Biochemical characterization of the DNA helicase activity of the escherichia coli RecQ helicase.
We demonstrate that RecQ helicase from Escherichia coli is a catalytic helicase whose activity depends on the concentration of ATP, free magnesium ion, and single-stranded DNA-binding (SSB) protein. Helicase activity is cooperative in ATP concentration, with an apparent S(0.5) value for ATP of 200 microm and a Hill coefficient of 3.3 +/- 0.3. Therefore, RecQ helicase utilizes multiple, interacting ATP-binding sites to mediate double-stranded DNA (dsDNA) unwinding, implicating a multimer of at least three subunits as the active unwinding species. Unwinding activity is independent of dsDNA ends, indicating that RecQ helicase can unwind from both internal regions and ends of dsDNA. The K(M) for dsDNA is 0.5-0.9 microm base pairs; the k(cat) for DNA unwinding is 2.3-2.7 base pairs/s/monomer of RecQ helicase; and unexpectedly, helicase activity is optimal at a free magnesium ion concentration of 0.05 mm. Omitting Escherichia coli SSB protein lowers the rate and extent of dsDNA unwinding, suggesting that RecQ helicase associates with the single-stranded DNA (ssDNA) product. In agreement, the ssDNA-dependent ATPase activity is reduced in proportion to the SSB protein concentration; in its absence, ATPase activity saturates at six nucleotides/RecQ helicase monomer and yields a k(cat) of 24 s(-1). Thus, we conclude that SSB protein stimulates RecQ helicase-mediated unwinding by both trapping the separated ssDNA strands after unwinding and preventing the formation of non-productive enzyme-ssDNA complexes.
Harmon
F G
FG
Division of Biological Sciences, Section of Microbiology, University of California, Davis, California 95616, USA.
Kowalczykowski
S C
SC
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D006690:Bisbenzimidazole / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008954:Models, Biological; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D053484:RecQ Helicases; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; C506724:RecQ protein, E coli; D004265:DNA Helicases; D053484:RecQ Helicases; D008274:Magnesium; D006690:Bisbenzimidazole