The G-quadruplex (G4) resolvase DHX36 efficiently and specifically disrupts DNA G4s via a translocation-based helicase mechanism - PubMed (original) (raw)

DHX36-mediated disruption of tetramolecular DNA G4s under steady-state and single-turnover conditions. A, illustration showing disruption of the tetramolecular G-quadruplex substrate 5G-A15, which includes five G-quartets and a 3′ single-stranded extension consisting of 15 adenosine nucleotides (left), into single strands (right). B, native gel image showing time-dependent disruption of 5G-A15 (upper band) to single strands (lower band) in steady-state experiments with 10 n

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DHX36 and G4 concentrations as indicated. In lane C1, the same ssDNA is shown with the G4 formation step omitted. In lane C2, the G4 formation step was performed with LiCl replacing KCl. C, initial rates for steady-state disruption of G4s with varying numbers of G-quartets: 4G-A15 (blue, _k_cat = 36 (±3) min−1 and Km = 180 (±6) n

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); 5G-A15 (red, _k_cat = 6.0 (±0.2) min−1 and Km = 40 (±2) n

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); and 6G-A15 (black, _k_cat = 0.6 (±0.1) min−1 and Km = 4.5 (±0.1) n

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). Solid blue squares show reactions of DHX36 with 4G-A15 in the absence of ATP. D, progress curves showing time-dependent disruption of the 5G-A15 G-quadruplex under single-turnover experiments with no DHX36 (triangles) and with 0.1 n

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(squares) or 1 n

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(diamonds) DHX36. E, plot of observed rate constants versus DHX36 concentration for disruption of 4G-A15 (2.8 (±0.3) × 108

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−1 min−1), 5G-A15 (2.4 (±0.2) × 108

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−1 min−1), and 6G-A15 (1.6 (±0.2) × 108

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−1 min−1) under single-turnover conditions (with the same color code as panel C). Reactions here and in subsequent figures were performed under standard conditions unless otherwise indicated (see “Experimental procedures,” DNA G4 disruption).