Publication highlights

DNA in our cells must be copied only once in the life cycle of a cell to maintain gene copy number and prevent genome instability. To make sure that this happens, loading of the enzyme (MCM), which separates two strands of the double helix, is separated in time from its activation. Once activated, MCM uses the energy derived from ATP hydrolysis to move along one DNA strand and physically separate the other strand, achieving DNA unwinding. Before activation, MCM is recruited by a loader onto the double helix. Researchers knew that, to complete loading, ATP hydrolysis by MCM is required but did not know why. Here researchers at the Crick show that MCM uses ATP hydrolysis to move along duplex DNA away from its loader. Their study also provides new mechanistic information about how polymer translocases (like DNA motors or the proteasome) use ATP hydrolysis to drive movement.

The MCM helicase enzyme separates the two strands of the double helix, enabling DNA replication, with two copies of MCM (a “double hexamer”) marking where replication can start. We have a clear understanding of how double hexamers are loaded in yeast, but not in human cells. Researchers from the Crick reconstituted double hexamer loading with purified human proteins and determined the atomic structures. They found that, unlike in yeast, loading of a human double hexamer is sufficient to start opening DNA. Two alternate loading pathways exist to load human double hexamers. Loading factors that are essential in yeast only play a dispensable, regulatory role in the human system. Thus, while the double-hexamer loaders are conserved throughout evolution, how they function is different. This work begins to unravel how human cells regulate initiation of DNA replication, ensure that their genome is duplicated only once, and prevent chromosome instability and cancer.

The ring-shaped MCM helicase enzyme is loaded around double-stranded DNA as a structure called a 'double hexamer' which is inactive before DNA is replicated. Upon activation, two split MCM single hexamers transition to encircling single-stranded DNA, establishing divergent replication forks. Researchers at the Crick investigated how this happens, by visualising helicase activation under the electron microscope. They found that Mcm10, the factor promoting initiation, wedges itself in between two MCM rings so that the double hexamer is broken in two. Mcm10 also constricts the MCM ring pore, forcing one of the two DNA strands to escape. These mechanisms ensure that chromosomes are replicated only once per cell cycle, preventing chromosome instability and cancer.

Researchers at the Francis Crick Institute discovered the mechanism for a protein that starts DNA replication, in a process that supports the propagation of life. Their findings, published in Molecular Cell, show that a protein called DONSON changes the structure of the minichromosome maintenance (MCM) helicase, which is the enzyme that opens the DNA double helix at the start of replication. Before replication starts, two copies of the MCM helicase form a double ring around the double helix. As replication starts, the MCM splits into two rings that separate the two filaments of the DNA, which become ready to be copied. To establish how helicases can split, researchers used a cryo-electron microscope to analyse material extracted from frog eggs, whose replication proteins are very similar to those present in humans. They found that two DONSON molecules are recruited at the same time, causing the MCM double ring to break and other components of the replication machinery to be recruited. Changes in the sequence of the DONSON gene negatively affect interactions between proteins and DNA replication processes in human cells, and are also found mutated in patients affected by primordial dwarfism, a condition that severely limits life expectancy.

Researchers in the Costa and Diffley labs have visualised the full process of the DNA double helix being untwisted and opened using cryo EM. The team observed that, after embracing the DNA, two enzymes drift away from each other in a process that deforms the double helix, initiating the process of DNA opening.