In situ structure of the replication machinery imaged inside a vertebrate nucleus

About the project

About the Project A 2027 Crick PhD project with Alessandro Costa. Project background and description DNA must be replicated only once per cell cycle to maintain chromosome integrity and prevent the onset of cancer. Eukaryotic cells have evolved to achieve this by temporally separating the loading of the replicative helicase from its activation. Over the last decade, biochemical reconstitution combined with cryo-electron microscopy has led to an exhaustive description of how the assembly and activation of the eukaryotic replication machinery is achieved. When it comes to metazoan DNA replication, however, we remain in the dark. Although orthologs of all yeast replication initiation proteins have been identified in higher eukaryotes, emerging evidence indicates that metazoans possess essential replication factors that yeast lost during evolution. Biochemical isolation of endogenous replication complexes from human cells and from frog egg extracts revealed the identity of essential initiation factors and started to explain the structural mechanisms that start replication. However, these protein complexes are fragile and tend to come apart as they are being purified. New approaches are required to study the intact replication machinery to understand how it enables activation. Our lab has recently devised methods to visualise the replicative helicase bound to chromatin inside replicating nuclei. This is achieved through a combination of correlative microscopy, cryo-FIB milling and cryo-tomography. Using methods to synchronise replicating nuclei, the PhD student will study the assembly and activation of the replication machinery. The aim is to identify all components required and define the order of events that lead to DNA replication initiation. As part of the project, the PhD student will learn to express and purify eukaryotic proteins, label them with fluorescent probes and reconstitute replicating nuclei using the Xenopus egg extract system. The student will also be trained in fluorescence microscopy, cryo-electron microscopy and cryo-electron tomography. The project will include a strong computational component, including reconstruction of cryo-tomograms, subtomogram averaging and atomic modelling. The structures obtained will be validated using site-directed mutagenesis combined with fluorescence-based DNA duplication assays conducted in replicating nuclei. Candidate background This project is suitable for a student who has a keen interest in molecular mechanisms of fundamental cellular processes. Experience in molecular biology and biochemistry is required. Knowledge of cell biology, cryo-electron microscopy and/or coding is not necessary but will be an advantage. Lab-specific question Describe a moment when learning the molecular mechanism behind a biological process transformed it from an abstract textbook fact into something you could picture happening. What was the mechanism, and why did understanding it change how you saw the bigger process?

Research areas