Biophysical Society Thematic Meeting| Lima 2019

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

Poster Abstracts

38-POS Board 38 SINGLE-MOLECULE IMAGING REVEALS NOVEL INTERACTIONS IN THE ASSEMBLY KINETICS OF THE HUMAN NHEJ REPAIR MACHINERY Michael J Morten 1 ; Maria X Benitez-Jones 1 ; Michael R Lieber 2 ; Dale A Ramsden 3 ; Mauro Modesti 4 ; Jean-Baptiste Charbonnier 5 ; Eli Rothenberg 1 ; 1 NYU School of Medicine, Department of Biochemistry and Molecular Pharmacology, New York, NY, USA 2 University of Southern California Keck School of Medicine, Norris Comprehensive Cancer Center, Los Angeles, CA, USA 3 University of North Carolina, Lineberger Comprehensive Cancer Center, Chapel Hill, NC, USA 4 Aix-Marseille Université, Cancer Research Center of Marseille, Marseille, France 5 Université Paris-Sud, Institute for Integrative Biology of the Cell, Paris, France Broken chromosomes, known as double strand breaks (DSBs), are arguably the most dangerous form of DNA damage to the cell; and their aberrant repair can cause massive genomic rearrangements, accelerated tumorigenesis and cell death. Nonhomologous end joining (NHEJ) is the main pathway for the repair of DSBs in mammalian cells, a process facilitated by the assembly of a multicomponent repair machinery. It relies on the efficient detection of broken DNA ends by the heterodimer Ku and the formation of a stable multi-protein complex that is composed of several enzymes and structural proteins, which mediates the synapsis, alignment and ligation of the broken ends. Despite the crucial importance of this complex in facilitating faithful DSB repair, its initial assembly and stability remain undefined. We use single molecule co-localization and smFRET microscopy to measure the assembly of the entire NHEJ complex and associated factors on DNA ends, and determine how its stability depend on various interaction among the different repair factors and DNA substrates. In contrast to current models, our studies show that the assembly process is highly dynamic and reversible, with the core NHEJ proteins exhibiting a novel cooperativity when assembling on exposed DNA ends. Importantly, increased molecular crowding reinforces the stability of these proteins assembled on DNA. Overall, our study directly correlates the stability of the pre-synaptic complex and the efficiency of repair. Thus, our approach of monitoring the formation of individual synapses during DNA repair in real time offers novel mechanistic insights into both the successful NHEJ repair and the dysfunctional regulation of DSBs.

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