ReconSil: An electron microscopy toolbox to study helicase function at an origin of replication.

Autor:	Pühringer T; Macromolecular Machines Laboratory, The Francis Crick Institute, London, United Kingdom., Greiwe JF; Macromolecular Machines Laboratory, The Francis Crick Institute, London, United Kingdom., Miller TCR; Center for Chromosome Stability, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark. Electronic address: tmiller@sund.ku.dk., Costa A; Macromolecular Machines Laboratory, The Francis Crick Institute, London, United Kingdom. Electronic address: alessandro.costa@crick.ac.uk.
Jazyk:	angličtina
Zdroj:	Methods in enzymology [Methods Enzymol] 2022; Vol. 672, pp. 203-231. Date of Electronic Publication: 2022 Apr 22.
DOI:	10.1016/bs.mie.2022.03.016
Abstrakt:	The loading of the MCM replicative helicase onto eukaryotic origins of replication occurs via a sequential, symmetric mechanism. Here, we describe a method to study this multistep reaction using electron microscopy. Tools presented include protein expression and purification protocols, methods to produce asymmetric replication origin substrates and bespoke image processing strategies. DNA templates include recognisable protein roadblocks that help to orient DNA replication factors along a specific origin sequence. Detailed electron microscopy image processing protocols are provided to reposition 2D averages onto the original micrograph for the in silico reconstitution of fully occupied origins of replication. Using these tools, a chemically trapped helicase loading intermediate is observed sliding along origin DNA, showcasing a key feature of the MCM loading mechanism. Although developed to study replicative helicase loading, this method can be employed to investigate the mechanism of other multicomponent biochemical reactions, occurring on a flexible polymeric substrate. (Copyright © 2022 Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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