Periodic Reporting for period 2 - CRYOREP (Chromosome Replication Visualised by Cryo-EM) Reporting period: 2020-09-01 to 2022-02-28 Summary of the context and overall objectives of the project Chromosome replication is essential for the propagation of life. If things go wrong in this process the physical stability of the genome is disrupted, which can lead to the development of cancer. With our project we aim to reconstitute chromosome replication reactions by mixing purified yeast proteins and DNA, and image these preparations using cryo-electron microscopy (cryo-EM). Employing established image processing procedures we can reconstruct near-atomic resolution structures of the DNA replication machinery captured as it is performing its work.Our achievements will hopefully lead to describing the molecular mechanism of DNA replication at atomic resolution. By understanding how the replication machinery works, we will be able to explain how to intervene when things go wrong in this process. Describing a the cascade of events that lead to chromosome replication is important in and of itself, as it explains the process which stands at the core of the perpetuation of life. Importantly, these studies will also provide new knowledge that can be used for the development of anti-cancer therapeutic agents. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Since the start of our project we have established the mechanism by which the replicative helicase is loaded at replication start sites. This helicase, named Minichromosome maintenance (MCM) complex, is the enzyme that separates the two strands in the DNA double helix, and provides the template for replication by dedicated "molecular typewriters" named replicative polymerases alpha, delta and epsilon. To understand helicase loading onto DNA we have have combined cryo-EM methods with time-resolved approaches, taking different snapshots of the process of helicase binding to DNA. We discovered that two helicases form a dimeric complex in a sequential, yet symmetric manner. Our findings resolve a long-standing controversy in the field. We are now studying how the DNA loaded MCM becomes activated, in a process that requires multiple phosphorylation events, and how the replication fork is established. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The work performed so far radically changed the way we use cryo-electron microscopy to understand DNA replication. Unlike what is done in traditional structural biology programmes, we are not only studying the structure of one macromolecular assembly but instead use cryo-EM to image a full reaction as it occurs in solution. We developed new computational approaches named "in silico reconstitution", which allow us to reconstruct the full context of a concerted reaction occurring at multiple sites on the same DNA molecule. This approach allows us to understand how different enzymes work together to facilitate DNA replication, which has been previously observed to progress symmetrically in opposite directions. The use of time-resolved approaches is another element of novelty in our study. With these methods we can understand how the structure of the molecular machines that duplicate our genome changes as time progresses, while the DNA is being duplicated.We now are making significant progress towards understanding how the MCM helicase is activated to start opening the double helix. These achievements will put us in an excellent position to understand how DNA is synthesised by a large macromolecular machine including the MCM helicase and the replicative polymerases. Likewise, we will now start studying how DNA, which in our cells is wrapped around structural proteins conferring physical stability to our genome, can become accessible to the helicase to allow unwinding. The end result of our study will be a molecular movie of chromosome replication.