Journal list menu

Volume 221, Issue 1 p. 435-443
Free Access

Single-stranded-DNA-binding proteins from human mitochondria and Escherichia coli have analogous physicochemical properties

Ute CURTH

Ute CURTH

Medizinische Hoschule, Zentrum Biochemie, Institut für Biophysikalische Chemie, Hannover, Germany

Search for more papers by this author
Claus URBANKE

Corresponding Author

Claus URBANKE

Medizinische Hoschule, Zentrum Biochemie, Institut für Biophysikalische Chemie, Hannover, Germany

Correspondence to C. Urbanke, Medizinische Hochschule, Zentrum Biochemie, Institut für Biophysikalische Chemie, D-30623 Hannover, Germany
Fax: +49 511 532 5966.Search for more papers by this author
Joachim GREIPEL

Joachim GREIPEL

Medizinische Hoschule, Zentrum Biochemie, Institut für Biophysikalische Chemie, Hannover, Germany

Search for more papers by this author
Holger GERBERDING

Holger GERBERDING

Universität Göttingen, Institut für Mikrobiologie, Göttingen, Germany

Search for more papers by this author
Valeria TIRANTI

Valeria TIRANTI

Istituto Nazionale Neurologico ‘Carlo Besta’, Milano, Italy

Search for more papers by this author
Massimo ZEVIANI

Massimo ZEVIANI

Istituto Nazionale Neurologico ‘Carlo Besta’, Milano, Italy

Search for more papers by this author
First published: April 1994
Citations: 82

Note. This article is dedicated to Prof. Dr Günter Maass on the occasion of his 60th birthday.

Abstract

The gene for the mature human mitochondrial single-stranded-DNA binding protein (HsmtSSB) has been transferred into a protein-overproducing vector and expressed in Escherichia coli. The protein was purified to homogeneity and its physicochemical properties were investigated. From sequence comparison, HsmtSSB shows some similarities to the N-terminal part of the single-stranded DNA-binding protein (SSB) from E. coli (EcoSSB). Hydrodynamic measurements show the protein to be tetrameric and give a sedimentation coefficient of 4.1 S corresponding to a C-terminally shortened EcoSSB. Electron-microscopic images of the free protein show a globular tetrahedral structure. Binding of poly(desoxythymidylic acid) [poly(dT)] leads to a reduction of the tryptophan fluorescence of the protein up to 96%. Fluorescence titrations with poly(dT) show apparent binding-site sizes of 50–70 nucleotides/tetramer between 0.05 M and 2 M NaCl. Binding to poly(dT) proceeds in a nearly diffusion-controlled reaction with an association-rate constant kass of 4 · 108 M−1S−1. The rate-limiting step is the formation of a transient complex where less than four binding sites on the protein are involved and the reshuffling of the protein of the protein on the linear matrix is fast. Electron microscopy of the complex with poly(dT) using negative staining shows a nearly random distribution of the protein between the individual poly(dT) strands. This leads to the conclusion that the binding cooperativity is low (ω < 150). The two tryptophans of HsmtSSB were replaced by threonine and tyrosine. The environment of both residues is influenced by nucleic acid binding with mutations of Trp68 strongly reducing the DNA-binding affinity of the protein.

Abbreviations

  • SSB
  • Single-stranded-DNA-binding protein
  • HsmtSSB
  • mitochondrial SSB from Homo sapiens
  • EcoSSB
  • SSB from Escherichia coli