RecQ DNA helicase of Escherichia coli. Characterization of the helix-unwinding activity with emphasis on the effect of single-stranded DNA-binding protein

J Mol Biol. 1993 Apr 20;230(4):1145-50. doi: 10.1006/jmbi.1993.1231.

Abstract

RecQ protein of Escherichia coli is a DNA helicase implicated in the RecF pathway of genetic recombination. To gain insight into the mode of its action, the effect of single-stranded DNA-binding proteins (SSBs) on the RecQ-mediated unwinding reaction was investigated. When the unwinding of M13-based, circular partially duplex substrates was measured as a function of the enzyme dose, a markedly sigmoidal relation was revealed, with relatively large amounts of the enzyme being necessary for substantial unwinding to occur. For instance, unwinding 50% of a 71 base-pair (bp) partial duplex substrate in ten minutes required an enzyme-to-substrate molar ratio of about 60. However, these features, indicating the enzyme's "inefficiency", were reversed by SSBs: in the presence of a saturating amount of E. coli SSB the sigmoidal relation was converted to a typically hyperbolic one, and the enzyme-to-substrate molar ratio at 50% unwinding of the 71 bp substrate was reduced to as low as 0.5. Phage T4 gene 32 protein also showed similar stimulatory activity. Further, the single-stranded DNA-dependent ATPase activity of RecQ was found to be relatively insensitive to E. coli SSB; its large excess brought about only a 60% inhibition. It is postulated that RecQ helicase is highly adapted to an SSB-rich environment, where the strand exchange reaction mediated by RecA protein, perhaps coupled closely with the RecQ reaction, should also take place.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / classification
  • Adenosine Triphosphatases / metabolism*
  • Adenosine Triphosphate / metabolism
  • DNA Helicases / classification
  • DNA Helicases / metabolism*
  • DNA, Bacterial / metabolism*
  • DNA, Circular / metabolism
  • DNA, Single-Stranded / metabolism
  • DNA-Binding Proteins / metabolism*
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • RecQ Helicases
  • Recombination, Genetic
  • Viral Proteins / metabolism

Substances

  • DNA, Bacterial
  • DNA, Circular
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Viral Proteins
  • gp32 protein, Enterobacteria phage T4
  • Adenosine Triphosphate
  • Adenosine Triphosphatases
  • RecQ protein, E coli
  • DNA Helicases
  • RecQ Helicases