Revealing the mechanism of replication initialization with a sequential microscopy-based approach

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Before cell division, each mitotic cell has to create an exact duplicate of its DNA to ensure genome stability and the faithful transmission of the full genetic information. Incomplete or excessive DNA replication could otherwise severely diminish the viability of the daughter cells. In eukaryotes, DNA replication takes place at specific nuclear locations during distinct time intervals. This regulation is linked to the chromatin conformation, but the exact mechanisms are yet poorly understood.Replication of eukaryotic genomes is initiated at thousands of genomic segments in parallel, so-called origins of replication. In a single cell, only a subset of these origins is used while all origins can initiate replication when looking at a population of cells. As a consequence, a large cell-to-cell variability in the origin usage exists and a study of the mechanisms controlling replication initiation would considerably profit from the use of single-cell techniques. Interestingly, despite this flexibility, replication domains emerge as 0.5-1 Mb regions of DNA duplicated at the same time and spanning several active origins. This synchronicity implies mechanisms to simultaneously activate several origins. The prevalent model predicts that the underlying chromatin conformation brings origins into spatial proximity which results in their synchronous activation. However, directly testing this model so far was not possible due to technological limitations.Here, I propose to use a super-resolution microscope (in particular 3D-STED) together with Hi‑M, a method recently developed by the host lab that permits the sequence-specific, sequential labeling and imaging of all potential origins in a replication domain (~20 in a 150-500 nm sized cluster). This unique approach will allow us to determine the position of the origins with nanometric resolution and to correlate their distribution with the overall chromatin conformation in a replication domain in mouse cells. The proposed project will permit a direct observation of the spatial organization of all potential origins in a replication domain, while origin activation takes place. Gaining a deeper insight into the role of chromatin conformation on origin organization will help to better understand the regulation of genome replication, a vital process for life.