Separation of the two single strands leads to the build-up of superhelical tension, which, if left unresolved, exerts extreme forces in the form of single duderstadt that disrupts critical enzymatic events on chromosomes. In this project, recently discovered topology sensing proteins will be fluorescently labeled and used in single-molecule imaging experiments to directly visualize DNA topology dynamics.
These tools will then be used to characterize how topological energy migrates around the replication fork and where overwinding accumulates leading to replication fork collapse.
In this project protein biochemistry techniques will be used to characterise the DNA binding properties of the labeled topology sensors. DNA substrates will be generated with defined topologies suitable for imaging.
Single immobilized DNA molecules together with labeled fluorescent sensors will be imaged using multiwavelength TIRF microscopy. Dynamics will be quantified using custom written Fiji plugins and python notebooks. During each stage of the project supervision and needed expertise will be provided.