Biophysical Society Thematic Meeting| Lima 2019

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

Friday Speaker Abstracts

SINGLE-MOLECULE TRACKING OF DNA REPAIR FACTORY DYNAMICS IN LIVE CELLS María X Benítez-Jones 1 ; Gergely Róna 1,2 ; Yandong Yin 1,2 ; Sarah Keegan 1,3 ; Timothée Lionnet 3 ; Gaëlle Legube 4 ; David Fenyö 1,3 ; Eli Rothenberg 1,2 ; 1 New York University School of Medicine, Department of Biochemistry and Molecular Pharmacology, New York, NY, USA 2 New York University School of Medicine, Perlmutter Cancer Center, New York, NY, USA 3 New York University School of Medicine, Institute for Systems Genetics, New York, NY, USA 4 Université de Toulouse, LBCMCP, Centre de Biologie Integrative (CBI), CNRS, Toulouse, France DNA double-stranded breaks (DSBs) are regarded as the most cytotoxic DNA lesions and failure to repair DSBs can lead to genetic disorders, aging, and cancer. In mammalian cells, DSBs are repaired via two vital pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Formation of DSBs initiates an elaborate DNA Damage Response (DDR) signaling cascade, which changes the chromatin environment around DSBs and propagates global cellular signaling events. Central to DDR signaling is the recruitment of DSB-related chromatin modulators and repair factors, such as p-53 binding protein-1 (53BP1). Together, these form a distinct macromolecular assembly known as the repair foci, within which the repair process occurs. Although the proper progression and regulation of the DSB repair process is central to cellular viability, little is known about the recruitment and exclusion of DNA repair factors to the repair foci. How do these dynamics correlate with the DDR and the choice between repair pathways? To address this knowledge gap, we have developed single-molecule imaging assays that enable us to track the dynamics of individual repair proteins at DSB repair foci in living cells. Using this approach, we have monitored the recruitment and retention of key repair factors to the liquid condensate structure of 53BP1 foci for perturbed and unperturbed states inside the nucleus of a living cell. Our study revealed that 53BP1 forms biomolecular liquid condensates at DSB foci via distinctive phase separation dynamics and observed that repair factors exhibit novel modes of diffusion at the surface and within the 53BP1 condensates for unperturbed and perturbed states. We conclude that the liquid-like dynamic properties of these 53BP1 condensates are essential for the effective repair of DSBs.

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