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Articles

Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3′-to-5′ Helicase That Unwinds 3′-Tailed RNA Duplexes and RNA:DNA Hybrids

Maria Loressa Uson, Heather Ordonez, Stewart Shuman
R. L. Gourse, Editor
Maria Loressa Uson
Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA
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Heather Ordonez
Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA
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Stewart Shuman
Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA
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R. L. Gourse
Roles: Editor
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DOI: 10.1128/JB.00418-15
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  • FIG 1
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    FIG 1

    M. smegmatis HelY is homologous to eukaryal Mtr4 and Ski2. The amino acid sequence of the 912-aa M. smegmatis HelY polypeptide is aligned to the sequences of the S. cerevisiae Mtr4 (1,073-aa) and Ski2 (1,287-aa) proteins. Positions of amino acid side chain identity/similarity in all three polypeptides are denoted by dots. Gaps in the alignments are denoted by dashes; numbers in brackets indicate the number of amino acids inserted. The distal margin of the NTPase domain is demarcated by the blue bracket. The superfamily II ATPase motifs Q, I, II, III, and VI of the NTPase domain are labeled and highlighted in gold shading. The two segments comprising the split winged helix domain are underscored by gray bars. The constituents of the CCCH zinc-binding motif in Ski2 are highlighted in green. The start of the C-terminal ratchet domain is indicated by the red bracket.

  • FIG 2
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    FIG 2

    HelY is an ATPase/dATPase. (A) Aliquots (5 μg) of purified wild-type (WT) HelY and K48A and D135A mutant HelY were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (in kilodaltons) of marker polypeptides are indicated on the left. (B) ATP hydrolysis. Reaction mixtures (10 μl) containing 20 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 1 mM [α-32P]ATP (10 nmol ATP), 200 nM poly(U) (as UMP nucleotide), and 10 pmol (1 μM) wild-type or mutant HelY, as specified, were incubated at 37°C for 20 min. The extents of ATP hydrolysis are plotted. Each datum is the average ± SEM from three separate experiments. (C) NTP specificity. Reaction mixtures (10 μl) containing 20 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 200 nM poly(U), 10 pmol HelY, and 1 mM the indicated NTP or dNTP were incubated at 37°C for 20 min. The reactions were quenched with 990 μl of malachite green reagent (Biomol Research Laboratories). Phosphate release was quantified by measuring the A620 and interpolating the value into a phosphate standard curve. The values were corrected for the low levels of phosphate measured in control reaction mixtures containing 1 mM NTP/dNTP but no added enzyme. Data are the averages ± SEMs from three separate experiments.

  • FIG 3
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    FIG 3

    Divalent cation specificity and pH profile. (A) Reaction mixtures (10 μl) containing 20 mM Tris-HCl, pH 8.0, 1 mM [α-32P]ATP, 200 nM poly(U), 10 pmol HelY, and either no added divalent cation (−), no added divalent cation plus 5 mM EDTA (EDTA), or 5 mM the indicated divalent cation (chloride salt) were incubated at 37°C for 20 min. (B) Reaction mixtures (10 μl) containing 20 mM Tris buffer (Tris acetate at pH 4.5, 5.0, 5.5, 6.0 or 6.5; Tris-HCl at pH 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5), 5 mM MgCl2, 1 mM [α-32P]ATP, 200 nM poly(U), and 10 pmol HelY were incubated for 20 min. The extents of ATP hydrolysis are plotted. Data are the averages ± SEMs from three separate experiments.

  • FIG 4
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    FIG 4

    Characterization of HelY ATPase activity. (A) Enzyme titration. Reaction mixtures (10 μl) containing 20 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 1 mM [α-32P]ATP, 200 nM poly(U), and HelY as indicated were incubated at 37°C for 20 min. (B) Time course. A reaction mixture containing 20 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 1 mM [α-32P]ATP, 200 nM poly(U), and 1 μM HelY was incubated at 37°C. Aliquots (10 μl containing 10 nmol ATP) were withdrawn at the times specified and quenched with formic acid. (C) Product inhibition by ADP. Reaction mixtures (10 μl) containing 20 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 1 mM [α-32P]ATP, 200 nM poly(U), 10 pmol HelY, and ADP at the indicated concentration were incubated at 37°C for 20 min. The extents of ATP hydrolysis are plotted. Each datum is the average ± SEM from three separate experiments.

  • FIG 5
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    FIG 5

    Nucleic acid cofactor dependence of ATP hydrolysis. Reaction mixtures (10 μl) containing 20 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 1 mM [α-32P]ATP, 10 pmol HelY, and 24-mer single-strand DNA or RNA oligonucleotide, as specified, were incubated at 37°C for 20 min.

  • FIG 6
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    FIG 6

    HelY helicase activity. Helicase reaction mixtures (10 μl) contained 20 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 1 pmol (100 nM) of the indicated 3′-tailed RNA:RNA, RNA:DNA, DNA:RNA, or DNA:DNA duplex substrates (depicted at the bottom, with the 5′ 32P label denoted by •), 1 mM ATP, and either no enzyme (lanes −) or 10 pmol (1 μM) HelY (lanes +). The mixtures were analyzed by native PAGE, and radiolabeled nucleic acids were visualized by autoradiography. Reaction mixtures lacking enzyme that were heat denatured prior to PAGE were analyzed in lanes Δ.

  • FIG 7
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    FIG 7

    Requirement for a 3′ single-strand tail on the RNA strand. Helicase reaction mixtures (10 μl) contained 20 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 1 mM ATP, 1 pmol (100 nM) of the indicated 3′-tailed, 5′-tailed, or blunt 32P-labeled DNA:RNA hybrid substrates, and either no enzyme (lanes −) or 10 pmol (1 μM) HelY (lanes +). The mixtures were analyzed by native PAGE, and radiolabeled nucleic acids were visualized by autoradiography. Reaction mixtures lacking enzyme that were heat denatured prior to PAGE were analyzed in lanes Δ.

  • FIG 8
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    FIG 8

    HelY titration and mutational inactivation of HelY helicase. (A) Titration. Reaction mixtures (10 μl) contained 20 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 1 mM ATP, 1 pmol (100 nM) 3′-tailed 32P-labeled DNA (24-mer):RNA (39-mer) substrate, and HelY as specified. (B) Mutational inactivation. Reaction mixtures (10 μl) contained 20 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 1 mM ATP, 1 pmol (100 nM) 3′-tailed 32P-labeled DNA:RNA substrate, and 10 pmol (1 μM) wild-type or mutant HelY, as specified. HelY was omitted from the control reaction mixture in lane −. The mixtures were analyzed by native PAGE, and radiolabeled nucleic acids were visualized by autoradiography.

  • FIG 9
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    FIG 9

    NTP requirement of the HelY helicase. Reaction mixtures (10 μl) contained 20 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 1 pmol (100 nM) 3′-tailed 32P-labeled DNA (24-mer):RNA (39-mer) substrate, 10 pmol (1 μM) HelY, and either no added NTP (lane −) or 1 mM the indicated NTP or dNTP. The reaction products were analyzed by native PAGE and visualized by autoradiography.

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Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3′-to-5′ Helicase That Unwinds 3′-Tailed RNA Duplexes and RNA:DNA Hybrids
Maria Loressa Uson, Heather Ordonez, Stewart Shuman
Journal of Bacteriology Sep 2015, 197 (19) 3057-3065; DOI: 10.1128/JB.00418-15

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Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3′-to-5′ Helicase That Unwinds 3′-Tailed RNA Duplexes and RNA:DNA Hybrids
Maria Loressa Uson, Heather Ordonez, Stewart Shuman
Journal of Bacteriology Sep 2015, 197 (19) 3057-3065; DOI: 10.1128/JB.00418-15
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