Assembly, function and molecular architecture of the eukaryotic replicative helicase.

Perhaps the most fundamental property of all living things is the ability to replicate their DNA, which allows cells to pass on their genetic material from parent to progeny. Five years ago, the first paper from my lab was published, describing the reconstitution of eukaryotic replicative helicase loading and the identification of the MCM2-7 double-hexamer as a novel eukaryotic DNA replication complex. This reconstitution had been a major aim for the DNA replication community over a timeframe of >15 years. To succeed with this within 3 years of establishing my group was a major success. This led to a series of discoveries (7 publications): the identification of a multi-step MCM2-7 double-hexamer assembly pathway and its regulatory circuits, the first cryo-EM structures of a eukaryotic helicase loading intermediate, and the structural and functional characterisation of the MCM2-7 double-hexamer. We are now in a strong position to take this research to a completely new level by addressin g two of the most fundamental questions in eukaryotic DNA replication: how the replicative helicase becomes loaded onto DNA and activated for DNA synthesis. It is by now clear that the MCM2-7 helicase represents a major hub of the eukaryotic DNA replication fork and that MCM2-7 loading onto DNA, interaction with DNA and unloading from DNA is central to its function in replication. Indeed, the MCM2-7 complex is known to form a hexameric ring structure that must be opened for DNA insertion and for removal from DNA, but equally MCM2-7 ring-opening is of major importance during helicase activation and DNA repair. Indeed, I believe that there is a significant gap in our understanding of eukaryotic DNA replication, as we have no knowledge about the way the MCM2-7 ring-opens or closes for regulated DNA replication. Last year, I published the identification of the MCM2-7 DNA-entry gate, which is made up by the Mcm2 and Mcm5 subunits, and in this application I provide preliminary da ta that describe the crucial MCM2-7 ring-opening mechanism. Therefore, the central question of my proposal is: What is the nature of the MCM2-7 ring-opening during DNA replication? Discovering the mechanism of replicative helicase ring-opening will allow me to open up a completely new research field covering helicase-loading, helicase-activation and regulated DNA unwinding at the replication fork during normal DNA synthesis, DNA repair or homologous recombination. My aims are: (A) How is the MCM2-7 ring-opened during helicase loading? (A.1) Identification of the MCM2-7 ring-opening mechanism. (A.2) Are the first and second MCM2-7 hexamers loaded by the same mechanism? (B) How is the MCM2-7 double-hexamer transformed into the replication fork? (B.1) How does MCM2-7 ring-opening and closing regulate its function? (B.2) How is the MCM2-7 helicase activated? DNA replication is a fundamental process and the questions I am proposing are deliberately ambitious. T o answer them I will use Saccharomyces cerevisiae, a genetically traceable organism with well understood DNA replication origins, and I will employ chemical-biology tools and in vitro reconstitution approaches that are ideally suited to identify the function of replication factors (as shown by Nobel Laureate Arthur Kornberg in bacteria). Crucially, we will takeadvantage of the recent advances in elctron microscopy to obtain the first high resolution EM structures of large DNA replicaiton complexes, which will provide unprecedented insights into the function of these molecular machines.