Binding and wrapping of E. coli single stranded binding protein (SSB) C-terminal tail variants measured using force spectroscopy.

Abstract

E. coli single-stranded DNA (ssDNA) binding protein (SSB) binds and wraps ssDNA in multiple conformations, plays an important role in DNA replication and repair, and has been utilized in biochemical applications such as increasing PCR specificity and yield. Structurally, SSB consists of an oligonucleotide binding domain (OB) that forms stable tetramers, and an unstructured C-terminal tail with an acidic tip. We use optical tweezers to measure the structure of a long ssDNA saturated with SSB. While low SSB concentrations stably contract the ssDNA with large amplitude, higher concentrations force bound SSB into a less wrapped conformation, decreasing binding site size and extending the substrate. This is consistent with the conformation of the SSB-ssDNA complex regulated through competitive binding, wherein increased protein density destabilizes higher order wrapped states and promotes the dissociation of excess SSB from the substrate. To determine the role of the C-terminal domain in this regulatory behavior, we measure the binding and wrapping of two C-terminal tail truncations, lacking either the 8-residue acidic tip (SSB-∆C8) or the entire C-terminal tail (SSB-OB). We find that removal of the acidic tip increases the binding affinity, which further increases with the removal of the whole C-terminal tail. Removal of the entire C-terminal tail results in increased biphasic binding as well as a greatly reduced direct dissociation rate. These results suggest that the C-terminal tails do not facilitate the SSB-SSB interactions that are responsible for cooperativity, but instead moderate the binding to and wrapping of ssDNA by competing for OB binding sites.