RecQ DNA helicase of Escherichia coli. Characterization of the helix-unwinding activity with emphasis on the effect of single-stranded DNA-binding protein.
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.
Umezu
K
K
Department of Microbiology, Faculty of Dentistry, Kyushu University, Fukuoka, Japan.
Nakayama
H
H
D000251:Adenosine Triphosphatases / Q000145:classification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D004265:DNA Helicases / Q000145:classification / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D053484:RecQ Helicases; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000378:metabolism
D004269:DNA, Bacterial; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C506724:RecQ protein, E coli; D004265:DNA Helicases; D053484:RecQ Helicases