A biochemistry platform to incorporate replication forks, DNA lesions, and nucleosome arrays to single‐molecule experiments

Abstract

Fundamental biological processes such as DNA replication, DNA repair, and chromatin organization are precisely regulated in space and time by sophisticated multiprotein molecular machinery. The high spatiotemporal resolution of optical tweezers combined with single molecule fluorescence microscopy enables real‐time visualization of these processes while simultaneously measuring their enzymatic activity via detection of mechanical manipulation of the DNA substrates. Our biochemistry platform provides a set of tools to incorporate replication forks, DNA lesions, and specific sequences (e.g. nucleosome arrays) in DNA molecules tethered between two beads of the optical tweezers. Importantly, our tools also allow labeling of exact positions within these DNA substrates with fluorophores. This enables precise localization of the imaged proteins in the DNA sequence during processes including DNA scanning, as well as recognition and enzymatic manipulation of specific DNA structures or lesions. Our next developments aim to combine multiple DNA features in a single DNA molecule, such as a replication fork in the presence of DNA lesions or in the context of a nucleosome array. These advances will allow more complex experimental set ups and increase the capability to mimic cellular biological processes at a single molecule level.