E. coli Single Stranded Binding Protein (SSB) Self-Regulates Wrapping of SSDNA through Competitive Binding

Abstract

E. coli single-stranded DNA (ssDNA) binding protein (SSB) is a stable homotetramer that wraps ssDNA in multiple conformations depending on protein concentration and solution conditions. SSB plays an important role in DNA replication and repair and has been utilized in biochemical applications such as increasing PCR specificity and yield. Here, we use an optical tweezers system to measure the binding and wrapping of a single ssDNA substrate by SSB. At saturating concentrations (>1 nM), SSB binds to ssDNA in a biphasic manner, in which a rapid contraction of the ssDNA is followed by a slower partial elongation. Subsequent removal of free SSB from solution results in further contraction of the ssDNA and addition of free SSB again elongates the ssDNA. Oscillations between the two states are controlled by free protein concentration and are repeatable over many cycles. In contrast, low SSB concentrations (<1 nM) stably contract the ssDNA with increased amplitude. We show that these results are consistent with a generalized model in which the conformation of the SSB-ssDNA complex is regulated through competitive binding, where increased protein density destabilizes higher order wrapped states and promotes the dissociation of excess SSB from the substrate. Furthermore, the ability of SSB to form stable tetramers and interactions between the disordered C-terminal tails are critical to these functions. Our results are consistent with a functional model of SSB that maximizes its ability to protect transiently exposed ssDNA without inhibiting replication and repair processes.