Super-resolution imaging reveals changes in Escherichia coli SSB localization in response to DNA damage.

Abstract

The E.coli single-stranded DNA-binding protein (SSB) is essential to viability. It plays key roles in DNA metabolism where it binds to nascent single strands of DNA and to target proteins known as the SSB interactome. There are >2,000 tetramers of SSB per cell with 100-150 associated with the genome at any one time, either at DNA replication forks or at sites of repair. The remaining 1,900 tetramers could constantly diffuse throughout the cytosol or be associated with the inner membrane as observed for other DNA metabolic enzymes. To visualize SSB localization and to ascertain potential spatiotemporal changes in response to DNA damage, SSB-GFP chimeras were visualized using a novel, super-resolution microscope optimized for the study of prokaryotic cells. In the absence of DNA damage, SSB localizes to a small number of foci and the excess protein is associated with the inner membrane where it binds to the major phospholipids. Within five minutes following DNA damage, the vast majority of SSB disengages from the membrane and is found almost exclusively in the cell interior. Here, it is observed in a large number of foci, in discreet structures or, in diffuse form spread over the genome, thereby enabling repair events.