The process of copying DNA, or DNA replication, is fundamental to all domains of life. DNA replication is carried out by a multi-protein complex referred to as the replisome. Errors occurring during DNA-replication can lead to genomic instability and promote diseases such as cancer. Errors can occur due to the presence of roadblocks on DNA like UV-induced lesions or DNA binding proteins. Both of these types of roadblocks are universally present throughout the genome. While DNA-repair pathways in cells have been studied extensively, the initial response mechanisms of the replisome to a roadblock remain poorly understood.
To shed light on the mechanistic details of how the replisome deals with roadblocks we use single-molecule fluorescence microscopy. We use microfluidic devices to stretch out single DNA molecules on functionalised cover-slips. To this DNA template we can introduce the two types of roadblocks. First, single UV-lesions can be inserted into the template DNA at well-defined positions. Second, using the crispr-cas9 system, a catalytically inactive mutant of the cas9 protein (dcas9) can be used to create a programmable protein roadblock. DNA replication by a fully reconstituted Saccharomyces cerevisiae replisome is then visualised on these DNA-substrates. With multiple fluorescent markers, we are able to simultaneously visualise the newly synthesised DNA and single CMG helicases, in real time. Tracking with sub-pixel accuracy allows us to measure the localisation of single components of the replisome with respect to each other and the roadblocks. Furthermore, we can quantify rates of CMG translocation and DNA synthesis. Using this assay we will elucidate the dynamic behaviour and function of the CMG helicase upon encountering roadblocks.