title: Is a fully wrapped SSB-DNA complex essential for Escherichia coli survival?
abstract: Escherichia coli single-stranded DNA binding protein (SSB) is an essential homotetramer that binds ssDNA and recruits multiple proteins to their sites of action during genomic maintenance. Each SSB subunit contains an N-terminal globular oligonucleotide/oligosaccharide binding fold (OB-fold) and an intrinsically disordered C-terminal domain. SSB binds ssDNA in multiple modes in vitro, including the fully wrapped (SSB)65 and (SSB)56 modes, in which ssDNA contacts all four OB-folds, and the highly cooperative (SSB)35 mode, in which ssDNA contacts an average of only two OB-folds. These modes can both be populated under physiological conditions. While these different modes might be used for different functions, this has been difficult to assess. Here we used a dimeric SSB construct with two covalently linked OB-folds to disable ssDNA binding in two of the four OB-folds thus preventing formation of fully wrapped DNA complexes in vitro, although they retain a wild-type-like, salt-dependent shift in cooperative binding to ssDNA. These variants complement wild-type SSB in vivo indicating that a fully wrapped mode is not essential for function. These results do not preclude a normal function for a fully wrapped mode, but do indicate that E. coli tolerates some flexibility with regards to its SSB binding modes.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, 660 S. Euclid Avenue, Box 8231, 63110-1093, USA.
authors.forename: Vincent M
authors.initials: VM
authors.lastname: Waldman
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, 660 S. Euclid Avenue, Box 8231, 63110-1093, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Weiland
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, 660 S. Euclid Avenue, Box 8231, 63110-1093, USA.
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Kozlov
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, 660 S. Euclid Avenue, Box 8231, 63110-1093, USA lohman@biochem.wustl.edu.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Lohman
mesh_terms: D001665:Binding Sites; D004249:DNA Damage; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000502:physiology*; D050296:Microbial Viability; D011485:Protein Binding
chemical_list: D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C476430:SSB protein, E coli