Structure, function, and therapeutic targeting of SSB protein interactions

DNA unwinding creates single-stranded (ss) DNA intermediates that serve as templates for diverse cellular functions. Exposed ssDNA results in two specific problems for the cell; first, ssDNA is thermodynamically less stable than dsDNA, which leads to spontaneous formation of duplex secondary structures that impede genome maintenance processes. Second, relative to dsDNA, ssDNA is hypersensitive to chemical and nucleolytic attacks that can cause damage to the genome. These potential problems are solved by encoding specialized ssDNA-binding proteins (SSBs) that bind to and stabilize ssDNA structures required for essential genomic processes. The SSB protein in bacteria contains two functionally distinct regions; the N-terminal oligonucleotide/oligosaccharide binding (OB) fold is responsible for oligomerization and ssDNA binding, while the flexible amphipathic C-terminal tail (SSB-Ct) is the site of its essential interactions with many proteins involved in DNA processing and genome maintenance.;SSB is directly involved with the replication process in bacteria. In Escherichia coli, SSB's association with the chi subunit of the DNA polymerase III holoenzyme had been proposed to confer stability to the replisome and to aid delivery of primers to the lagging-strand DNA polymerase. In this work I crystallographically identified the SSB-binding site on chi. This enabled me to create a series of chi variants that destabilized the chi/SSB interface and conduct biochemical and cellular studies to delineate the role of the interaction in replication. Sequence changes in chi that block complex formation with SSB lead to salt-dependent replication defects in vitro, highlighting the roles of the chi/SSB complex in maintaining the replisome. Destabilization of the chi/SSB complex in vivo produced temperature-dependent cell cycle defects likely arising from replisome instability.;In addition to its role in DNA replication, SSB interacts with many heterologous proteins involved in nucleic acid processing and these interactions are essential to bacteria. I have shown that compounds that disrupt SSB interactions in vitro act as potent antibiotics in vivo. In addition, I have shown that essential protein/protein interactions in bacteria are a strong target for development of novel antibacterial therapeutics.