Characterization of a single-stranded DNA-binding protein from Klebsiella pneumoniae: mutation at either Arg73 or Ser76 causes a less cooperative complex on DNA

Abstract

Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism, such as in processes like DNA replication, repair and recombination, and is essential for cell survival. Here, we characterized the ssDNA-binding properties of Klebsiella pneumoniae SSB (KpSSB) by using fluorescence-quenching measurements, electrophoretic mobility shift analysis (EMSA) and site-directed mutagenesis. Analysis of purified KpSSB by gel-filtration chromatography showed a stable tetramer in solution. In fluorescence titrations, KpSSB bound to 25-40nucleotides (nt) per tetramer depending on the salt concentration. Using EMSA, we characterized the stoichiometry of KpSSB complexed with a series of ssDNA homopolymers, and the size of the binding site was determined to be 26±1nt. Mutation at either Arg73 or Ser76 of KpSSB caused a less cooperative complex on DNA. Arg73 forms an intermolecular hydrogen bond with Ser76, and this appears to be a likely driving force that directs the self-assembly of SSB on DNA. © 2012 The Authors. Journal compilation © 2012 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.