Using Atomic Force Microscopy to Study the Real Time Dynamics of DNA Unwinding by Mitochondrial Twinkle Helicase

Materials and Reagents

1. 100 mM ATP stock solution in deionized (DI) water buffered to pH 7.5 (aliquoted and stored at -80°C) (Sigma-Aldrich, catalog number: A7699)

2. Mica (SPI Chem Mica Grade V-1 50 × 25 mm × 0.15 mm, catalog number: 12001-26-2) (Figure 1)

   
   

   
   Figure 1. SPI Chem Mica used for Atomic Force Microscopy (AFM) experiments




3. Aminopropyl silatrane (APS) (Shlyakhtenko et al., 2013)

4. HEPES (Fisher Bioreagent, catalog number: 7365-45-9)

5. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 7647-14-5)

6. Magnesium chloride (MgCl₂) (Fisher Chemical, catalog number: 7791-18-6)

7. Glycerol (EMD Millipore, catalog number: 356350)

8. Nonidet P-40 (NP-40) (ThermoFisher Scientific, catalog number: 85124)

9. Nt.BstNBI (NEB, catalog number: R0607S)

10. Amicon Ultrafiltration (Millipore, catalog number: C82301, MW 100K)

11. ScaI restriction enzyme (NEB, catalog number: R3122S)

12. pscwo1 plasmid (Addgene, catalog number: 72300)

13. Helicase Imaging Buffer (see Recipes)

14. APS working solution (see Recipes)

15. Buffer 3.1: 1× Buffer components: NEB (see Recipes)

Equipment

1. NanoDrop 2000C (Thermo Scientific, catalog number: ND-2000C)

2. AFM tips for air imaging – Pointprobe PPP-FMR probes (Nanosensors, spring constant k = 2.8 N/m, Resonant frequency f_r = 70 kHz).

   AFM tips for Buffer imaging – BL-AC40TS (Biolever Mini) probe (Asylum Research, spring constant k = 0.09 N/m, Resonant Frequency f_r = 110 kHz)

3. Centrifuge (to fit Eppendorf 1.5 ml tubes)

4. Cylinder of Nitrogen gas with a pressure regulator and nozzle.

5. Eppendorf tubes (1.5 ml) (Eppendorf, catalog number: 022431081)

6. Scotch Tape

7. MFP-3D – Bio AFM (Figures 2-4) (Asylum Research, Oxford Instruments)

   
   

   
   Figure 2. AFM instrument details showing basic AFM components inside the AFM noise canceling chamber with the AC2 controller

   
   

   
   Figure 3. AFM head with labeled controls

Software

1. IGOR Pro 6 (https://www.wavemetrics.com/software/igor-pro-802, WaveMetrics, Portland, OR, USA)

2. MFP3D AFM imaging software or Gwydiion software (http://gwyddion.net/) (Nečas and Klapetek, 2012)

Procedures

1. Proteins used for unwinding assays

   Recombinant human Twinkle helicase with a C-terminal His6-affinity tag was overexpressed in E. coli BL21 (DE3) Codon Plus-RIPL (Stratagene). The Twinkle helicase was purified to >95% purity using successive nickel-affinity, anion exchange, and heparin-affinity chromatography (Longley et al., 2010; Kaur et al., 2020). Twinkle helicase protein concentration was evaluated using Coomassie-stained SDS-PAGE gels with BSA standards (Krieg et al., 2005; He, 2011). For a detailed purification protocol, see Longley et al. (2010) . Human mtSSB protein was overexpressed in E. coli and purified as described previously (Longley et al., 2009).




2. DNA substrates for the unwinding assays

   DNA substrate – Gapped circular DNA substrate (4,060 bp) – was prepared by introducing two clusters of four closely spaced nicks in one strand of pscwo1 plasmid (Addgene: plasmid # 72300) using Nt.BstNBI. These circular substrates were incubated with complementary oligos (Integrated DNA Technologies, IDT) (oligo: plasmid – 10:1 ratio) at 68°C for 30 min, followed by slow cooling to room temperature. Amicon Ultrafiltration was used to remove excess oligonucleotides and short duplex DNA. Diagnostic restriction digestion was used to evaluate the gapping efficiency (85-95%). Linear gapped DNA substrate for AFM studies was generated by digesting the circular gapped DNA with ScaI restriction enzyme using Buffer 3.1 (Countryman et al., 2018; Kaur et al., 2018; Kaur et al., 2020).




3. AFM imaging in air to study DNA unwinding by Twinkle helicase

   Using AFM imaging in air, protein-DNA samples are incubated and deposited on a sample surface. These samples are then imaged in air, and the scanning AFM tip captures a snapshot of the protein-DNA static structure-function information at the incubated time. Furthermore, time titration of the protein-DNA sample can provide information on how the protein-DNA interaction changes with respect to incubated time.

   1. Prepare freshly cleaved mica surfaces by peeling a layer of mica off using scotch tape.

   2. Measure the concentration of the purified gapped DNA using a NanoDrop spectrophotometer. DNA concentration is estimated by measuring the absorbance at a wavelength of 260 nm (A₂₆₀) using a spectrophotometer. The absorbance of double-stranded DNA at a concentration of 50 µg/ml is 1 and follows a linear relationship with concentration. Using this relationship, we can formulate the unknown DNA concentration as:

      
      

      DNA concentration (to be measured in µg/ml) = 50 µg/ml × A₂₆₀

   
   

   3. Incubate Twinkle helicase (71.2 nM hexamers) with circular gapped DNA (3.6 nM) in Helicase Imaging Buffer for 1 min at room temperature. Increasing the Twinkle helicase: circular gapped DNA ratio can lead to an increased molar ratio of Twinkle per DNA molecule, causing more Twinkle helicase hexamers to unwind the same DNA molecule. Based on the extent of unwinding required and the number of unwinding sites, the ratio and incubation time can be adjusted.

   4. Dilute the incubated sample 10-fold in Helicase Imaging Buffer (final volume 20 µl) and deposit the sample directly onto the freshly cleaved mica surface. There is no limitation on the volume of the sample, but there is a limitation on the concentration of the sample as you are performing single molecule studies. Overcrowded samples cannot provide clear information as it is difficult to observe single molecules trajectories. Therefore, the concentration of the sample on the surface should be adjusted accordingly, so that single molecules are clearly identifiable.

   5. Wash the deposited samples three times with Helicase Imaging Buffer (300-500 µl) followed by three times with DI water (300-500 µl) and dry the sample with streams of N₂ gas before AFM imaging.

   6. Open the MFP3D software and select the standard tab > AC air topography mode from the mode master tab (Figure 3).

   7. Load the AFM probe (Pointprobe PPP-FMR probes) on the cantilever holder (Figure 4) and align the position of the laser plot to get maximum SUM signal (laser intensity) and zero deflection. While mounting the AFM tip on the cantilever holder, do not push too far back inside as this can result in misalignment of the AFM tip.

      
      

      
      Figure 4. Mode Master tab with AC air topography mode selected (red square)

   
   

   8. Autotune the mounted probe to the resonance frequency (frequency with greatest oscillation amplitude) with a target amplitude of a 1 V free air amplitude voltage for operation in AAC mode. Adjust the Feedback filter on the Master Panel > Advanced Scan Parms to 1,500 Hz (Figures 3, 5, 6, and 7).

      
      

      
      Figure 5. Labeled cantilever holder parts with mounted AFM tip

      
      

      
      Figure 6. Cantilever tune and Tune Panel tab of the Master Panel showing target amplitude of 1 V selected (red square)

      
      

      
      Figure 7. Main tab and Advanced Scan Parms tab of the Master Panel showing Feedback filter at 1.5 kHz (red square) in AAC air imaging mode

   
   

   9. Image the sample deposited on mica surface using repulsive mode by adjusting the target percent to -5%-10% of the free air amplitude, enforcing the tip to remain in the repulsive regime (phase signal < 90°) while scanning the surface.

   10. Capture the images at a scan rate of 1-2 Hz and a resolution of 512 × 512 pixels.




4. AFM Imaging for DNA unwinding by Twinkle helicase facilitated by mtSSB

   1. Incubate Twinkle helicase (71.2 nM hexamers) and increasing concentrations of mtSSB tetramer (17.8 nM, 35 nM, 54 nM, and 142 nM) with circular gapped DNA (3.6 nM) in Helicase Imaging Buffer for 1 min at room temperature. Follow this order of addition to the incubated samples: Helicase Imaging Buffer, circular gapped DNA, and Twinkle and mtSSB simultaneously. Higher concentrations of mtSSB are observed to facilitate the unwinding reaction.

   2. Dilute the samples 10-fold in Helicase Imaging Buffer (final volume 20 µl) and deposit directly onto the freshly cleaved mica surface.

   3. Wash the deposited samples with Helicase Imaging Buffer (300-500 µl) three times followed by DI water (300-500 µl) three times and dry the sample with streams of N₂ gas prior to AFM imaging.

      Image the sample deposited onto a mica surface using AC mode on MFP-3D-Bio AFM with Pointprobe PPP-FMR probes (Nanosensors, k = 2.8 N/m, f_r = 70 kHz) as described in the previous section (Figures 1-3).

   4. Capture the images at a scan rate of 1-2 Hz and a resolution of 512 × 512 pixels.




5. AFM imaging in liquids to capture real-time dynamics of Twinkle helicase on circular gapped DNA

   To elucidate the dynamics of protein-DNA interactions, time lapse buffer imaging of the protein-DNA is done. In this mode of AFM imaging, the protein and DNA are not bound strongly to the surface and have restricted freedom to move around while imaging of the sample by the AFM tip in a layer of respective buffer (100-200 µl). This helps to visualize the dynamics of the protein-DNA interactions in real time as the tip is scanning across the sample surface in the native state.

   1. Prepare a freshly cleaved mica surface by peeling a layer of mica off the scotch tape.

   2. Deposit 30 µl of the APS working solution and incubate for 15 min.

   3. Wash the APS solution with DI water (300-500 µl) three times and dry with streams of N₂ gas.

   4. Check the concentration of the purified gapped DNA using a NanoDrop spectrophotometer.

   5. Incubate 0.7 nM circular gapped DNA onto the APS treated mica surface for 15 min. Wash the deposited DNA with Helicase Imaging Buffer (300-500 µl). Add 100-200 µl of Helicase Imaging Buffer to the washed sample.

   6. Select a low spring constant AFM imaging probe for imaging in liquids (AFM probes used in this experiment – BL-AC40TS, Asylum Research, spring constant k = 0.09 N/m, Resonant Frequency f_r = 110 kHz).

   7. Open the MFP3D software and select standard > Topography > AC water Topography mode (Figure 8).

      
      

      
      Figure 8. Mode Master tab with AC water Topography mode selected (red square)

   
   

   8. Add 2-3 droplets of Helicase Imaging Buffer on the AFM probe before mounting the AFM head onto the base.

   9. Align the laser spot on the cantilever for maximum SUM signal (laser intensity) and zero deflection. The SUM signal obtained in liquids was observed to be lower than the SUM signal for the same AFM probe in the air.

   10. In the Master panel, select capture Thermal Data and Do Thermal. Zoom in on the broad peak and fit it. Right-click on the fit to move the fit to the tune (Move Freq and Phase to the tune) (Figure 9).

       Note: The resonance frequency in liquids is 1/3^rd of its value in air.

       
       

       
       Figure 9. Main tab and Advanced Scan Parms tab of the Master Panel showing Thermal tab and Feedback filter at 0.5 kHz (red square) in AAC water imaging mode

   
   

   11. Select the Tune tab in the Master panel and turn on ‘Append Thermal.’ Set the sweep width to 30 kHz. Click on the ‘One Tune.’ Choose the peak that overlaps the most with the thermal and set it as Drive frequency.

   12. Center the phase at resonance to 90° by clicking on the ‘Center Phase’ button.

       Adjust the Feedback filter on the Master Panel > Advanced Scan Parms to 500 Hz (Figure 5).

   13. Increase the Drive Amplitude to get 0.5-0.7 V of the free air oscillation amplitude.

   14. For imaging biological samples, typically use 80% of the free amplitude as setpoint. The setpoint was adjusted approximately between 0.4-0.55 V.

   15. Images were captured at a scan rate of 2-4 Hz and a resolution of 512 × 512 pixels. A typical sample DNA coverage includes 8-10 DNA molecules at a 1 µm × 1 µm scan size.

   16. Incubate Twinkle helicase (71.2 nM hexamers, 10 µl) in Helicase Imaging Buffer for 5 min at room temperature.

   17. Dilute the Twinkle helicase 10-fold in Twinkle Helicase Buffer and very slowly add the diluted Twinkle helicase (50-70 µl) into the liquids on top of the deposited DNA using a pipette tip while it is being scanned by the AFM tip.

Data analysis

Measure the unwinding DNA length from images collected from dried samples

1. Measure the height of ssDNA (200 ± 104 pm, Gaussian peak) and dsDNA (371 ± 116 pm, Gaussian peak) using cross-sectional analysis in the MFP-3D AFM imaging software to identify dsDNA and ssDNA product (Kaur et al., 2020) (Figure 10).

   Steps for measuring the height of the dsDNA:

   1. Open the Analyze Panel window and select Draw with Mode – Free Hand (refer to Figures 10 and 11).

   2. Trace lines perpendicular to the contour of the imaged DNA.

   3. Adjust one pointer towards the peak of the cross-section of the DNA and the second pointer to the mica surface.

   4. The dZ value indicates the height of the DNA being measured.

   
   

   
   Figure 10. Height measurement steps of a (A) DNA AFM image (B) with its cross-sectional analysis showing a height measured in the (C) analyze panel (dZ) (red square)




2. Measure the contour length of the unwound ssDNA, characterizing it with the height of the ssDNA and make a histogram to plot the average unwinding length. The average unwinding rate is the average DNA unwinding length divided by the sample incubation time before deposition onto the mica surface (Kaur et al., 2020) (Figure 11).

   Steps for measuring the contour length of any DNA:

   1. Open the Analyze Panel window and select Draw with Mode – Free Hand (refer to Figures 10 and 11).

   2. Trace lines overlapping the contour of the imaged DNA.

   3. Adjust one pointer to the starting point of the DNA and the second pointer to the end of the DNA in the cross-section.

   4. The dXY value gives the contour length of the DNA being measured.

   Note: Before measuring the length of the unwinded DNA, measure the height of the DNA to make sure it is ssDNA. The height of ssDNA is lower than the height of dsDNA, as mentioned above.

   
   

   
   Figure 11. Contour length measurement steps of a (A) traced DNA AFM image (B) with its cross-sectional analysis showing a height measured in the (C) analyze panel (dXY) (red square)

Recipes

1. Helicase Imaging Buffer (Kaur et al., 2020)

   20 mM HEPES (pH 7.5)

   100 mM NaCl

   7.5 mM MgCl₂

   7% Glycerol

   0.2% NP-40

   4.5 mM ATP

2. APS working solution (Shlyakhtenko et al., 2013)

   1. Prepare 50 mM APS solution by dissolving 58 mg in 5 ml DI water.

   2. Dilute the stock solution to a ratio of 1:300 to make a working solution.

   3. Seal the stock solution in an Eppendorf tube with parafilm and store at room temperature.

3. Buffer 3.1: 1× Buffer components: NEB

   100 mM NaCl

   50 mM Tris-HCl (pH 7.9)

   10 mM MgCl₂

   100 µg/ml BSA

Acknowledgments

This work was supported by the National Institutes of Health (R01GM107559 to H.W.), Grant P30ES025128 from the North Carolina State University Center for Human Health and the Environment Pilot Project (to P. K.), and Grant ES065078 from the Intramural Research Program of the NIEHS, National Institutes of Health (to W. C. C.). This protocol was adapted from Kaur P., Longley, M. J., Pan, H., Wang, W., Countryman, P., Wang, H. and Copeland, W. C. (2020). Single-molecule level structural dynamics of DNA unwinding by human mitochondrial Twinkle helicase. J Biol Chem 295(17): 5564-5576.

Competing interests

The authors declare no competing financial interests.

References

1. Bhatti, J. S., Bhatti, G. K. and Reddy, P. H. (2017). Mitochondrial dysfunction and oxidative stress in metabolic disorders - A step towards mitochondria based therapeutic strategies. Biochim Biophys Acta Mol Basis Dis 1863(5): 1066-1077.
2. Bogenhagen, D. F., Rousseau, D. and Burke, S. (2008). The layered structure of human mitochondrial DNA nucleoids. J Biol Chem 283(6): 3665-3675.
3. Countryman, P., Fan, Y., Gorthi, A., Pan, H., Strickland, J., Kaur, P., Wang, X., Lin, J., Lei, X., White, C., et al. (2018). Cohesin SA2 is a sequence-independent DNA-binding protein that recognizes DNA replication and repair intermediates. J Biol Chem 293(3):1054-1069.
4. Goffart, S., Cooper, H. M., Tyynismaa, H., Wanrooij, S., Suomalainen, A. and Spelbrink, J. N. (2009). Twinkle mutations associated with autosomal dominant progressive external ophthalmoplegia lead to impaired helicase function and in vivo mtDNA replication stalling. Hum Mol Genet 18(2): 328-340.
5. He, F. (2011). Bradford Protein Assay. Bio-101: e45.
6. Kaur, P., Longley, M. J., Pan, H., Wang, H., and Copeland, W. C. (2018) Single-molecule DREEM imaging reveals DNA wrapping around human mitochondrial single-stranded DNA binding protein. Nucleic Acids Res 46(21):11287-11302.
7. Kaur, P., Longley, M. J., Pan, H., Wang, W., Countryman, P., Wang, H. and Copeland, W. C. (2020). Single-molecule level structural dynamics of DNA unwinding by human mitochondrial Twinkle helicase. J Biol Chem 295(17): 5564-5576.
8. Korhonen, J. A., Pham, X. H., Pellegrini, M. and Falkenberg, M. (2004). Reconstitution of a minimal mtDNA replisome in vitro. EMBO J 23(12): 2423-2429.
9. Krieg, R. C., Dong, Y., Schwamborn, K. and Knuechel, R. (2005). Protein quantification and its tolerance for different interfering reagents using the BCA-method with regard to 2D SDS PAGE. J Biochem Biophys Methods 65(1): 13-19.
10. Longley, M. J., Humble, M. M., Sharief, F. S. and Copeland, W. C. (2010). Disease variants of the human mitochondrial DNA helicase encoded by C10orf2 differentially alter protein stability, nucleotide hydrolysis, and helicase activity. J Biol Chem 285(39): 29690-29702.
11. Longley, M. J., Smith, L. A. and Copeland, W. C. (2009) Preparation of human mitochondrial single-stranded DNA-binding protein. Methods Mol Biol 554:73-85.
12. Nečas, D. and Klapetek, P. (2012). Gwyddion: an open-source software for SPM data analysis. Cent Eur J Phys 10(1): 181-188.
13. Sen, D., Nandakumar, D., Tang, G. Q. and Patel, S. S. (2012). Human mitochondrial DNA helicase TWINKLE is both an unwinding and annealing helicase. J Biol Chem 287(18): 14545-14556.
14. Suomalainen, A. and Battersby, B. J. (2018). Mitochondrial diseases: the contribution of organelle stress responses to pathology. Nat Rev Mol Cell Biol 19(2): 77-92.
15. Tyynismaa, H., Mjosund, K. P., Wanrooij, S., Lappalainen, I., Ylikallio, E., Jalanko, A., Spelbrink, J. N., Paetau, A. and Suomalainen, A. (2005). Mutant mitochondrial helicase Twinkle causes multiple mtDNA deletions and a late-onset mitochondrial disease in mice. Proc Natl Acad Sci U S A 102(49): 17687-17692.
16. Ylikallio, E. and Suomalainen, A. (2012). Mechanisms of mitochondrial diseases. Ann Med 44(1): 41-59.