Multiprotein E. coli SSB–ssDNA complex shows both stable binding and rapid dissociation due to interprotein interactions

Author:

Naufer M Nabuan1,Morse Michael1,Möller Guðfríður Björg1,McIsaac James2,Rouzina Ioulia3,Beuning Penny J2,Williams Mark C1ORCID

Affiliation:

1. Department of Physics, Northeastern University, Boston, MA 02115, USA

2. Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA

3. Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH 43210, USA

Abstract

Abstract Escherichia coli SSB (EcSSB) is a model single-stranded DNA (ssDNA) binding protein critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations to facilitate DNA replication and repair. Here we measure the binding and wrapping of many EcSSB proteins to a single long ssDNA substrate held at fixed tensions. We show EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as ssDNA-bound protein density passes critical saturation and high free protein concentration increases the fraction of EcSSBs in less-wrapped conformations. By destabilizing EcSSB wrapping through increased substrate tension, decreased substrate length, and protein mutation, we also directly observe an unstable bound but unwrapped state in which ∼8 nucleotides of ssDNA are bound by a single domain, which could act as a transition state through which rapid reorganization of the EcSSB–ssDNA complex occurs. When ssDNA is over-saturated, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped complexes. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers are rapidly removed from ssDNA to allow for DNA maintenance and replication functions, while still fully protecting ssDNA over a wide range of protein concentrations.

Funder

National Science Foundation

Publisher

Oxford University Press (OUP)

Subject

Genetics

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