Domain organization of M. smegmatis RqlH. The 691-amino acid RqlH polypeptide is depicted in the top panel as a linear array (with the N-terminus at left and the C-terminus at right) and the known or imputed domains drawn as cylinders spanning their segments of the primary structure. RqlH is composed of an N-terminal ATPase domain (beige) flanked by a downstream tetracysteine zinc-binding domain (green) and a C-terminal ComF domain (magenta) that includes motifs found in amidophosphoribosyltransferase enzymes. The N-terminal 505-amino acid segment of RqlH that includes the ATPase and zinc-binding domains and suffices for helicase activity is indicated below the domain cartoon. The amino acid sequences of the RqlH ATPase and zinc-binding domains are aligned to the homologous domains of E. coli RecQ in the middle and bottom panels, respectively. Positions of side chain identity/similarity are indicated by dots above the sequences. Gaps in the alignments are denoted by dashes. The superfamily II ATPase motifs I, II, III and VI are highlighted in beige in the middle panel; the Lys49 and Asp148 residues that were mutated to alanine are denoted by |. The four putative zinc-binding cysteines are highlighted in green in the bottom panel.

Glycerol gradient sedimentation of RqlH-(1–505). (A) RqlH-(1–505) was sedimented through a 15–30% glycerol gradient as described under Experimental Procedures. Aliquots (20 μl) of the odd numbered fractions (fraction 1 being the bottom of the gradient) were analyzed by SDS–PAGE. The Coomassie-stained gel is shown; the positions and sizes (kDa) of marker polypeptides are indicated on the left. (B) ATPase reaction mixtures containing 20 mM Tris–HCl, pH 8.0, 1 mM DTT, 5 mM MgCl₂, 10 nmol [α³²P]ATP, 2 μg sonicated salmon sperm DNA and 1 μl of the indicated glycerol gradient fractions were incubated at 37°C for 30 min. The activity profile is plotted. The peak positions of the internal standards catalase, BSA and cytochrome C analyzed in a parallel glycerol gradient are indicated by vertical arrows.

Nucleotide substrate specificity and kinetic parameters. (A) Reaction mixtures (10 μl) containing 20 mM Tris–HCl, pH 8.0, 1 mM DTT, 5 mM MgCl₂, 2 μg sonicated salmon sperm DNA, 0.5 pmol (50 nM) RqlH-(1–505) and 1 mM of the indicated NTP/dNTP were incubated at 37°C for 30 min. The reactions were quenched with 990 μl of malachite green reagent (Biomol Research Laboratories). Phosphate release was quantified by measuring A₆₂₀ and interpolating the value to a phosphate standard curve. The values were corrected for the low levels of phosphate measured in control reaction mixtures containing 1 mM of the indicated NTP/dNTP but no added enzyme. Each datum is the average of three separate experiments ±SEM. (B) Reaction mixtures (60 μl) containing 20 mM Tris–HCl, pH 8.0, 1 mM DTT, 5 mM MgCl₂, 1.25 μM 44-mer oligonucleotide (shown in Figure 4B), 200 nM RqlH-(1–505) and either 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 or 4.0 mM [α³²P]ATP were incubated at 37°C. Aliquots (5 μl) were withdrawn at 1, 2, 3 and 5 min and quenched immediately with formic acid. The extents of ATP hydrolysis were plotted as a function of time for each ATP concentration and the initial rates were derived by linear regression analysis in Prism. The initial rates (pmol/s) were divided by the molar amount of input enzyme to obtain a turnover number v (per second), which is plotted in the figure as a function of ATP concentration. Each datum is the average of three separate time course experiments ± SEM. A non-linear regression curve fit of the data to the Michaelis-Menten equation (in Prism) is shown. The K_m and k_cat values are indicated.

RqlH-(1–505) unwinds a 3′-tailed DNA duplex. Helicase reaction mixtures (10 μl) contained 1 pmol of 3′-tailed duplex substrate (depicted at bottom, with the 5′ ³²P-labeled denoted by filled circle) and the indicated amounts of the RqlH-(1–505), RqlH-(1–505)-K49A, or RqlH-(1–505)-D148A proteins. The reaction products were analyzed by native PAGE and visualized by autoradiography.

RqlH and RqlH-(1–505) are DNA-dependent ATPases. (A) Purification. Aliquots (2.5 μg) of full-length wild-type RqlH and full-length mutants RqlH-K49A and RqlH-D148A were analyzed by SDS–PAGE in parallel with aliquots (2.5 µg) of the truncated proteins RqlH-(1–505), RqlH-(1–505)-K49A and RqlH-(1–505)-D148A. The Coomassie blue-stained gel is shown. The positions and sizes (kDa) of marker are indicated on the left. (B) ATPase reaction mixtures containing 20 mM Tris–HCl, pH 8.0, 1 mM DTT, 5 mM MgCl₂, 1 mM (10 nmol) [α³²P]ATP, 2 μg sonicated salmon sperm DNA and RqlH or RqlH-(1–505) proteins as specified were incubated for 30 min. Reaction products were analyzed by PEI-cellulose TLC. Each datum is the average of three protein titration experiments ± SEM.

DNA dependence of ATP hydrolysis. (A) ATPase reaction mixtures containing 20 mM Tris–HCl (pH 8.0), 1 mM DTT, 5 mM MgCl₂, 1 mM (10 nmol) [α³²P]ATP, 1 pmol (100 nM) RqlH-(1–505) and 1 μg of the indicated salmon sperm or pUC19 DNAs were incubated at 37°C for 30 min. Blunt-end linear pUC19 was generated by digesting circular pUC19 with SmaI. Heat-denaturation of the DNAs was performed by incubating a 1 mg/ml DNA solution at 95°C for 10 min followed by quenching on ice. The extents of ATP hydrolysis are plotted. Each datum is the average of three separate experiments ± SEM. (B) ATPase reaction mixtures containing 20 mM Tris–HCl (pH 8.0), 1 mM DTT, 5 mM MgCl₂, 1 mM (10 nmol) [α³²P]ATP, 1 pmol (100 nM) RqlH-(1–505) and increasing amounts of 44-, 36-, 24-, 12- or 6-mer DNA oligonucleotides as specified were incubated at 37°C for 30 min. The nucleobase sequences of the oligonucleotides are shown at bottom. The extents of ATP hydrolysis are plotted as a function of added DNA (pmol). Each datum is the average of three separate DNA titration experiments ± SEM.

Directionality of RqlH-(1–505) translocation on ssDNA. Shown in the right panel is a schematic representation of directional 5′ SA displacement by the RqlH-(1–505) motor acting as a cowcatcher to pry apart the otherwise stable SA–biotin interaction. Translocase assays were performed as described under Experimental Procedures. Native PAGE analysis of the reaction products is shown in the left panel. The species corresponding to SA–DNA complex and free DNA are indicated. The nucleobase sequences of the 5′ ³²P-labeled 5′ or 3′ biotinylated 34-mer single-stranded DNAs are shown at bottom with (B) signifying the position of the biotin spacer. The complete translocase reaction mixtures in lanes 5 and 12 contained 1 mM ATP, 5 mM MgCl₂, 0.5 pmol ³²P-labeled 3′ or 5′ biotinylated DNA attached to SA, and 1 pmol of RqlH-(1–505). Enzyme was omitted from control reactions in lanes 2 and 9. Enzyme and SA were both omitted from control reactions in lanes 1 and 8. ATP was omitted from the reactions in lanes 3 and 10. Magnesium was omitted from the reactions in lanes 4 and 11. The complete reaction mixtures in lanes 6 and 13 contained 1 pmol of the RqlH-(1–505)-K49A mutant protein in lieu of RqlH-(1–505). The complete reaction mixtures in lanes 7 and 14 contained 1 pmol of the RqlH-(1–505)-D148A mutant protein instead of RqlH-(1–505).

Directionality of duplex unwinding and requirement for a loading strand. Helicase reaction mixtures (10 μl) contained 1 pmol of the indicated ³²P-labeled DNA substrate and (where indicated by plus symbol) 4 pmol RqlH-(1–505). Enzyme was omitted from the reaction mixtures in lanes –. A reaction mixture lacking enzyme that was heat denatured prior to PAGE is included in lane Δ. The reaction products were analyzed by native PAGE and visualized by autoradiography (top panel). The 5′-tail, 3′-tail, blunt, Y form, and bubble substrates are shown in the bottom panel, with the with the 5′ ³²P-labeled denoted by filled circle.