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. Having a dedicated and relevant DNA substrate is critical to determining the relevant molecular mechanisms in a single-molecule experiment. Here, we will share our biochemical solutions for various applications on double-stranded and single-stranded DNA. We also present our newly developed biochemical tools to incorporate replication forks, DNA lesions, and specific sequences in DNA molecules tethered between two beads. 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.