Single molecule manipulation and imaging of complex DNA-protein interactions.

Abstract

The genetic information of an organism is encoded in the base pair sequence of its DNA. Many specialized proteins are involved in organizing, preserving and processing the vast amounts of information on the DNA. In order to do this swiftly and correctly these proteins have to move quickly and accurately along and/or around the DNA constantly rearranging it. In this presentation I will show (Super-resolution) Correlative Tweezers-Fluorescence Microscopy (CTFM), a single-molecule approach capable of visualizing individual DNA-binding proteins on densely covered DNA and in presence of high protein concentrations. Moreover, proteins on DNA can be visualized on multiple DNA strand. Next, using this instrument we have investigated human non-homologous end joining (NHEJ). NHEJ is the primary pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells. Here we show that the XRCC4-XLF complexes robustly bridge two independent DNA molecules and that these bridges are able to slide along the DNA. These observations suggest that XRCC4-XLF complexes form mobile sleeve-like structures around DNA that can reconnect the broken ends very rapidly and hold them together. I will also show how we can use this instrument for the study of mitotic chromosomes. These structures are highly dynamic throughout the cell cycle, and undergo compaction during mitosis to adopt the characteristic “X-shape”. Here I introduce a workflow to interrogate the organization of human chromosomes based on optical trapping and manipulation. This allows high-resolution force measurements and fluorescence visualization of native metaphase chromosomes to be conducted under tightly controlled experimental conditions. The methods described here open the door to a wide array of investigations into the structure and dynamics of both normal and disease-associated chromosomes.