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. 2018 Nov 16;293(46):17792-17802.
doi: 10.1074/jbc.RA118.004499. Epub 2018 Sep 26.

Pif1 Helicase Unfolding of G-quadruplex DNA Is Highly Dependent on Sequence and Reaction Conditions

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Free PMC article

Pif1 Helicase Unfolding of G-quadruplex DNA Is Highly Dependent on Sequence and Reaction Conditions

Alicia K Byrd et al. J Biol Chem. .
Free PMC article

Abstract

In addition to unwinding double-stranded nucleic acids, helicase activity can also unfold noncanonical structures such as G-quadruplexes. We previously characterized Pif1 helicase catalyzed unfolding of parallel G-quadruplex DNA. Here we characterized unfolding of the telomeric G-quadruplex, which can fold into antiparallel and mixed hybrid structures and found significant differences. Telomeric DNA sequences are unfolded more readily than the parallel quadruplex formed by the c-MYC promoter in K+ Furthermore, we found that under conditions in which the telomeric quadruplex is less stable, such as in Na+, Pif1 traps thermally melted quadruplexes in the absence of ATP, leading to the appearance of increased product formation under conditions in which the enzyme is preincubated with the substrate. Stable telomeric G-quadruplex structures were unfolded in a stepwise manner at a rate slower than that of duplex DNA unwinding; however, the slower dissociation from G-quadruplexes compared with duplexes allowed the helicase to traverse more nucleotides than on duplexes. Consistent with this, the rate of ATP hydrolysis on the telomeric quadruplex DNA was reduced relative to that on single-stranded DNA (ssDNA), but less quadruplex DNA was needed to saturate ATPase activity. Under single-cycle conditions, telomeric quadruplex was unfolded by Pif1, but for the c-MYC quadruplex, unfolding required multiple helicase molecules loaded onto the adjacent ssDNA. Our findings illustrate that Pif1-catalyzed unfolding of G-quadruplex DNA is highly dependent on the specific sequence and the conditions of the reaction, including both the monovalent cation and the order of addition.

Keywords: DNA helicase; DNA unwinding; DNA-protein interaction; G-quadruplex; Pif1; enzyme kinetics; enzyme mechanism; pre-steady-state kinetics; secondary structure.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Quadruplex stability in the presence of a complementary strand is affected by both the sequence and the monovalent cation. A, schematic illustration of the trapping reaction. Folded G4DNA (2 nm) is mixed with a complementary strand (120 nm) as a DNA trap and reactions are quenched by adding an excess of unlabeled G4DNA (750 nm) to sequester the trap. Products were separated by electrophoresis (B). Melting of T15_c-MYC (C) and T15_hTEL (D) by the complementary strand was measured in 50 mm K+ (red), 50 mm Na+ (blue), and physiological salt (green). Data were fit to a single exponential to obtain rate constants for melting of 1.7 ± 0.3 s−1 for c-MYC in Na+, 0.19 ± 0.02 s−1, 1.2 ± 0.4 s−1, and 0.16 ± 0.06 s−1, for melting of hTEL in 50 mm K+, 50 mm Na+, and physiological salt, respectively. Errors bars represent the standard deviation of three independent experiments.
Figure 2.
Figure 2.
hTEL reporter substrates are partially melted during the preincubation with Pif1. A, G4DNA unfolding by Pif1 is monitored by measuring the rate of unfolding of a short reporter duplex in the presence of protein and annealing traps. After unfolding the G4DNA, Pif1 unwinds the dsDNA, which is forked due to Pif1's preference for forked duplexes. The unlabeled strand is trapped with excess complementary reporter trap, and the products are separated by electrophoresis. B, Pif1 (400 nm) was preincubated for 5 min with 2 nm radiolabeled DNA before loading into one sample port of a rapid chemical quench flow instrument. Time points were initiated by mixing with 5 mm ATP, 10 mm Mg2+, 60 nm reporter trap, and 10 μm T50 protein trap from the other sample port and quenched with 400 mm EDTA. The incubation time of the enzyme/DNA mixture increased with each successive time point in the reaction. C, unwinding of T15 reporter in K+ (black squares) and Na+ (green triangles) was fit to a 4-step sequential mechanism (D) using KinTek Explorer (85) to obtain rate constants for unwinding of 46.0 ± 3.9 and 43.2 ± 3.9 s−1, respectively. Data for unwinding the T15_hTEL reporter in K+ (red circles) and Na+ (blue diamonds) could not be fit to a simple n-step sequential mechanism. Data are the result of a single experiment due to the variable preincubation times. D, the reaction scheme describes unwinding by the helicase in a series of n sequential steps. ES, enzyme-substrate complex; EI, intermediate; EP, enzyme-product complex; E, free enzyme; S, substrate; P, ssDNA product. Each step is defined by an unwinding rate constant, ku, and a dissociation rate constant, kd. E and F, Pif1 (400 nm) was preincubated with 2 nm T15_hTEL reporter in 50 mm K+ (red) or 50 mm Na+ (blue) for varying amounts of time before mixing with 5 mm ATP, 10 mm Mg2+, 60 nm reporter trap, and 10 μm T50 protein trap for 10 s before quenching with 400 mm EDTA. Samples were separated by electrophoresis (G) and plotted to determine the quantity of G4 reporter unwound. The quantity of product formed in a 10-s reaction with ATP increased 19 ± 3% as the preincubation time increased from 15 s to 1 h in K+ (red) and 25 ± 2% as the preincubation time increased from 15 s to 1 h in Na+ (blue). Data are the average and standard deviation of three independent experiments.
Figure 3.
Figure 3.
Model for ATP independent partial melting of G4DNA. When Pif1 is preincubated with less stable G4DNA structures, particularly those stabilized by Na+, ATP independent trapping of thermally melted G4DNA structures can occur during the time in which the enzyme is preincubated with the substrate, resulting in a long ssDNA overhang on a partial duplex substrate. After addition of ATP, rapid unwinding of the duplex can occur. This can lead to the appearance of increased product formation when an unstable G4DNA structure is present relative to experiments with stable G4DNA structures performed in K+ where the G4DNA structure is unaffected by preincubation with the enzyme.
Figure 4.
Figure 4.
Unwinding hTEL reporter substrates and dsDNA without preincubation with Pif1. A, radiolabeled DNA (2 nm), 5 mm ATP, and 10 mm Mg2+ were loaded in one sample port of a rapid chemical quench flow instrument and time points were initiated by mixing with 400 nm Pif1 from the other sample port and quenched with 400 mm EDTA, 60 nm reporter trap, and 10 μm T50 protein trap. Samples were separated by electrophoresis (B) and data (C) was fit to a 4-step sequential mechanism, which includes binding steps (D) to obtain rate constants of 120 ± 40, 110 ± 20, 28 ± 7, and 23 ± 2 s−1 for unwinding of the T15 reporter in K+ (black squares) and Na+ (green triangles) and T15_hTEL reporter in K+ (red circles) and Na+ (blue diamonds), respectively. Data are the average and standard deviation of three independent experiments.
Figure 5.
Figure 5.
Pif1 ATP hydrolysis kinetics on ssDNA and G4DNA. The rate of ATP (5 mm) hydrolysis by 50 nm Pif1 was measured with varying quantities of T15 or T15_hTEL DNA cofactors in physiological salt and the specific activity was determined by dividing the ATP hydrolysis rate by the enzyme concentration. A plot of the ATPase activity versus the concentration of DNA was fit to a hyperbola to obtain the kcat of 55 ± 4 s−1 on T15 and 33 ± 7 s−1 on T15_hTEL. The Kact was 130 ± 9 nm on T15 and 39 ± 17 nm on T15_hTEL. Data are the average and standard deviation of three independent experiments.
Figure 6.
Figure 6.
Melting does not occur during preincubation when Pif1 is not in excess with hTEL reporter substrates in K+. A, Pif1 (100 nm in red; 50 nm in blue) was preincubated with 100 nm T15_hTEL reporter in 50 mm K+ for varying amounts of time before mixing with 5 mm ATP, 10 mm Mg2+, 3 μm reporter trap, and 10 μm T50 protein trap for 10 s before quenching with 400 mm EDTA. Samples were separated by electrophoresis (B) and plotted (C) to determine the quantity of G4 reporter unwound. The quantity of product formed in a 10-s reaction with ATP was essentially unchanged as the preincubation time increased from 15 s to 1 h. Data are the average and standard deviation of three independent experiments.
Figure 7.
Figure 7.
Pif1 unfolds hTEL G4DNA with slower rate but more bases are traversed compared with dsDNA. A, radiolabeled DNA (100 nm) and 100 nm Pif1 were loaded in one sample port of a rapid chemical quench flow instrument and time points were initiated by mixing with 5 mm ATP, 10 mm Mg2+, 3 μm reporter trap, and 10 μm T50 protein trap from the other sample port and quenched with 400 mm EDTA. Samples were separated by electrophoresis and plotted (B) for the T15 reporter in K+ (black squares), the T15_hTEL reporter in K+ (red circles), and T15_cMYC reporter in K+ (blue diamonds). C, data were fit using nonlinear least squares analysis to an n-step sequential mechanism (Fig. 2D) to obtain rate constants for unwinding (ku) and dissociation (kd) and the number of apparent steps (n). Data are the average and standard deviation of three independent experiments. The calculated processivity using Equation 3 is 0.72 for the T15 reporter and 0.69 for the T15_hTEL reporter.
Figure 8.
Figure 8.
Unfolding of c-MYC G4DNA substrates with varying length ssDNA overhangs under single turnover conditions by Pif1. A, products of unfolding 2 nm G4DNA by 400 nm Pif1 were separated by electrophoresis. B, data were fit to a single exponential to obtain rate constants for unwinding of the T15_c-MYC reporter (open red circles), T24_c-MYC reporter (open blue squares), T32_c-MYC reporter (open green diamonds), and T40_c-MYC reporter (open black triangles) of 0.29 ± 0.08, 0.075 ± 0.012, 0.059 ± 0.004, and 0.049 ± 0.009 s−1, respectively. Data represent the average and standard deviation of three independent experiments. Data shown in closed symbols had the protein trap (T50) preincubated with the Pif1 to test the efficiency of the trapping strand.

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