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bioRxiv · 10.1101/2019.12.20.885368

Self-regulation of single-stranded DNA wrapping dynamics by E. coli SSB promotes both stable binding and rapid dissociation

Naufer, M. N.·Morse, M.·Möller, G. B.·McIsaac, J.·Rouzina, I.·Beuning, P. J.·Williams, M.
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Abstract

E. coli SSB (EcSSB) is a model protein for studying functions of single-stranded DNA (ssDNA) binding proteins (SSBs), which are critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations in order to protect these transiently formed regions during processes such as replication and repair. Using optical tweezers, we measure the binding and wrapping of a single long ssDNA substrate under various conditions and free protein concentrations. We show that EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as protein density on the substrate passes a critical saturation. Increasing free EcSSB concentrations increase the fraction of EcSSBs in less-wrapped conformations, including a previously uncharacterized EcSSB8 bound state in which [~]8 nucleotides of ssDNA are bound by a single domain of the tetramer with minimal substrate deformation. When the ssDNA is over-saturated with EcSSB, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped EcSSB-ssDNA complexes. We develop a multi-step kinetic model in which EcSSB tetramers transition through multiple wrapped conformations which are regulated through nearest neighbor interactions and ssDNA occupancy. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers can be rapidly removed from an ssDNA substrate to allow for DNA maintenance and replication functions while still fully protecting ssDNA over a wide range of protein concentrations.

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Naufer, M. N., Morse, M., Möller, G. B., McIsaac, J., Rouzina, I., Beuning, P. J., Williams, M.. 2019-12-21. Self-regulation of single-stranded DNA wrapping dynamics by E. coli SSB promotes both stable binding and rapid dissociation. https://doi.org/10.1101/2019.12.20.885368

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