Abstract
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.
Highlights
DNA helicases are the motor proteins responsible for separating the individual strands of dsDNA1 to provide single-stranded DNA (ssDNA) for key cellular processes such as DNA repair, recombination, and replication
RecQ helicase does not display a preference for initiating unwinding from double-stranded DNA (dsDNA) ends compared with internal regions of dsDNA: the kinetic parameters for DNA unwinding are similar when the helicase was provided with three separate DNA substrates that differed in the ratio of the total number of base pairs to dsDNA ends
The standard Hoechst 33258 (H33258) dye concentration of 300 nM was empirically determined by analyzing the effect of dye concentration on the unwinding activity of RecQ helicase; this concentration of H33258 dye was chosen because the unwinding activity of RecQ helicase, as measured by DNA strand separation in agarose gel electrophoresis assays, was unaffected by the presence of the dye, and this amount of dye was sufficient to determine accurately the initial rate of unwinding [32]
Summary
Reagents—Chemicals were reagent grade, and all solutions were prepared using Barnstead NanoPure water. Standard conditions were reactions (300 l total volume) having 1 mM magnesium-acetate, 1 mM ATP, 1 M SSB protein, 2.5 M (bp) linear dsDNA, and 100 nM RecQ helicase. The total change in fluorescent signal corresponding to 100% unwinding (⌬FT) was determined for each assay by subtracting the value for the free H33258 dye from the fluorescence obtained for the dye-dsDNA complex This is possible since the fluorescence of the H33258 dye is not altered by ssDNA [32]. The standard reaction buffer consisted of 25 mM Tris acetate (pH 7.5), 1 mM magnesium acetate, 0.1 mM DTT, 1 mM P-enolpyruvate, 25 units mlϪ1 pyruvate kinase, 25 units mlϪ1 lactate dehydrogenase, 150 M NADH, and 1 mM ATP. Reactions had the indicated concentration of RecQ helicase, and 1.0 M (nt) heat-denatured HindIII-cut pUC19 linear DNA. Except for SSB protein and RecQ helicase, were incubated together at 37 °C for 2 min prior to initiation of the reaction by the addition of a mixture of SSB protein and RecQ helicase
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