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. 2015 May 8;290(19):11948-68.
doi: 10.1074/jbc.M114.619395. Epub 2015 Mar 23.

Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis

Shivendra Singh Dewhare  1 , T G Umesh  1 , K Muniyappa  2
Affiliations

Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis

Shivendra Singh Dewhare et al. J Biol Chem. .

Abstract

The annotated whole-genome sequence of Mycobacterium tuberculosis revealed the presence of a putative recD gene; however, the biochemical characteristics of its encoded protein product (MtRecD) remain largely unknown. Here, we show that MtRecD exists in solution as a stable homodimer. Protein-DNA binding assays revealed that MtRecD binds efficiently to single-stranded DNA and linear duplexes containing 5' overhangs relative to the 3' overhangs but not to blunt-ended duplex. Furthermore, MtRecD bound more robustly to a variety of Y-shaped DNA structures having ≥18-nucleotide overhangs but not to a similar substrate containing 5-nucleotide overhangs. MtRecD formed more salt-tolerant complexes with Y-shaped structures compared with linear duplex having 3' overhangs. The intrinsic ATPase activity of MtRecD was stimulated by single-stranded DNA. Site-specific mutagenesis of Lys-179 in motif I abolished the ATPase activity of MtRecD. Interestingly, although MtRecD-catalyzed unwinding showed a markedly higher preference for duplex substrates with 5' overhangs, it could also catalyze significant unwinding of substrates containing 3' overhangs. These results support the notion that MtRecD is a bipolar helicase with strong 5' → 3' and weak 3' → 5' unwinding activities. The extent of unwinding of Y-shaped DNA structures was ∼3-fold lower compared with duplexes with 5' overhangs. Notably, direct interaction between MtRecD and its cognate RecA led to inhibition of DNA strand exchange promoted by RecA. Altogether, these studies provide the first detailed characterization of MtRecD and present important insights into the type of DNA structure the enzyme is likely to act upon during the processes of DNA repair or homologous recombination.

Keywords: ATPase; Atomic Force Microscopy (AFM); Bioinformatics; Cloning; DNA Helicase; DNA Recombination; Enzyme Kinetics; Infectious Disease; Mycobacteria; Mycobacterium tuberculosis.

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Figures

FIGURE 1.
FIGURE 1.
Sequence alignment of RecD subunit from different bacterial species. Protein sequences deduced from the nucleotide sequences were aligned using the ClustalW2 program and displayed by Jalview. The amino acid sequences of the proteins are shown in the single-letter code. Gaps are indicated by dashes. The positions of the amino acids are indicated at the top of the figure. The conserved amino acid residues are shaded as follows: red, hydrophobic residues; magenta, basic residues; blue, acidic residues; and green, hydroxyl/amines/glutamine. Highly conserved motifs are enclosed in a rectangle. Accession numbers of amino acid sequence used in this analysis are as follows: P96919, M. tuberculosis RecD subunit; A1KGA7, Mycobacterium bovis (strain BCG/Paris 1173P2) RecD; A0PLZ2, M. ulcerans RecD; A0QL87, M. avium RecD; A0QS28, M. smegmatis RecD; P04993, E. coli (strain K-12) RecD.
FIGURE 2.
FIGURE 2.
Expression and purification of MtRecD and its variant, MtRecDK179A. A, SDS-PAGE analysis showing induced expression of MtRecD and at various stages during its purification. Lane 1, molecular mass markers; lane 2, uninduced whole-cell lysate; lane 3, induced whole-cell lysate; lane 4, eluate from Ni2+-NTA matrix; lane 5, eluate from Superdex 200 gel filtration chromatography. The sizes of the marker proteins in kDa are indicated on the left. B, SDS-PAGE analysis showing induced expression of MtRecDK179A and at various stages during its purification. Lane 1, molecular mass markers; lane 2, uninduced whole-cell lysate; lane 3, induced whole-cell lysate; lane 4, eluate from Ni2+-NTA matrix; lane 5, eluate from Superdex 200 gel filtration chromatography. The sizes of the marker proteins in kDa are indicated on the left.
FIGURE 3.
FIGURE 3.
Determination of native molecular mass of MtRecD. A, chromatogram depicting the elution profile of MtRecD. The overlapping chromatographic elution profile corresponds to MtRecD at different concentrations (0.5 μm, black trace; 1 μm, green trace, and 1.5 μm, red trace, respectively). B, graphic representation of molecular mass as a function of Kav versus log molecular mass of marker proteins. Molecular mass of MtRecD calculated by interpolation is ∼120 kDa (green line). C, upper panel, AFM image of MtRecD; lower panel, three-dimensional surface plot of the image in C, upper panel. The red and green arrows indicate dimeric and monomeric forms of MtRecD, respectively. D, chemical cross-linking of MtRecD. Reactions were performed as described under “Experimental Procedures.” Lane 1, molecular mass markers; lane 2, MtRecD incubated in the absence of glutaraldehyde; lanes 3–7, MtRecD in the presence of increasing concentrations of glutaraldehyde (0.005, 0.0075, 0.010, 0.015, and 0.02%, respectively). E, AFM image of S. cerevisiae Hop1. F, three-dimensional surface plot of the image of Hop1. M, molecular mass markers; C, reaction performed in the absence of glutaraldehyde.
FIGURE 4.
FIGURE 4.
MtRecD exhibits higher binding affinity for linear duplex with a 5′ ssDNA overhang. Reaction mixtures containing the indicated 32P-labeled DNA substrate (1 nm) were incubated in the absence (lane 1) or presence of 10, 25, 50, 75, 100, 150, 200, 300, 400, and 500 nm MtRecD (lanes 2–11), respectively. After incubation at 37 °C for 25 min, samples were subjected to electrophoresis on native polyacrylamide gels and visualized as described under “Experimental Procedures.” Filled triangles on top of the gel images indicate increasing concentrations of MtRecD. The positions of the free DNA and MtRecD-DNA complexes are indicated on the right. A–F, reactions performed with the indicated DNA substrates. G, graphic representation of the extent of the formation of MtRecD-DNA complexes. The amount of MtRecD-DNA complexes in A–F was quantified and plotted against increasing concentrations of MtRecD; ●, MtRecD, linear duplex with 5′ overhang; ■, MtRecD, partial duplex with 3′ overhang; ▴, MtRecD, single-stranded DNA; ▾, MtRecD, partial duplex with biotinylated 5′ overhang; ♦, MtRecD, partial duplex with biotinylated 3′ overhang; *, MtRecD, blunt-ended duplex DNA. CI and CII denote complex I and complex II, respectively. The ● on linear duplex DNA overhangs in C and D corresponds to the position of biotinylated nucleotide. An asterisk denotes the 32P-labeled end. The error bars in G represent the deviation from the mean for data from three separate experiments.
FIGURE 5.
FIGURE 5.
MtRecD binds robustly to Y-shaped DNA structures. Reaction mixtures containing the indicated 32P-labeled DNA (1 nm) was incubated in the absence (lane 1) or presence of 10, 25, 50, 75, 100, 150, 200, 300, 400, and 500 nm MtRecD (lanes 2–11), respectively. After incubation at 37 °C for 25 min, samples were subjected to electrophoresis on native polyacrylamide gels and visualized as described under “Experimental Procedures.” Filled triangle on top of the gel images represents increasing concentrations of MtRecD. The positions of the free DNA and MtRecD-DNA complexes are indicated on the right. A–F, reactions performed with the indicated DNA substrates. G, graphic representation of the extent of the formation of MtRecD-DNA complexes with different substrates. The amount of MtRecD-DNA complexes in A–F was quantified and plotted against increasing concentrations of MtRecD. ▾, MtRecD, Y-shaped duplex having 40/18-nt overhangs; ○, MtRecD, Y-shaped duplex having 40/19-nt overhangs; ▴, MtRecD, Y-shaped duplex having 30/18-nt overhangs; ♦, MtRecD, Y-shaped duplex with 30/19-nt overhangs; ●, MtRecD, Y-shaped duplex having 18/19-nt overhangs; ■, MtRecD, Y-shaped duplex having 5/5-nt overhangs. CI and CII denotes complex I and complex II, respectively. An asterisk denotes the 32P-labeled end. The error bars in G represent the deviation from the mean for data from three independent experiments.
FIGURE 6.
FIGURE 6.
Effect of NaCl on the stability of MtRecD-DNA complexes. Reaction mixtures contained 1 nm 32P-labeled DNA substrate and 500 nm MtRecD. After incubation for 30 min, NaCl was added to a final concentration of 25, 50, 75, 100, 125, 150, 175, 200, and 250 mm (lanes 3–11), respectively. Lane 1, substrate alone; lane 2, complete reaction in the absence of NaCl. A–D, DNA substrates shown above the gel images. E, graphic representation of the extent of dissociation of MtRecD-DNA complex(s) plotted versus increasing concentrations of NaCl. ▴, MtRecD, single-stranded DNA; ●, MtRecD, duplex with 5′ overhang; ■, MtRecD, duplex with 3′ overhang; ▾, MtRecD, Y-shaped duplex. CI and CII represent complexes I and II, respectively. The intensity of bands was quantified using UVI-Band Map software version 97.4 and plotted against concentration of NaCl using GraphPad Prism version 5.0. The data points represent the mean of three independent experiments. An asterisk denotes the 32P-labeled end.
FIGURE 7.
FIGURE 7.
UV-catalyzed cross-linking of ATP to MtRecD. Assay was performed as described under “Experimental Procedures.” A, binding of 32P-labeled ATP to MtRecD as a function of protein concentration. Lane 1, MtRecD incubated in the absence of [γ-32P]ATP; lanes 2–9, MtRecD (0.4, 0.8, 1.2, 1.6, 2, 2.4, 2.8, and 3.2 μm) incubated with 1.6 pmol of [γ-32P]ATP. B, graph showing the extent of binding of 32P-labeled ATP by MtRecD. C, binding of 32P-labeled ATP to MtRecD as a function of ATP concentration. Lane 1, MtRecD incubated in the absence of [γ-32P]ATP; lanes 2–9, 1.5 μm MtRecD incubated with 5, 7.5, 10, 20, 30, 40, 50, and 75 μm [γ-32P]ATP, respectively. D, graph showing the extent of binding of 32P-labeled ATP by MtRecD. The intensity of bands was quantified and plotted against concentration of protein (A) and ATP (C).
FIGURE 8.
FIGURE 8.
Characterization of MtRecD-catalyzed ATPase activity. A, time course of ATPase activity by MtRecD. Reaction mixtures containing 5 μm [γ-32P]ATP, 20 μm ssDNA, and MtRecD (300 nm) were incubated for 5, 10, 20, 30, 60, 90, 120, and 180 min (lanes 2–9), respectively. Lane 1, reaction mixture incubated for 180 min in the absence of MtRecD. B, graphic representation of time course of ATP hydrolysis by MtRecD. C, ATP hydrolysis as a function of MtRecD concentration. Assay was performed with 5 μm [γ-32P]ATP in the absence (lane 1) or presence of (lanes 2–8) 25, 50, 100, 200, 300, 400, and 500 nm MtRecD, respectively. D, graphic representation of the extent of ssDNA-dependent ATPase activity as a function of increasing MtRecD concentration. E, graphic representation of single- or double-stranded DNA-dependent ATPase activity. Assay was performed with a fixed concentration of MtRecD (300 nm) in the absence or presence of 2.5, 5, 7.5, 10, 15, 20, and 30 μm single- or double-stranded DNA, respectively. The error bars represent the deviation from the mean for data from three independent experiments. Asterisks denote radiolabeled ATP or inorganic phosphate (Pi). C, reaction performed in the absence of MtRecD.
FIGURE 9.
FIGURE 9.
ATP hydrolysis catalyzed by MtRecD follows Michaelis-Menten kinetics. A, assay was performed with MtRecD (0.5 μm) in the presence of 10 μm ssDNA, 5 mm MgCl2, and 0.75, 1, 2.5, 5, 7.5, 10, and 12.5 μm [γ-32P]ATP (lanes 2–8), respectively. Reaction products were separated as described under “Experimental Procedures.” The rate of the reaction was calculated from the slopes of such plots. B, shows 1/[v] versus 1/[S] in the form of a Lineweaver-Burk plot. Data from multiple kinetic experiments are included in this plot. The kinetic parameters were determined in reactions performed in the presence of excess of ssDNA and ATP. For all concentrations of ATP, we determined the initial velocities from multiple time courses over time ranges giving linear hydrolysis of ATP. Velocities were plotted as functions of ATP concentration and fitted to the Michaelis-Menten equation.
FIGURE 10.
FIGURE 10.
MtRecDK179A binds partial duplex with a 5′ overhang but contains weak ATPase activity. A, reactions were performed with 1 nm 32P-labeled DNA and increasing concentrations of MtRecDK179A in the absence (lane 1) or presence of 10, 25, 50, 75, 100, 150, 200, 300, 400, and 500 nm MtRecDK179A (lanes 2–11), respectively. B, graphic representation of the extent of the formation of MtRecDK179A-DNA complexes (CI and CII). The data points represent the mean of three independent experiments. C, reactions were performed with a fixed concentration of ATP and of increasing concentrations of MtRecDK179A in the absence (lane 1) or presence of 25, 50, 100, 200, 300, 400, and 500 nm MtRecDK179A (lanes 2–8), respectively. C, reaction performed in the absence of MtRecD mutant protein. D, graphic representation of the extent of ATPase activity as a function of increasing concentration of MtRecDK179A. The intensity of bands was quantified and plotted against the indicated protein concentrations.
FIGURE 11.
FIGURE 11.
DNA unwinding activity of MtRecD as a function of time and concentrations of ATP. A, DNA unwinding as a function of increasing concentrations of ATP. Reaction mixtures containing 0.3 μm MtRecD and 1 nm linear duplex with a 5′ overhang were incubated in the absence (lane 3) or presence of 1, 1.5, 2, 2.5, 3, 3.5, 5, 7.5 and 10 mm ATP (lanes 4–12), respectively. Lane 1, heat-denatured substrate; lane 2, substrate alone. B, graphic representation of DNA unwinding activity as a function of ATP concentration. The data points represent the mean of three independent experiments. C, kinetics of MtRecD-catalyzed DNA unwinding. Reaction mixtures containing 1 nm linear duplex with a 5′ overhang and 0.3 μm MtRecD was incubated for a period of 0, 5, 10, 15, 20, 25, 30, and 35 min (lanes 3–10), respectively. Lane 1, heat-denatured substrate; lane 2, substrate alone. D, graphic representation of the kinetics of unwinding. The intensity of bands in A and C was quantified after correcting the background using UVI-Band Map software version 97.4 and plotted using GraphPad Prism version 5.0. The data points represent the mean of three independent experiments. C, reaction performed in the absence of MtRecD.
FIGURE 12.
FIGURE 12.
MtRecD exhibits preferential 5′ overhang unwinding activity. Assay was performed with 1 nm of the indicated 32P-labeled DNA substrate in the absence (lane 2) or presence of 50, 75, 100, 200, 300, or 500 nm MtRecD (lanes 5–10), respectively. Lane 2, substrate alone. Lanes 3 and 4 represent reactions performed in the absence of MgCl2 and ATP, respectively. Reaction mixtures were preincubated at 37 °C for 5 min. The reaction was started by the addition of 3.5 mm ATP and assayed as described under “Experimental Procedures.” A–H, gel images depicting MtRecD catalyzed unwinding of the indicated DNA substrate. I, graphic representation of the extent of unwinding of DNA substrates in A–H plotted versus increasing concentrations of MtRecD. Because the unwinding of seven Y-shaped DNA structures were in the range of 18–36%, the curves are stacked one above the other, an expanded view of I in the region of 0–40% is shown in J. The intensity of the bands was quantified and plotted against the indicated concentrations of MtRecD. Vertical bars represent standard deviation of three independent experiments. A–H, lane 1, heat-denatured substrate; lane 2, substrate alone; lanes 3 and 4, reaction mixtures lacking ATP and MgCl2, respectively. Filled triangles on top of the gel images represent increasing concentrations of MtRecD. An asterisk denotes the 32P-labeled end. The positions of the substrate DNA and unwound DNA product are indicated on the right. C, reaction performed in the absence of MtRecD.
FIGURE 13.
FIGURE 13.
MtRecD possesses bipolar DNA unwinding activity. A and B, gel images depicting MtRecD catalyzed unwinding of linear duplex having 5′ and 3′- ssDNA overhang. Assay was performed with 1 nm 32P-labeled DNA substrate in presence of 50, 75, 100, 200, 300, and 500 nm MtRecD subunit (lanes 5–10), respectively. Lane 1, heat-denatured substrate; lane 2, substrate alone; lanes 3 and 4, reaction mixtures lacking MgCl2 and ATP, respectively. Filled triangles on top of the gel images indicate increasing concentrations of MtRecD. A closed circle on DNA overhangs denotes the position of biotinylated nucleotide. An asterisk denotes the 32P-labeled end. The positions of the substrate DNA and unwound DNA product are indicated on the right. C and D, gel images depicting MtRecD-catalyzed unwinding of linear duplex having 5′ and 3′ ssDNA overhang in the presence of streptavidin. Assay was performed with 1 nm 32P-labeled DNA substrate in the absence (lane 3) or presence of 50, 75, 100, 200, 300, and 500 nm MtRecD (lanes 4–9), respectively. Lane 1, heat-denatured substrate; lane 2, substrate alone, respectively. Reaction mixtures were preincubated at 37 °C for 5 min. The reaction was started by the addition of 3.5 mm ATP and assayed as described under “Experimental Procedures.” Filled triangle on top of the gel images represents increasing concentrations of MtRecD. An asterisk denotes the 32P-labeled end. The positions of the substrate DNA and unwound DNA product are indicated on the right. E, graphic representation of the extent of unwinding of DNA substrates in A–D plotted versus increasing concentrations of MtRecD. ●, MtRecD, partial duplex with biotinylated 5′ overhang; ■, MtRecD, partial duplex with 3′ overhang; ▴, MtRecD, partial duplex with biotinylated streptavidin 5′ overhang; ▾, MtRecD, partial duplex with biotinylated streptavidin 3′ overhang. The black circle on linear duplex DNA overhangs in C and D corresponds to the position of biotinylated nucleotide. The PacMan symbol corresponds to streptavidin.
FIGURE 14.
FIGURE 14.
MtRecD and MtRecA directly and specifically interact with each other in vivo and in vitro. A, far Western analysis with increasing concentrations (1–4 μm) of wild-type and mutant MtRecD, MtSSB, and BSA (lanes 1–4, respectively). B, far Western analysis with increasing concentrations (1- 4 μm) of MtRecA or BSA (lanes 1–4, respectively). C, panel i, MtRecD was immunoprecipitated (IP) with anti-MtRecA antibody and immunoblotted with anti-MtRecD antibody; (panel ii) MtRecDK179A was immunoprecipitated with anti-MtRecA antibody and immunoblotted with anti-MtRecD antibody; and panel iii, MtRecA was immunoprecipitated with anti-MtRecD antibody and immunoblotted with anti-MtRecA antibody. Lane 1, MtRecD, MtRecDK179A. or MtRecA; lane 2, Sepharose bead control; lane 3, nonspecific antibody control; lane 4, immunoprecipitate. Assays were performed as described under “Experimental Procedures.”
FIGURE 15.
FIGURE 15.
MtRecD inhibits DNA strand exchange promoted by MtRecA. A, schematic depiction of the experimental design. B, representative experiment showing the effect of increasing concentrations of MtRecD on strand exchange promoted by MtRecA. Assay was performed with 5 μm ssDNA, 2.5 μm MtRecA, and 1 μm 32P-labeled linear double-stranded DNA in the absence (lanes 2) or in the presence of 0.05, 0.75, 0.1, 0.2, 0.3, 0.5, 0.75, and 1.0 μm MtRecD (lanes 3–10), respectively, as described under the “Experimental Procedures.” Lane 1 shows reactions performed in the absence of MtRecA. The positions of double-stranded DNA and displaced ssDNA are indicated on the left. C, graphic representation of the extent of inhibition of strand exchange by MtRecD. The data points represent the mean ± S.D. of three independent experiments.
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