ReconSil: An electron microscopy toolbox to study helicase function at an origin of replication
Authors listThomas Puehringer Julia Greiwe Thomas Miller Alessandro Costa
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.
Journal Methods in Enzymology