Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Sunday Speaker Abstracts

SINGLE MOLECULE MANIPULATION AND IMAGING OF COMPLEX DNA- PROTEIN TRANSACTIONS Gijs Wuite 1 ; 1 vrije universiteit amsterdam, physics, amsterdam, The Netherlands 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 order to elucidate these kind of processes we perform single-molecule experiments on model systems such as restriction enzymes, DNA polymerases and repair proteins. In this presentation I will show (Super-resolution) Quadruple Trap 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. 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. Such breaks are formed, for example, during gene-segment rearrangements in the adaptive immune system or by cancer therapeutic agents. Although the core components of the NHEJ machinery are known, it has remained difficult to assess the specific roles of these components and the dynamics of bringing and holding the fragments of broken DNA together. I will present data using dual and quadruple-trap optical tweezers combined with fluorescence microscopy, on how human XRCC4, XLF and XRCC4– XLF complexes interact with DNA in real time. We find that 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. (Brouwer et al., Nature, doi:10.1038/nature18643 , 2016)

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