Single-molecule analysis reveals two separate DNA-binding domains in the Escherichia coli UvrA dimer

Nucleic Acids Res. 2009 Apr;37(6):1962-72. doi: 10.1093/nar/gkp071. Epub 2009 Feb 10.

Abstract

The UvrA protein is the initial damage-recognizing factor in bacterial nucleotide excision repair. Each monomer of the UvrA dimer contains two ATPase sites. Using single-molecule analysis we show that dimerization of UvrA in the presence of ATP is significantly higher than with ADP or nonhydrolyzable ATPgammaS, suggesting that the active UvrA dimer contains a mixture of ADP and ATP. We also show that the UvrA dimer has a high preference of binding the end of a linear DNA fragment, independent on the presence or type of cofactor. Apparently ATP binding or hydrolysis is not needed to discriminate between DNA ends and internal sites. A significant number of complexes could be detected where one UvrA dimer bridges two DNA ends implying the presence of two separate DNA-binding domains, most likely present in each monomer. On DNA containing a site-specific lesion the damage-specific binding is much higher than DNA-end binding, but only in the absence of cofactor or with ATP. With ATPgammaS no discrimination between a DNA end and a DNA damage could be observed. We present a model where damage recognition of UvrA depends on the ability of both UvrA monomers to interact with the DNA flanking the lesion.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Diphosphate / analysis
  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / metabolism
  • Adenosine Triphosphatases / ultrastructure*
  • Adenosine Triphosphate / analysis
  • Adenosine Triphosphate / metabolism
  • DNA / metabolism
  • DNA Damage*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism
  • DNA-Binding Proteins / ultrastructure*
  • Dimerization
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / metabolism
  • Escherichia coli Proteins / ultrastructure*
  • Microscopy, Atomic Force
  • Protein Multimerization
  • Protein Structure, Tertiary

Substances

  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • Adenosine Diphosphate
  • Adenosine Triphosphate
  • DNA
  • UvrA protein, E coli
  • Adenosine Triphosphatases