Cryo-Electron Microscopy Analysis of the Replicative Helicase

• Applicant: Dr. Sarah Schneider
• Subject Area: Structural Biology ; Biochemistry ; Cell Biology
• Term: from 2018 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 394139458

During eukaryotic DNA replication, the replicative helicase MCM2-7 becomes loaded onto double-stranded DNA (dsDNA) to form a pre-replicative complex (pre-RC), which serves as the platform for replication fork assembly. Efficient and regulated helicase loading is essential for DNA replication, but has also high relevance for genome stability, stem cell homeostasis, aging and tumorigenesis. Structural changes from a spiral shaped MCM2-7 complex towards a perfectly shaped ring with DNA inserted represent the critical steps during the helicase loading, as this process physically links the enzyme to DNA. However, the underlying mechanisms of this reaction are completely unknown. My overall aim is to use single-particle cryo-electron microscopy (cryo-EM) to fully elucidate how these structural changes and the DNA insertion reaction works at an atomic level. I will take advantage of unique expertise of the group, which developed highly efficient MCM2-7 in vitro assembly protocols and has a long track record of working at the interface of biochemistry and electron microscopy. Therefore, I can rely on an already obtained a 3.9 Å structure of the related helicase loading complex to obtain the elusive structure of the helicase loading complex arrested before DNA insertion. Moreover, I will investigate the loading complex containing an essential factor of helicase loading, Cdt1, which has been modified taking advantage of the knowledge from the recently solved helicase loading complex. Solving these structures will provide extensive mechanistic insight into the structural changes the eukaryotic helicase undergoes during the critical stages of helicase loading. Importantly, this work will serve as a paradigm for related processes involving regulated MCM2-7 ring opening during DNA synthesis, DNA repair or potentially also DNA recombination, generating a wide interest. In the long-term I would like to translate this knowledge to the human system, as this will allow me to identify unique regulatory principles of human helicase loading regulation, which are very important for genomic stability. Furthermore, the structural knowledge could be important for the development of small molecule DNA replication inhibitors.

• DFG Programme: Research Fellowships
• International Connection: United Kingdom
• Host: Professor Dr. Christian Speck