Journal
NUCLEIC ACIDS RESEARCH
Volume 49, Issue 3, Pages 1532-1549Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkaa1267
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Funding
- National Science Foundation [MCB-1817712, MCB-1615946]
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The study found that EcSSB binds to ssDNA in a biphasic manner, leading to incomplete wrapping at high concentrations. Destabilizing EcSSB wrapping through increased substrate tension, decreased substrate length, and protein mutation can disrupt the wrapping. When ssDNA is oversaturated, stimulated dissociation can rapidly remove excess EcSSB.
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 similar to 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.
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