title: Mechanism of Exonuclease I stimulation by the single-stranded DNA-binding protein.
abstract: Bacterial single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during cellular DNA replication, recombination and repair reactions. SSBs also form complexes with an array of genome maintenance enzymes via their conserved C-terminal tail (SSB-Ct) elements. In many cases, complex formation with SSB stimulates the biochemical activities of its protein partners. Here, we investigate the mechanism by which Escherichia coli SSB stimulates hydrolysis of ssDNA by Exonuclease I (ExoI). Steady-state kinetic experiments show that SSB stimulates ExoI activity through effects on both apparent k(cat) and K(m). SSB variant proteins with altered SSB-Ct sequences either stimulate more modestly or inhibit ExoI hydrolysis of ssDNA due to increases in the apparent Michaelis constant, highlighting a role for protein complex formation in ExoI substrate binding. Consistent with a model in which SSB stabilizes ExoI substrate binding and melts secondary structures that could impede ExoI processivity, the specific activity of a fusion protein in which ExoI is tethered to SSB is nearly equivalent to that of SSB-stimulated ExoI. Taken together, these studies delineate stimulatory roles for SSB in which protein interactions and ssDNA binding are both important for maximal activity of its protein partners.
authors.affiliation: Department of Biomolecular Chemistry, 550 Medical Science Center, 1300 University Avenue, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706-1532, USA.
authors.forename: Duo
authors.initials: D
authors.lastname: Lu
authors.forename: Angela R
authors.initials: AR
authors.lastname: Myers
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: George
authors.forename: James L
authors.initials: JL
authors.lastname: Keck
mesh_terms: D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D007700:Kinetics; D017384:Sequence Deletion; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C476430:SSB protein, E coli; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I