggest that MCM is stably loaded onto DNA in multiple forms.
Eukaryotic DNA replication is regulated to ensure copying of the genome (only) once per cell cycle. Here the authors, using optical trapping and confocal microscopy, demonstrate the dynamics of the origin recognition complex and subsequent intermediates that lead up to the loading of an MCM helicase onto DNA.
Databáze: OpenAIRE