Discovery of a new complex formed before DNA replication
Like a football match that can’t begin until all the players have arrived, got changed, and warmed up, DNA replication can’t start until all of the necessary elements have gathered and assembled themselves in the proper formation. Before the process kicks into action, molecular machinery forms on the DNA strands, ready to pull them apart so each can be used as a template for a new copy. It is known that cancer cells are particularly susceptible to inhibition of this process, and now new research from the CSC may have revealed a key weakness that could allow us to halt cancer in its tracks.
In a paper published in Molecular Cell, Christian Speck (DNA Replication Group) and his team discovered a new step in the loading of the helicase – a double-ringed complex that latches to DNA before replication. Their previous work had shown that the main players in the loading process – ORC (origin recognition complex), Cdc6 and Cdt1 – work together to load two MCM2-7 hexamers onto the DNA one at a time. “After that, we wanted to uncover the mechanism,” explained Speck. “How do you go from one single hexamer to a double?” The first question centred on Cdt1 – a relatively small molecule compared to the big MCM2-7 and ORC/Cdc6 complexes. “We found that Cdt1 binds to a specific place on the MCM surface, and this reshapes the whole complex.”
The shape-shifting effectively hides an inhibitory zone on the protein’s surface, which allows it to bind to ORC and load itself onto the DNA. “This guarantees that Cdt1 is present in the final complex. Cdt1 helps regulate each step of the reaction, from bringing the MCM into the nucleus, then allowing complex formation, to finally activating ATP hydrolysis [the spark of energy that powers the reaction].”
Once the first MCM2-7 hexamer has bound to the DNA, a second hexamer is drawn to the site, so that the completed double-ring helicase can get to work splitting the DNA strands. “This is where the other new finding comes in,” says Speck. “People thought that after ATP hydrolysis a double hexamer would form. But the product of ATP hydrolysis is actually a new complex – an ORC/Cdc6/MCM2-7 complex (called OCM).” This process releases Cdt1 from the complex, and OCM then draws in a second MCM2-7 hexamer, and acts as a platform for the final assembly.
If hydrolysed at the wrong time, the intermediate OCM complex can fall apart, halting initiation of DNA replication. “This could open up an avenue in the future for generating inhibitors against the reaction,” hints Speck. “People have found that cancer cells are very sensitive to inhibition of this reaction, so finding its Achilles’ heel could lead the way to new treatments. Perhaps Mother Nature has shown us one of these vulnerabilities, where a small modification of ORC can lead to the complex disassembling.” As the molecular details of each step become progressively clearer, the possibilities for practical application will continue to grow.
Reference: Fernández-Cid, A., Riera, A., Tognetti, S., Herrera, M. C., Samel, S., Evrin, C., Winkler, C., Gardenal, E., Uhle, S., Speck, C., (2013). An ORC/Cdc6/MCM2-7 complex is formed in a multistep reaction to serve as a platform for MCM Double-Hexamer assembly. Molecular cell.
Image: A 3D printed model of the pre-replication complex.