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May 2020
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Characterize the Interaction of the DNA Helicase PriA with the Stalled DNA Replication Fork Using Atomic Force Microscopy
用原子力显微镜描述DNA解旋酶PriA与停止的DNA复制叉的相互作用   

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Abstract

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. ssDNA-binding protein (SSB) is typically present at the abandoned forks, protecting the ssDNA from nucleases. Research that is based on the assays for junction dissociation, surface plasmon resonance, single-molecule FRET, and x-ray crystal structure has revealed the helicase activity of PriA, the SSB-PriA interaction, and structural information of PriA helicase. Here, we used Atomic Force Microscopy (AFM) to visualize the interaction between PriA and DNA substrates with or without SSB in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. The protocol describes the steps to obtain high-resolution AFM images and the details of data analysis and presentation.

Keywords: Atomic force microscopy (原子力显微镜), Protein-DNA interaction (蛋白质和核酸相互作用), DNA purification (DNA纯化), DNA replication (DNA复制), PriA helicase (PriA解旋酶), SSB protein (单链结合蛋白)

Background

When DNA replication encounters roadblocks or breakage, it needs to be repaired and restarted afterward (Kogoma, 1997; Cox et al., 2000; McGlynn and Lloyd, 2002; Gabbai and Marians, 2010; Michel et al., 2018). In bacteria, the DNA helicase PriA mediates this process by recognizing the abandoned DNA replication fork, facilitating the reassembly of the replisome and loading of the helicase DnaB (Wickner and Hurwitz, 1975; Zavitz and Marians, 1992; Sandler and Marians, 2000; Michel et al., 2004; Windgassen et al., 2018b). Studies based on the assays for junction dissociation, surface plasmon resonance, and single-molecule FRET have elaborated on the preference of PriA to the fork DNA structures based on the helicase activity and the protein-protein interaction of PriA with other proteins (Zavitz and Marians, 1992; Cadman and McGlynn, 2004; Bhattacharyya et al., 2014; Yu et al., 2016). The x-ray crystal structure described the structural mechanism of how PriA recognizes and processes the branched DNA replication forks (Bhattacharyya et al., 2014; Windgassen et al., 2018a; Windgassen et al., 2019). In this work, we applied Atomic Force Microscopy (AFM) to visualize the PriA-DNA complex topographically. The protocol describes how to use AFM to characterize the complexes based on the analyses of AFM images.


Materials and Reagents

  1. Amicon Ultra-0.5 ml centrifugal filters (Millipore-Sigma, UFC503008, pore size: 30 kDa NMWCO)

  2. Nonwoven cleanroom wipes: TX604 TechniCloth (TexWipe, catalog number: TX604)

  3. Petri dish (Fisher Scientific, catalog number: 08-757-100A)

  4. Standard disposal cuvette (Perfector Scientific, catalog number: 9002)

  5. Distilled deionized H2O (DDI H2O)

  6. pUC19 Vector (New England Biolabs, catalog number: N3041S)

  7. PCR primers (IDT, custom order)

    F364: 5’-GAGTTCTTGAAGTGGTGGCC-3’

    R364: 5’-GGTAACTGTCAGACCAAGTTTACTC-3’

    F480: 5’-GCGATTAAGTTGGGTAAC-3’

    R480: 5’-GTTCTTTCCTGCGTTATC-3’

  8. DreamTaq polymerase (Thermo Fisher Scientific, catalog number: EP0701)

  9. Deoxynucleotide (dNTP) Solution Mix (New England Biolabs, catalog number: N0447S)

  10. PCR purification kit (Qiagen, catalog number: 28104)

  11. Restriction endonuclease: DdeI (New England Biolabs, catalog number: R0175S)

  12. Restriction endonuclease: BspQI (New England Biolabs, catalog number: R0712S)

  13. CutSmart® Buffer (New England Biolabs, catalog number: B7204S)

  14. Oligonucleotide (IDT, custom order)

    O30: 5’-TCATCTGCGTATTGGGCGCTCTTCCGCTTCCTATCT-3’

    O31: 5’-TCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCATA-3’

    O32: 5’-GCTTATGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCTTGCGCAGCGAGTCAGTGAGATAGGAAGCGGAAGAGCGCCCAATACGCAGA-3’

    O33: 5’-CACTGACTCGCTGCGCAAGGCTAACAGCATCACACACATTAACAATTCTAACATCTGGGTTTTCATTCTTTGGGTTTCACTTTCTCCAC-3’

    O34: 5’-CTAACAGCATCACACACATTAACAATTCTAACATCTGGGTTTTCATTCTTTGGGTTTCACTTTCTCCACCACTGACTCGCTGCGCAAGG-3’

    O36: 5’-TACGTGTAGGAATTATATTAAAGAGAAAGTGAAACCCAAAGAATGAAAAAGAAGATGTTAGAATTGTAAGCGGTATCAGCTCACTCATA-3’

    O37: 5’-GCTTATGAGTGAGCTGATACCGC-3’

    O42: 5’-TCATGACTCGCTGCGCAAGGCTAACAGCATCACACACATTAACAATTCTAACATCTGGG TTTTCATTCTTTGGGTTTCACTTTCTCCAC-3’

    O43: 5’-CCTTGCGCAGCGAGTCA-3’

  15. T4 Polynucleotide Kinase (New England Biolabs, catalog number: M0201S)

  16. T4 DNA Ligase (Thermo Fisher Scientific, catalog number: 15224090)

  17. DL-Dithiothreitol (Sigma-Aldrich, catalog number: 43819-5G)

  18. EDTA (Thermo Fisher Scientific, catalog number: 15576028)

  19. Tris base (Sigma-Aldrich, catalog number: 10708976001)

  20. Phenol:Chloroform:Isoamyl Alcohol 25:24:1, Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA (Sigma-Aldrich, catalog number: P3803-100ML)

  21. Isopropanol (Fisher Scientific, catalog number: A426P-4)

  22. Ethanol (Decon Labs, catalog number: 2701)

  23. Sodium acetate buffer solution, pH 5.2±0.1 (25 °C), 3 M, 0.2 μm filtered (Sigma-Aldrich, catalog number: S7899-100ML)

  24. Acetic acid (ACROS Organics, catalog number: AC124040010)

  25. HCl (Sigma-Aldrich, catalog number: 258148-25ML)

  26. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266-100G)

  27. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888-500G)

  28. Muscovite Block Mica (AshevilleMica, catalog number: Grade-1)

  29. 1-(3-Aminopropyl) silatrane (APS) [synthesized as described in ref. (Shlyakhtenko et al., 2013)]

  30. TESPA-V2 AFM probe (Bruker AFM Probes, catalog number: TESPA-V2)

  31. Platinum coated calibration grid, 1 µm × 1 µm period (Bruker AFM Probes, catalog number: PG)

  32. 10× binding buffer (see Recipes)

Equipment

  1. Aquamax Water Purification System (APS Water Services corporation)

  2. PCR Thermal cycler (Eppendorf, catalog number: 5332-54318)

  3. Votexer (Glas-Col, catalog number: 099A PV6)

  4. HPLC (Shimadzu, catalog numbers: 228-34350-92; 228-35555-92; 228-39001-92; 228-39001-92; 228-39005-92)

  5. TSKgel DNA-STAT column: 4.6 mm I.D. × 10 cm, 5 μm (Tosoh, catalog number: 821962)

  6. ND-1000 NanoDrop Spectrophotometers (ThermoFisher Scientific, listing number: E112352)

  7. VWR 1400E Vacuum Oven (VWR, model number: 1400E)

  8. MultiMode 8, Nanoscope V system (Bruker, model number: MMAFM-2)

Software

  1. FemtoScan Online (Advanced Technologies Center, Moscow, Russia, http://www.nanoscopy.net/en/Femtoscan-V.shtm)

  2. Origin (OriginLab Corporation, Northampton, MA, USA, https://www.originlab.com/)

Procedure

  1. Prepare the DNA substrates

    The tail DNA substrate (T3 or T5) was assembled from a duplex-DNA segment with a sticky end (the 224 bp segment for T3, the 356 bp segment for T5) and a tail-DNA segment. The fork DNA substrate (F3 or F5) was assembled from two duplex-DNA segments with sticky ends (the 224 bp segment and the 356 bp segment) and a core fork segment.

    1. Assemble the T3 DNA substrate

      1. Prepare the duplex-DNA segment (224-bp duplex-DNA) for T3

        1. Run PCR reaction: Prepare the 364-bp PCR product for the 224-bp segment:

          Use plasmid pUC19 (1 ng/μl) along with the designed forward primer (F364, 25 μM) and the reverse primer (R364, 25 μM) and amplify the substrate DNA using PCR:

          1. Mix 680 μl of DDI H2O, 80 μl of Dream Taq buffer, 12 μl of F364, 12 μl of R364, 12 μl of dNTP solution mix, 2 μl pUC19, and 2 μl of Dream Taq (total volume 800 μl) in a 1.5 ml microfuge tube.

          2. Aliquot the mixture into the thin-walled PCR tubes (~100 μl for each tube) and place the reactions in a thermal cycler.

          3. Run the following program for 33 cycles after an initial denaturation for 2 min at 94 °C: 30 s denaturation at 94 °C, 30 s annealing at 62 °C, 70 s extension at 72 °C. Set a final extension at 72 °C for 10 min following the 33 cycles.

            Note: We usually take ~0.5 μl of the PCR product and run an agarose gel (~2%, w/v) to check the PCR reaction.

          4. Purification of the PCR product:

            Apply phenol-chloroform extraction or use Amicon Ultra-0.5 ml centrifugal filters.

            The phenol-chloroform extraction procedure:

            1. Collect the PCR product into several 1.5 ml microfuge tube, and keep the volume around 700 μl for each tube.

            2. Add an equal volume of Phenol:Chloroform:Isoamyl Alcohol 25:24:1 into each microfuge tube and vortex at ~2,000 rpm for 20 s, and then centrifuge at max speed for 5 min.

            3. Collect the upper phase accurately to a clean microfuge tube and keep the volume around 400 μl per tube.

            4. Add 0.1 volume of 3M potassium acetate, pH 5.2, and then add 2.5-3 volumes of ice-cold 95% EtOH to each tube.

            5. Mix thoroughly and then keep the tubes at -80 °C for 30 min.

            6. Place tubes in the centrifuge and orient it properly (we usually orient the tail of the cap out from the center). It will let you know the place on the bottom where the pellet is precipitated (it will be under the tail on the bottom of the tube, in this case). Spin them at max speed for 15 min in the cold room (4 °C).

            7. Remove supernatant with a pipette and add 300-500 μl of cold 70% EtOH. Orient tubes in the centrifuge in the exact same way as it was in the first spin. It will keep the pellet from sliding and give better attachment to the wall. Spin them at max speed for 5 min.

            8. Remove supernatant gently with a pipette, and dry the pellet completely by incubation at 37°C for 10-15 min. Dissolve the pellet before use.

        2. Cleave the PCR product with the restriction endonuclease (DdeI):

          Dissolve the purified PCR product into 180 μl of DDI H2O. Add 20 μl of the CutSmart® Buffer and 3 μl of the DdeI to the DNA in a 1.5 ml microfuge tube. Incubate the mixture at 37 °C overnight.

        3. Purify the restriction endonuclease product: run a 2% (w/v) agarose gel or apply HPLC.

          The HPLC purification procedure:

          1. Prepare buffer solutions:

            Buffer A: 30 mM Tris-HCl pH 9

            Buffer B: 30 mM Tris-HCl pH 9, 1.2 M NaCl

            Washing buffer: 40% Acetic acid

          2. Equilibrate the system as suggested by the manufacturer.

          3. Load the sample and run the separation method as listed below (Table 1):


            Table 1. Timetable for the purification of the 224-bp product

            Time (min) Module Event Value
            0.01 pumps B.Conc 20%
            0.01 pumps T.Flow 0.5 ml/min
            15 pumps B.Conc 40%
            20 pumps B.Conc 50%
            120 pumps B.Conc 80%
            150 pumps B.Conc 100%
            170 pumps B.Conc 20%
            174 pumps T.Flow 0.01 ml/min


          4. Collect the product:

            The retention time (RT) of the 224-bp product is at 95-97 min. During that time, we start to collect ~200 μl of the elution buffer (the dead volume of the tubing) when the OD goes above 100. Collect every two drops of the following elution solution into one microfuge tube until 1 min after the OD number goes below 10.

          5. Purify the product:

            The samples are collected in many tubes and in the high salt buffer. We need to desalt and concentrate the final product: Measure the OD value for each of the tubes, taking the first tube as background. Collect the tubes when the OD value is more than 20% of the high OD value. Load all the selected tubes into the Amicon Ultra-0.5 ml centrifugal filters (pore size: 30 kDa NMWCO, see Materials) and centrifuge. Determine the DNA concentration by the UV absorbance at 260 nm.

      2. Prepare the tail-DNA segment for T3

        1. Phosphorylate each oligonucleotide (O42, O43):

          Add the components to a 1.5 ml microfuge tube and mix well: 10 μl of the oligonucleotide (100 μM), 5 ul of the 10× T4 ligase buffer, 1 μl of the kinase, 34 μl of DDI H2O. Incubate it at 37 °C for 1 h.

        2. Anneal the oligonucleotides:

          Add the phosphorylated oligonucleotides (O42, O43) in equimolar ratio to a microtube and mix well. Put the tube in the boiled water bath (~100 °C) and incubate it in the water bath overnight, letting it cool down to room temperature (~20°C) gradually.

          Note: We usually run ~2 μl sample on an agarose gel (~2%, w/v) to check the annealing product.

      3. Ligate the duplex-DNA segment with the tail segment

        Add the components and the ligase into a microtube: the molar ratio of each segment is 1:1.4 (224 bp segment: tail segment). Incubate it at 16 °C overnight.

        Note: We usually run ~2 μl sample on an agarose gel (~2%, w/v) to check the ligation reaction.

      4. Purify the ligation product by HPLC. Collect the DNA and concentrate the solution using the Amicon Ultra-0.5 ml centrifugal filters.

      5. Determine the DNA concentration by measuring the absorbance of purified DNA at 260 nm.

    2. Assemble the T5 DNA substrate

      1. Prepare the duplex-DNA segment (356-bp duplex-DNA) for T5

        1. Run PCR reaction: Prepare the 480 bp PCR product for the 356-bp segment:

          Use plasmid pUC19 (1 ng/μl) along with the designed forward primer (F480, 25 μM) and the reverse primer (R480, 25 μM) and amplify the substrate DNA using PCR:

          1. Mix 680 μl of DDI H2O, 80 μl of Dream Taq buffer, 12 μl of F480, 12 μl of R480, 12 μl of dNTP solution mix, 2 μl pUC19, and 2 μl of Dream Taq (total volume 800 μl) in a 1.5 ml microfuge tube.

          2. Aliquot the mixture into the thin-walled PCR tubes. Place the tubes containing the reaction mixture into a thermal cycler.

          3. Run the following program for 33 cycles after an initial denaturation for 2 min at 94 °C: 30 s denaturation at 94 °C, 30 s annealing at 54 °C, 70 s extension at 72 °C. Set a final extension at 72 °C for 10 min following the 33 cycles.

            Note: We usually take ~0.5 μl of the PCR product and run an agarose gel (~2%, w/v) to check the PCR reaction.

          4. Purification of the PCR product: Apply phenol-chloroform extraction or use the Amicon Ultra-0.5 ml centrifugal filters.

            Follow the procedure described in 1.a.i.5).

        2. Cleave the PCR product with the restriction endonuclease (BspQI):

          Dissolve the purified PCR product into 180 μl of DDI H2O. Add 20 μl of the CutSmart® Buffer and 3 μl of the BspQI to the DNA in a 1.5 ml microfuge tube. Incubate the mixture at 50 °C overnight.

        3. Purify the restriction endonuclease reaction: run a 2% (w/v) agarose gel or apply HPLC:

          Follow the HPLC purification procedure as describe in 1.a.iii, but with the separation method below (Table 2):


          Table 2. Timetable for the purification of the 356-bp product

          Time (min) Module Event Value
          0.01 pumps B.Conc 20%
          0.01 pumps T.Flow 0.5 ml/min
          15 pumps B.Conc 60%
          120 pumps B.Conc 80%
          150 pumps B.Conc 100%
          170 pumps B.Conc 20%
          174 pumps T.Flow 0.01 ml/min
          The retention time (RT) of the 356-bp product is at ~75 min.


      2. Prepare the tail-DNA segment for T5

        1. Phosphorylate each oligonucleotide (O36, O37):

          Add the components to a 1.5 ml microfuge tube and mix well: 10 μl of the oligonucleotide (100 μM), 5 μl of the 10× T4 ligase buffer, 1 μl of the kinase, 34 μl of DDI H2O. Incubate it at 37 °C for 1 h.

        2. Anneal the oligonucleotides:

          Follow the procedure described in 1.b.ii.

      3. Ligate the duplex-DNA segment with the tail segment

        Add the components and the ligase into a microtube: the molar ratio of each segment is 1:1.4 (356 bp segment: tail segment). Incubate it at 16 °C overnight.

        Note: We usually run ~2 μl sample on an agarose gel (~2%, w/v) to check the ligation reaction.

      4. Purify the ligation product by HPLC. Collect the DNA and concentrate the solution using the Amicon Ultra-0.5 ml centrifugal filters.

      5. Determine the DNA concentration by measuring the absorbance of purified DNA at 260 nm.

    3. Assemble the F3 DNA substrate

      The duplex segments for the fork DNA are the 224-bp segment and the 356-bp segment used for the tail DNA substrate (shown in Figure 1A).



      Figure 1. The assembly process of the fork DNA substrates. A. The F3 DNA substrate: ligate two duplex-DNA segments with sticky ends (the 224-bp segment and the 356-bp segment) and a core fork segment (with a 3'-end, 69-nucleotide single-stranded region) together. B. The F5 DNA substrate: ligate two duplex-DNA segments with sticky ends (the same duplex-DNA segments used for F3) and a core fork segment (with a 5'-end, 69-nucleotide single-stranded region) together.


      1. Prepare the core segment for the F3 DNA

        1. Phosphorylate each oligonucleotide (O30, O31, O32, O33):

          Add the components to a 1.5 ml microfuge tube and mix well: 10 μl of the oligonucleotide (100 μM), 5 μl of the 10× T4 ligase buffer, 1 μl of the kinase, 34 μl of DDI H2O. Incubate it at 37 °C for 1 h.

        2. Anneal the oligonucleotides (core segment):

          For F3 fork substrate, add the phosphorylated oligonucleotides (O30, O31, O32, O33) in equimolar ratio to a microtube and mix well. Put the tube in the boiled water bath (~100 °C) and incubate it in the water bath overnight, letting it cool down to room temperature (~20 °C) gradually.

          Note: We usually run ~2 μl sample on an agarose gel (~2%, w/v) to check the annealing product.

      2. Ligate the duplex-DNA segments with the core segment

        Add the segments and the ligase into a microtube: the molar ratio of each segment is 1:1:1.2 (224-bp segment: 356-bp segment: core segment). Incubate it at 16 °C overnight.

        Note: We usually run ~2 μl sample on an agarose gel (~2%, w/v) to check the ligation reaction.

      3. Purify the ligation product by HPLC. Collect the DNA and concentrate the solution using the Amicon Ultra-0.5 ml centrifugal filters (pore size: 30 kDa NMWCO).

      4. Determine the DNA concentration by measuring the absorbance of the purified DNA at 260 nm.

    4. Assemble the F5 DNA substrate (see schematic in Figure 1B)

      1. Prepare the core segment for the F5 DNA

        1. Phosphorylate each oligonucleotide (O30, O31, O32, O34):Add the components to a 1.5 ml microfuge tube and mix well: 10 μl of the oligonucleotide (100 μM), 5 μl of the 10× T4 ligase buffer, 1 μl of the kinase, 34 μl of DDI H2O. Incubate it at 37 °C for 1 h.

        2. Anneal the oligonucleotides:

          Add the phosphorylated oligonucleotides (O30, O31, O32, O34) in equimolar ratio to a microtube and mix well. Follow the same procedure that was described in 3.a.ii.

          Note: We usually run ~2 μl sample on an agarose gel (~2%, w/v) to check the annealing product.

      2. Ligate the duplex-DNA segments with the core segment: Follow the same procedure that was described in 3.b.

      3. Purify the ligation product by HPLC. Collect the DNA and concentrate the solution using the Amicon Ultra-0.5 ml centrifugal filters (pore size: 30 kDa NMWCO).

      4. Determine the DNA concentration by measuring the absorbance of purified DNA at 260 nm.


  2. Functionalize the mica surface

    1. Prepare a 50 mM 1-(3-Aminopropyl) silatrane APS stock solution in DDI H2O as described (Shlyakhtenko et al., 2013). The stock solution can be kept for more than a year at 4 °C. Prepare 15 ml of the working APS (167 μM) from the APS stock.

    2. Cut 1 × 3 cm strips of mica from high-quality mica sheets. Check that the piece fits when placed diagonally in a cuvette. A schematic of the process to prepare APS functionalized mica for AFM imaging is shown in Stumme-Diers et al. (2019). Use a razor blade to cleave layers of the mica until both sides are freshly cleaved, and the piece should be thin (~0.1 mm). Immediately place the mica piece into the APS filled cuvette and incubate for 30 min.

    3. Rinse the mica piece under running DDI H2O droplets or slow fluid for ~10 s. Completely dry both sides of the APS-mica strip under the gentle argon flow.

      Note: Use the lock tweezer to take the mica piece. A non-woven cellulose and polyester wipe (recommended wipe detailed in Materials) can be used to aid in wicking water from the edge of the mica when drying; keep the mica piece perpendicular to the wipe to avoid damage to the functionalized mica surface.

    4. The APS-functionalized mica is ready to use. For storage, place the dry mica strip into a clean cuvette and keep it in a vacuum chamber. The functionalized mica can be stored in the vacuum chamber or in the argon atmosphere for at least a week.


  3. Prepare the protein-DNA complex

    1. Prepare the binding solution from the 10× binding buffer (see Recipes). Take 1 μl of the 10× binding buffer and make up the volume to 10 μl with DDI H2O. The binding solution contains 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, and 1 mM DTT.

    2. Prepare the Protein-DNA mixture

      1. Prepare the PriA-DNA complex. Mix 3.6 μl of the PriA monomer (molar concentration: 100 nM) with 1 μl of DNA substrate (molar concentration: 45 nM) in a molar ratio of 8:1. Add 5.4 μl of the binding buffer to the mixture and incubate at room temperature (~20 °C) for 10 min.

      2. Prepare the PriA-SSB-DNA complex. Mix 10 μl of the SSB tetramer (molar concentration: 50 nM) with 10 μl of the PriA monomer, and the mixture was kept on ice for 30 minutes before use. Take 3.6 μl of the protein-mixture and add it to the fork DNA substrate in a 1:2:4 (DNA substrates: SSB: PriA) molar ratio. Make up the volume to 10 µl with the binding buffer and incubate the mixture for 10 min at room temperature (~20 °C).

    3. After incubation, dilute the complex-solution to achieve lower DNA concentration (~2 nM), which is ready for deposition onto the APS functionalized mica.

      Note: Arrange the experiment to have the APS-mica ready-to-use before this step. Once the mixture is diluted, it should be immediately deposited onto the mica.


  4. Prepare the protein-DNA samples on the APS-mica

    1. Apply double-faced adhesive tape to several magnetic pucks and place them to the side.

    2. Cut the APS-mica substrate to 1 × 1 cm squares. Place these pieces in a clean petri dish and keep them covered.

    3. Prepare a dilution of the assembled protein-DNA complexes (keep the final DNA concentration at 1.0 to 2.0 nM) using the binding buffer.

    4. Deposit 10 μl of the diluted protein-DNA sample at the center of the APS-mica piece and incubate for two minutes. Gently rinse the sample with DDI H2O droplets for ~10 s to remove all buffer components.

    5. Dry the deposited sample under a light flow of clean argon gas with the help of a clean wipe. Attach the sample to the magnetic puck and store it in a vacuum cabinet filled with argon for at least 3 h before imaging.

      Note: Be careful not to touch the mica surface after sample deposition.


  5. AFM imaging

    Images were acquired using a MultiMode 8, Nanoscope V system (Bruker, Santa Barbara, CA) operated in tapping mode in the air on TESPA probes (320 kHz nominal frequency and a 42 N/m spring constant) from the same vendor.

    1. Mount the probe into the cantilever holder. Be sure that it is in firm contact with the end of the groove. Mount the cantilever holder onto the end of the scanner head.

    2. Mount the sample on the AFM stage.

      Note: Be careful not to contact the sample surface.

    3. Adjust the laser until the sum is at the maximum so that it is on the cantilever. Adjust the photodetector and set the vertical and lateral deflection values to near zero.

    4. Tune the cantilever. Click 'AutoTune' to find the cantilever's free-air resonant frequency and adjust the peak offset to ~3%.

    5. Set Initial Scan Parameters. Set the initial Scan Size to 1 µm, X and Y Offset to 0, and Scan Angle to 0. Set Integral Gain to 1.0, Proportional Gain to 5, and Scan Rate to 1 Hz. Click the engage button to begin the approach.

    6. Once approached, gradually optimize the Amplitude Setpoint until the surface of the sample is clearly seen. Once the scan parameters are optimized, increase the resolution to 1024 × 1024 pixels. Check to see if Trace and Retrace are tracking each other well during the scan. Click the capture button followed by the engage button to begin image acquisition.

    7. Flatten the image. Click 'Flatten' in the tool panel and save as a copy in the same folder.


    Notes:

  1. AFM calibration is accomplished with the use of a calibration reference and the instruction manual provided by the manufacturer (NanoScope Software Version 5, 004-210-000). According to this protocol, use the Platinum Coated Calibration Grid (PG), 1 µm × 1 µm period [100 nm Depth (± 10%)] as the calibration reference and follow the instructions in the manual for the instrument. For Z-axis calibration, by measuring the vertical features of the image using Depth analysis of the same software to correct the Z sensitivity parameter. To accomplish this, divide the actual depth of features (100 nm for PG) by the measured depth (indicated in Depth analysis by the Peak-to-Peak value), multiply this quotient by the Z sensitivity value in the Z Calibration dialog box, and replace the former Z sensitivity value with the new result.

  2. During auto-tuning of the cantilever, the resonance frequency and drive amplitude are automatically selected for the largest signal-to-noise ratio. In our AFM imaging system (MultiMode 8, Nanoscope V system), the drive amplitude is usually 5-15 mV and yields 200-250 mV free amplitude. If needed, the free amplitude (in volts) can be converted to the deflection of the cantilever (in nm) with the calibrated spring constant and inverse optical lever sensitivity of the cantilever, following the detailed instruction provided by the manufacturer (NanoScope Software Version 5, 004-210-000). For the TESPA probe, the nominal spring constant is 37 nN/nm and nominal sensitivity is 60 nm/V, thus nominally, the free amplitude (200-250 mV) is ~12-15 nm.

  3. The setpoint values were manually adjusted to the maximal stable values during scanning to maintain the high-resolution image and minimize the tip sweeping effect. The operational amplitude varies on different instruments and or tips. In our AFM imaging system (MultiMode 8, Nanoscope V system), the amplitude setpoint is 150 mV, corresponding to ~80% of the free amplitude of the cantilever.

Data analysis

The AFM images were analyzed using the FemtoScan Online software package (Advanced Technologies Center, Moscow, Russia). Graphs were made by Origin software (OriginLab Corporation, Northampton, MA, USA).

  1. Measure the contour length. Open the flattened image in FemtoScan, measure and record the contour length of the free DNA from one end to the other. For the internal length calibration, use the measurements to generate a histogram and fit it with a normal (Gaussian) distribution. To obtain the calibration factor, divide the peak center (Xc) by the substrate length into base pairs.

    Note: The calibration factor should be ~0.34.

  2. Measure the position of the protein (Figure 2). Start from the end of the shorter arm on the DNA substrates towards the center of the protein to obtain the position of the protein (arm length 1, shown in Figure 2). Keep recording from the center of the protein towards the other end of the DNA substrate to obtain the contour length of the DNA (arm length 1 and arm length 2 together, shown in Figure 2). Divide each arm length by the calculated calibration factor to obtain arm lengths in DNA base pairs. A histogram for the distribution of the protein can be made from the dataset of arm length 1 (Figure 2C).



    Figure 2. Measurement of the protein position. A. Representative AFM image (0.5 × 0.5 µm) of F5 with PriA. The Z-scale is 3 nm. Arrow points to bound PriA on the F5 DNA substrate. B. Zoomed-in image (0.25 × 0.25 µm) with the dotted line showing the contour length measurement. The position of each protein is measured from the end of the shorter arm to the center of the protein (dotted green line), and the total length of the protein-bound DNA substrate was measured continuously from the center of the protein to the end of the other arm (dotted blue line). C. Histogram for PriA position on F5 DNA using the data of the short arm length. The histogram was fitted by Gaussian with a single peak centered at 254 ± 23 bp (S.D.), and with a bin size of 20 bp.


    For the double-feature complexes in the SSB-PriA-DNA results, measure the height of each feature using the cross-section feature of the software as described below, and assign the taller one to SSB. For the length measurement, start from the end closer to the SSB, continuously measure and record towards the center of SSB and PriA, keep on recording until the end of the other side of the DNA substrate. Plot these values as a chart by setting the DNA length as clustered column and protein position as scatter (Figure 3).



    Figure 3. Measurement of the protein distribution of the complexes containing PriA and SSB proteins. A. Representative AFM image of the PriA+SSB+fork DNA (0.5 × 0.5 µm). The Z-scale is 3 nm. B. The zoomed-in image (0.25 × 0.25 µm) of the double-protein complex with the dotted line showing the contour length measurement. The green arrow directs to PriA in the complex, while the SSB position is directed by the blue arrow. C. Map of the proteins on the F3 DNA substrate with the SSB position corresponding to zero value. Blue squares indicate the position of SSB and the red dots point to the PriA position.


  3. Measure the volume of the protein. For each protein, collect two sets of height (H) and full width at half maximum values (D) from orthogonal cross-section measurements (Figure 4A). Apply the measurements to the formula as described in Gilmore et al. (2009): V = 3.14 × H/6 × (0.75 x D1 x D2 + H2). Plot these values as a histogram and fit the peak(s) with a normal (Gaussian) distribution to obtain the volume of the protein population.

    Note: In our AFM-imaging system (MultiMode 8, Nanoscope V system, E scanner), the accuracy of the vertical measurement is at the sub-nm level (Lyubchenko, 2011). The measured height of DNA in the air is usually around 0.6 to 0.8 nm as performed in our and other laboratories (Thomson et al., 1996; Hansma et al., 1996; Maeda et al., 1999; Pietrasanta et al., 1999; Ye et al., 2000; Kato et al., 2002; Shlyakhtenko et al., 2003), and it can be taken as a control for the height measurements of the protein-DNA complexes. Additionally, the measured thickness of the supported phospholipid bilayer is 4-5 nm (Lv et al., 2018), which is in perfect agreement with the bilayer measurements obtained by other methods.



    Figure 4. Measurement of the size of the protein. A. Representative AFM image of the double-protein complex (0.2 × 0.2 µm). Cross-section profiles (green and blue lines) of a protein produce the height distribution curves shown in (B-E): (B) and (C) are the plots for SSB, and (D) and (E) show the height distributions for PriA. From these curves, height (H) and width (D) values are used for calculating the protein volume.

Recipes

  1. 10× binding buffer

    100 mM Tris-HCl (pH 7.5)

    500 mM NaCl

    50 mM MgCl2

    10 mM DTT

Acknowledgments

The work was supported by the National Institutes of Health grants R01 GM118006 to YLL and R01 GM100156 to PRB and YLL. This protocol was adapted from a previously described work (Wang et al., 2020).

Competing interests

There are no competing interests to declare.

References

  1. Bhattacharyya, B., George, N. P., Thurmes, T. M., Zhou, R., Jani, N., Wessel, S. R., Sandler, S. J., Ha, T. and Keck, J. L. (2014). Structural mechanisms of PriA-mediated DNA replication restart. Proc Natl Acad Sci U S A 111(4): 1373-1378.
  2. Cadman, C. J. and McGlynn, P. (2004). PriA helicase and SSB interact physically and functionally. Nucleic Acids Res 32(21): 6378-6387.
  3. Cox, M. M., Goodman, M. F., Kreuzer, K. N., Sherratt, D. J., Sandler, S. J. and Marians, K. J. (2000). The importance of repairing stalled replication forks. Nature 404(6773): 37-41.
  4. Gabbai, C. B. and Marians, K. J. (2010). Recruitment to stalled replication forks of the PriA DNA helicase and replisome-loading activities is essential for survival. DNA Repair (Amst) 9(3): 202-209.
  5. Gilmore, J. L., Suzuki, Y., Tamulaitis, G., Siksnys, V., Takeyasu, K. and Lyubchenko, Y. L. (2009). Single-molecule dynamics of the DNA-EcoRII protein complexes revealed with high-speed atomic force microscopy. Biochemistry 48(44): 10492-10498.
  6. Hansma, H. G., Revenko, I., Kim, K. and Laney, D. E. (1996). Atomic force microscopy of long and short double-stranded, single-stranded and triple-stranded nucleic acids. Nucleic Acids Res 24(4): 713-720.
  7. Kato, M., McAllister, C. J., Hokabe, S., Shimizu, N. and Lyubchenko, Y. L. (2002). Structural heterogeneity of pyrimidine/purine-biased DNA sequence analyzed by atomic force microscopy. Eur J Biochem 269(15): 3632-3636.
  8. Kogoma, T. (1997). Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription. Microbiol Mol Biol Rev 61(2): 212-238.
  9. Lv, Z., Banerjee, S., Zagorski, K. and Lyubchenko, Y. L. (2018). Supported Lipid Bilayers for Atomic Force Microscopy Studies. Methods Mol Biol 1814: 129-143.
  10. Lyubchenko, Y. L. (2011). Preparation of DNA and nucleoprotein samples for AFM imaging. Micron 42(2): 196-206.
  11. Maeda, Y., Matsumoto, T. and Kawai, T. (1999). Observation of single-and double-stranded DNA using non-contact atomic force microscopy.App Sur Sci 140(3-4): 400-405.
  12. McGlynn, P. and Lloyd, R. G. (2002). Recombinational repair and restart of damaged replication forks. Nat Rev Mol Cell Biol 3(11): 859-870.
  13. Michel, B., Grompone, G., Flores, M. J. and Bidnenko, V. (2004). Multiple pathways process stalled replication forks. Proc Natl Acad Sci U S A 101(35): 12783-12788.
  14. Michel, B., Sinha, A. K. and Leach, D. R. F. (2018). Replication Fork Breakage and Restart in Escherichia coli. Microbiol Mol Biol Rev 82(3): e00013-18.
  15. Pietrasanta, L. I., Thrower, D., Hsieh, W., Rao, S., Stemmann, O., Lechner, J., Carbon, J. and Hansma, H. (1999). Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)-binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy. Proc Natl Acad Sci U S A 96(7): 3757-3762.
  16. Sandler, S. J. and Marians, K. J. (2000). Role of PriA in replication fork reactivation in Escherichia coli. J Bacteriol 182(1): 9-13.
  17. Shlyakhtenko, L. S., Gall, A. A. and Lyubchenko, Y. L. (2013). Mica functionalization for imaging of DNA and protein-DNA complexes with atomic force microscopy.Methods Mol Biol 931: 295-312.
  18. Shlyakhtenko, L. S., Gall, A. A., Filonov, A., Cerovac, Z., Lushnikov, A. and Lyubchenko, Y. L. (2003). Silatrane-based surface chemistry for immobilization of DNA, protein-DNA complexes and other biological materials. Ultramicroscopy 97(1-4): 279-287.
  19. Stumme-Diers, M. P., Stormberg, T., Sun, Z. and Lyubchenko, Y. L. (2019). Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging. J Vis Exp(143). doi: 10.3791/58820.
  20. Thomson, N. H., Kasas, S., Smith, B., Hansma, H. G. and Hansma, P. K. (1996). Reversible binding of DNA to mica for AFM imaging. Langmuir 12(24): 5905-5908.
  21. Wang, Y., Sun, Z., Bianco, P.R. and Lyubchenko, Y.L. (2020). Atomic force microscopy–based characterization of the interaction of PriA helicase with stalled DNA replication forks. J Biol Chem 295(18): 6043-6052.
  22. Wickner, S. and Hurwitz, J. (1975). Association of phiX174 DNA-dependent ATPase activity with an Escherichia coli protein, replication factor Y, required for in vitro synthesis of phiX174 DNA.Proc Natl Acad Sci U S A 72(9): 3342-3346.
  23. Windgassen, T. A., Leroux, M., Sandler, S. J. and Keck, J. L. (2019). Function of a strand-separation pin element in the PriA DNA replication restart helicase. J Biol Chem 294(8): 2801-2814.
  24. Windgassen, T. A., Leroux, M., Satyshur, K. A., Sandler, S. J. and Keck, J. L. (2018a). Structure-specific DNA replication-fork recognition directs helicase and replication restart activities of the PriA helicase. Proc Natl Acad Sci U S A 115(39): E9075-E9084.
  25. Windgassen, T. A., Wessel, S. R., Bhattacharyya, B. and Keck, J. L. (2018b). Mechanisms of bacterial DNA replication restart. Nucleic Acids Res 46(2): 504-519.
  26. Ye, J. Y., Umemura, K., Ishikawa, M. and Kuroda, R. (2000). Atomic force microscopy of DNA molecules stretched by spin-coating technique.Anal Biochem 281(1): 21-25.
  27. Yu, C., Tan, H. Y., Choi, M., Stanenas, A. J., Byrd, A. K., K, D. R., Cohan, C. S. and Bianco, P. R. (2016). SSB binds to the RecG and PriA helicases in vivo in the absence of DNA. Genes Cells 21(2): 163-184.
  28. Zavitz, K. H. and Marians, K. J. (1992). ATPase-deficient mutants of the Escherichia coli DNA replication protein PriA are capable of catalyzing the assembly of active primosomes. J Biol Chem 267(10): 6933-6940.

简介

[摘要]在细菌中,停滞的DNA复制叉的重新启动需要DN A解旋酶PriA 。PriA可以识别并重塑废弃的DNA复制叉,在3'到5'方向展开DNA,并促进解旋酶DnaB加载到DNA上以重新开始复制​​。ssDNA结合蛋白(SSB)通常存在于废弃的叉子上,从而保护ssDNA免受核酸酶的破坏。该研究是基于所述测定法离解结,表面等离振子共振,单分子FRET,和x射线晶体结构已经揭示的解旋酶活性PRIA ,SSB- PriA相互作用以及PriA解旋酶的结构信息。在这里,我们使用原子力显微镜(AFM)可视化了在不存在ATP的情况下在有或没有SSB的情况下PriA和带有或不带有SSB的DNA底物之间的相互作用,以描绘PriA在其ATP催化的DNA解链反应之前的底物识别模式。该协议描述了获取高分辨率AFM图像的步骤以及数据分析和表示的细节。

[背景]当DNA复制遇到障碍或断裂时,需要对其进行修复并随后重新启动(Kogoma,1997; Cox等,2000; McGlynn和Lloyd,2002;G abbai和Marians,2010; Michel等,2018)。 )。在细菌中,DNA解旋酶PRIA通过识别废弃DNA复制叉,从而便于重新组装的介导这一过程复制体的解旋酶和装载DNAB (Wickner和赫维茨,1975; Zavitz和Marians,1992; Sandler和Marians,2000;米歇尔等人,2004; Windgassen等人,2018b )。圣基于所述udies测定法结解离,表面等离振子共振,和单分子FRET已经阐述的偏好PRIA基于所述解旋酶的活性和蛋白质-蛋白质相互作用的叉DNA结构PRIA与其它蛋白质(Zavitz和Marians ,1992;Cadman和McGlynn ,2004; Bhattacharyya等,2014; Yu等,2016)。的x射线晶体结构描述的如何的结构机构PRIA识别和处理的分支DNA复制叉(巴氏等人,2014; Windgassen等人升。,2018A ; W indgassen 。等人,2019) 。在这项工作中,我们应用了原子力显微镜(AFM)在地形上可视化了PriA -DNA复合物。该协议描述了如何使用AFM基于对AFM图像的分析来表征复合物。

关键字:原子力显微镜, 蛋白质和核酸相互作用, DNA纯化, DNA复制, PriA解旋酶, 单链结合蛋白



材料和[R eagents


1. Amicon Ultra-0.5 ml离心过滤器(Millipore- S Sigma,UFC503008,孔径:30 kDa NMWCO)     

2.无纺布洁净室湿巾:TX604 TechniCloth (TexWipe ,目录号:TX604)     

3.培养皿(Fisher Scientific,目录号:08-757-100A)     

4.标准比色杯(Perfector Scientific,目录号:9002)     

5.蒸馏去离子d H 2 O(DDI H 2 O)     

6. pUC19载体(新英格兰生物实验室,目录号:N3041S)     

7. PCR引物(IDT,定制订单)     

F364:5'-GAGTTCTTGAAGTGGTGGCC-3'


R364:5'-GGTAACTGTCAGACCAAGTTTACTC-3'


F480:5'-GCGATTAAGTTGGGTAAC-3'


R480:5'-GTTCTTTCCTGCGTTATC-3'


8. DreamTaq聚合酶(Thermo Fisher Scientific,目录号:EP0701)     

9.脱氧核苷酸(dNTP )溶液混合物(New England Biolabs ,目录号:N0447S)     

10. PCR纯化试剂盒(Qiagen ,目录号:28104) 

11.限制性核酸内切酶:DdeI (新英格兰生物实验室,目录号:R0175S) 

12.限制性核酸内切酶:BspQI (新英格兰生物实验室,目录号:R0712S) 

13. CutSmart ®缓冲液(新英格兰生物实验室,目录号:B7204S) 

14.寡核苷酸(IDT,定制) 

O30:5'-TCATCTGCGTATTGGGCGCTCTTCCGCTTCCTATCT-3'


O31:5'-TCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCATA-3'


O32:5'-GCTTATGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCTTGCGCAGCGAGTCAGTGAGATAGGAAGGAGAGAAGAGCGCCCAATACGCAGA-3'


O33:5'-CACTGACTCGCTGCGCAAGGCTAACAGCATCACACACATTAACAATTCTAACATCTGGGTTTTCATTCTTTGGGTTTCACTTTCTCCAC-3'


O34:5'-CTAACAGCATCACACACATTAACAATTCTAACATCTGGGTTTTCATTCTTTGGGTTTCACTTTCTCCACCACTGACTCGCTGCGCAAGG-3'


O36:5'-TACGTGTAGGAATTATATTAAAGAGAAAGTGAAAAAACCAAAGAATGAAAAAGAAGATGTTAGAATTGTAAGCGGTATCAGCTCACTCATA-3'


O37:5'-GCTTATGAGTGAGCTGATACCGC-3'


O42:5'-TCATGACTCGCTGCGCAAGGCTAACAGCATCACACACATTAACAATTCTAACATCTGGG TTTTCATTCTTTGGGTTTCACTTTCTCCAC-3'


O43:5'-CCTTGCGCAGCGAGTCA-3'


15. T4多核苷酸e激酶(New England Biolabs ,目录号:M0201S) 

16. T4 DNA连接酶(Thermo Fisher Scientific,目录号:15224090) 

17. DL-二硫苏糖醇(Sigma-Aldrich公司,目录号:43819-5G) 

18. EDTA(Thermo Fisher Scientific,目录号:15576028) 

19. Tris base(Sigma-Aldrich,目录号:10708976001) 

20.苯酚:氯仿:异戊醇25:24:1,用10 mM Tris ,pH 8.0、1 mM EDTA饱和(Sigma-Aldrich,目录号:P3803-100ML) 

21.异丙醇(Fisher Scientific,目录号:A426P-4) 

22.乙醇(Decon Labs,目录号:2701) 

23.乙酸钠缓冲溶液,pH 5.2±0.1(25℃),3 M,0.2微米过滤(Sigma-Aldrich公司,目录号:S7899-100ML) 

24.乙酸(ACROS Organics,目录号:AC124040010) 

25. HCl (Sigma-Aldrich,目录号:258148-25ML) 

26.氯化镁(MgCl 2 )(Sigma-Aldrich,目录号:M8266-100G) 

27.钠Ç hloride (氯化钠)(SI GMA-Aldrich公司,目录号:S9888-500G) 

28.白云母块云母(AshevilleMica ,目录号:1级) 

29. 1-(3-氨基丙基)silatrane (APS)[如参考文献所述合成。(Shlyakhtenko等人,2013年)] 

30. TESPA-V2 AFM探针(Bruker AFM探针,目录号:TESPA-V2) 

31.镀铂校准栅,周期为1 µm × 1 µm(Bruker AFM探头,目录号:PG) 

32. 10 ×绑定缓冲区(请参阅食谱) 



设备


Aquamax水净化系统(APS水务公司)
PCR热循环仪(Eppendorf ,目录号:5332-54318)
Votexer (Glas -Col,目录号:099A PV6)
HPLC(Shimadzu,目录号s :228-34350-92; 228-35555-92; 228-39001-92; 228-39001-92; 228-39005-92)
TSKgel DNA-STAT色谱柱:4.6 mm ID × 10 cm,5μm (Tosoh,目录号:821962)
ND-1000 NanoDrop分光光度计(ThermoFisher Scientific,清单编号:E112352)
VWR 1400E真空烤箱(V W R,型号:1400E)
MultiMode 8,Nanoscope V系统(布鲁克,型号:MMAFM-2)


软件


Femto S can Online(俄罗斯莫斯科高级技术中心,http://www.nanoscopy.net/en/Femtoscan-V.shtm)
Origin(美国马萨诸塞州北安普顿市OriginLab Corporation,https://www.originlab.com/)


程序


准备DNA底物
尾部DNA底物(T3或T5)是从双链体DNA片段装配有粘性末端(224 bp的片段为T3,356 bp的片段为T5)和尾部-DNA片段。叉子DNA底物(F3或F5)由两个带有粘性末端的双链DNA片段(224 bp片段和356 bp片段)和一个核心叉子片段组装而成。


1.组装T3 DNA底物     

准备T3的双链体DNA片段(224 bp的双链体DNA)
运行PCR反应:为224-bp片段准备364-bp PCR产物:
使用的质粒pUC19(1纳克/微升与设计的正向沿)引物(F364,25 μM )和反向引物(R364,25 μM )和扩增用PCR的底物DNA:


1)混合680微升的DDIħ 2 O,80微升梦的Taq缓冲液,12微升的F364,12微升R364的,12微升的的dNTP溶液混合,2微升pUC19上,2微升梦的Taq (总体积800微升)放入1.5毫升微量离心管中。     

2)将混合物等分到薄壁PCR管中(每个管约100μl ),并将反应放入热循环仪中。     

3)在94°C初始变性2分钟后,运行以下程序33个循环:94°C变性30 s,62°C退火30 s,72°C延伸70 s。在33个循环后,在72°C下设置10分钟的最终延伸。     

注意:w ^ ë通常取0.5〜微升的PCR产物,并运行一个琼脂糖凝胶(〜2%,W / V)以检查PCR反应。


4)PCR产物的纯化:     

应用苯酚-氯仿萃取或使用Amicon Ultra-0.5 ml离心过滤器。


苯酚-氯仿萃取步骤:


一个)收集PCR产物分成几个1.5mL微管中,并保持约700体积微升每个管中。     

b)中加入等体积的苯酚:氯仿:异戊在〜2 1到每个微量离心管和涡流:酒精25:24 ,000的转速˚F或20秒,然后离心以最大速度5分钟。                   

c)中准确地收集上层相到一个干净的微量离心管中,并保持约400体积微升每管。     

d)向每个试管中添加0.1体积的3M乙酸钾,pH 5.2,然后添加2.5-3体积的冰冷的95%乙醇。     

e)充分混合,然后将试管在-80 °C下放置30分钟。     

f)将试管放入离心机中并正确定位(我们通常将瓶盖的尾部从中心定位)。它会让您知道沉淀物沉淀在底部的位置(在这种情况下,它将位于试管底部的尾部下方)。在冷藏室(4 °C)中以最大速度旋转15分钟。     

克)去除上清液用移液管并加入300-500微升冷的70%的乙醇。以与第一次旋转完全相同的方式对离心机中的试管进行定向。它将防止颗粒滑动,并更好地附着在壁上。以最大速度旋转它们5分钟。     

h)用移液管轻轻除去上清液,并通过在37°C下孵育10-15分钟将沉淀完全干燥。使用前溶解沉淀。   

用限制性核酸内切酶(DdeI )切割PCR产物:
溶解纯化的PCR产物为180微升DDI H的2 O.加入20微升所述的CutSmart ®缓冲液和3微升的的DdeI到DNA在1.5ml微离心管中。将混合物在37 °C下孵育过夜。


纯化限制性核酸内切酶产物:运行2%(w / v)琼脂糖凝胶或应用HPLC。
HPLC纯化程序:


1)准备缓冲溶液:     

缓冲液A:30 mM Tris-HCl pH 9


缓冲液B:30 mM Tris-HCl pH 9,1.2 M NaCl


洗涤缓冲液:40%乙酸


2)按照制造商的建议平衡系统。     

3)加载样品并运行下面列出的分离方法(表1):     



表1.纯化224bp产品的时间表


时间(分钟)


中号odule


Ë发泄


V ALUE


0.01


水泵





20%


0.01


水泵


流量


0.5毫升/分钟


15


水泵





40%


20


水泵





50%


120


水泵





80%


150


水泵





100%


170


水泵





20%


174


水泵


流量


0.01毫升/分钟


4)收集产品:     

的224-bp的产物的保留时间(RT)是在95 - 97分钟。在此期间,当OD值超过100时,我们开始收集约200μl的洗脱缓冲液(管道的死体积)。将每两滴以下洗脱溶液收集到一个微量离心管中,直到OD数后1分钟低于10


5)纯化产品:     

样品收集在许多试管中和高盐缓冲液中。我们需要脱盐并浓缩最终产品:以第一个试管为背景,测量每个试管的OD值。当OD值大于高OD值的20%时,收集试管。将所有选定的试管装入Amicon Ultra-0.5 ml离心过滤器(孔径:30 kDa NMWCO,请参见材料)并离心。通过260 nm处的UV吸光度确定DNA浓度。


准备T3的尾巴DNA片段
磷酸化每个寡核苷酸(O42,O43):
组分添加到1.5ml微量离心管中并充分混合:10微升的寡核苷酸(100 μM ),5微升的10 × T4连接酶缓冲液,1 μ升激酶,34微升DDI H的2 O.孵育它在37°C下放置1小时。


退火寡核苷酸:
将等摩尔比例的磷酸化寡核苷酸(O4 2,O43)加入微管中并充分混合。将试管放入开水浴(〜100°C)中,并在水浴中孵育过夜,使其逐渐冷却至室温(〜20°C)。


注意:w ^ ë通常运行约2微升上的样品的琼脂糖凝胶(〜2%,W / V)吨O检查退火产物。


用尾巴片段连接双链DNA片段
将组分和连接酶添加到微管中:每个片段的摩尔比为1:1.4(224 bp片段:尾部片段)。在16°C下孵育过夜。


注意:w ^ ë通常运行约2微升上的样品的琼脂糖凝胶(〜2%,W / V)以检查连接反应。


通过HPLC纯化连接产物。收集DNA并使用Amicon Ultra-0.5 ml离心过滤器浓缩溶液。
通过测量260 nm处纯化DNA的吸光度来确定DNA浓度。
2.组装T5 DNA底物     

准备T5的双链DNA片段(356-bp双链DNA)
运行PCR反应:准备356 bp片段的480 bp PCR产物:
使用的质粒pUC19(1纳克/微升与设计的正向沿)引物(F480,25 μM )和反向引物(R480,25 μM )和扩增用PCR的底物DNA:


1)混合680微升的DDIħ 2 O,80微升梦的Taq缓冲液,12微升的F480,12微升R480的,12微升的的dNTP溶液混合,2微升pUC19上,2微升梦的Taq (总体积800微升)放入1.5毫升微量离心管中。     

2)将混合物等分到薄壁PCR管中。将装有反应混合物的试管放入热循环仪中。     

3)在94°C初始变性2分钟后,运行以下程序33个循环:94°C变性30 s,54°C退火30 s,72°C延伸70 s。在33个循环后,在72°C下设置10分钟的最终延伸。     

注意:w ^ ë通常取0.5〜微升的PCR产物,并运行一个琼脂糖凝胶(〜2%,W / V)以检查PCR反应。


4)PCR产物的纯化:应用苯酚-氯仿萃取或使用Amicon Ultra-0.5 ml离心过滤器。     

请按照1中描述的步骤进行操作。ai5)。


用限制性核酸内切酶(BspQI )切割PCR产物:
溶解纯化的PCR产物为180微升DDI H的2 O. 20添加微升所述的CutSmart ®缓冲液和3微升的的BspQI到DNA在1.5ml微离心管中。将混合物在50 °C下孵育过夜。


纯化限制性核酸内切酶反应:运行2%(w / v)琼脂糖凝胶或应用HPLC:
遵循1.a.iii中所述的HPLC纯化程序,但采用以下分离方法(表2):


表2.纯化356bp产品的时间表


时间(分钟)


中号odule


Ë发泄


V ALUE


0.01


水泵





20%


0.01


水泵


流量


0.5毫升/分钟


15


水泵





60%


120


水泵





80%


150


水泵





100%


170


水泵





20%


174


水泵


流量


0.01毫升/分钟


356-bp产品的保留时间(RT)为〜75分钟。


准备T5的尾巴DNA片段
磷酸化每个寡核苷酸(O36,O37):
组分添加到1.5 ml间icrofuge管拌匀:10微升的寡核苷酸(100 μM ),5 μ升的10 × T4连接酶缓冲液,1 μ升激酶,34微升DDI H的2 O.在37 °C下孵育1小时。


退火寡核苷酸:
请按照1.b.ii中所述的步骤进行操作。


用尾巴片段连接双链DNA片段
将组分和连接酶添加到微管中:每个片段的摩尔比为1:1.4(356 bp片段:尾部片段)。在16 °C下孵育过夜。


注意:w ^ ë通常运行约2微升上的样品的琼脂糖凝胶(〜2%,W / V)到检查连接反应。


通过HPLC纯化连接产物。收集DNA并使用Amicon Ultra-0.5 ml离心过滤器浓缩溶液。
通过测量260 nm处纯化DNA的吸光度来确定DNA浓度。
3.组装F3 DNA底物     

叉状DNA的双链体区段是用于尾部DNA底物的224-bp区段和356-bp区段(如图1A所示)。






图1.叉子DNA底物的组装过程。A.将F3 DNA底物:用粘性末端结扎两种双工-DNA片段(224碱基对片段和356-bp的片段)和核心叉段(具有3'端,69个核苷酸的单链区域)一起。B.该F5 DNA底物:用粘性末端(用于F3相同的双工-DNA片段)和核心叉段(具有5'-末端,69个核苷酸的单链区域)一起结扎两种双工-DNA片段。


准备F3 DNA的核心片段
磷酸化每个寡核苷酸(O30,O31,O32,O33):
组分添加到1.5ml微量离心管中并充分混合:10微升的寡核苷酸(100 μM ),5 μ升的10 × T4连接酶缓冲液,1 μ升激酶,34微升DDI H的2 O.孵育将其在37 °C下放置1小时。


退火寡核苷酸(核心片段):
对于F3叉底物,将等摩尔比例的磷酸化寡核苷酸(O30,O31,O32,O33 )添加到微管中并充分混合。将试管放入开水浴(〜100°C)中,并在水浴中孵育过夜,使其逐渐冷却至室温(〜20 °C)。


注意:w ^ ë通常运行约2微升上的样品的琼脂糖凝胶(〜2%,W / V)以检查退火产物。


用核心片段连接双链DNA片段
将片段和连接酶添加到微管中:每个片段的摩尔比为1:1:1.2(224-bp片段:356-bp片段:核心片段)。在16°C下孵育过夜。


注意:w ^ ë通常运行约2微升上的样品的琼脂糖凝胶(〜2%,W / V)以检查连接反应。


通过HPLC纯化连接产物。收集DNA并使用Amicon Ultra-0.5 ml离心过滤器(孔径:30 kDa NMWCO)浓缩溶液。
通过测量纯化的DNA在260 nm处的吸光度来确定DNA浓度。
4.组装F5 DNA底物(请参见图1B中的示意图)     

准备F5 DNA的核心片段
磷酸化每个寡核苷酸(O30,O31,O32,O34):
将组分添加到1.5 ml微量离心管中并充分混合:10μ升的寡核苷酸(100 μM ),5 μ升的10 × T4连接酶缓冲液,1 μ升激酶,34 μ升DDI H的2 O.孵育它在37 ℃下1个小时。
退火寡核苷酸:
将等摩尔比例的磷酸化寡核苷酸(O30,O31,O32 ,O34 )添加到微管中并充分混合。遵循3.a.ii中描述的相同步骤。


注意:w ^ ë通常运行约2微升上的样品的琼脂糖凝胶(〜2%,W / V)到检查退火产物。


将双链DNA片段与核心片段连接:遵循3.b中描述的相同步骤。
通过HPLC纯化连接产物。收集DNA并使用Amicon Ultra-0.5 ml离心过滤器(孔径:30 kDa NMWCO)浓缩溶液。
通过测量260 nm处纯化DNA的吸光度来确定DNA浓度。


使云母表面功能化
如所述(Shlyakhtenko et al。,2013),在DDI H 2 O中制备50 mM 1-(3-氨基丙基)甲硅烷基APS储备溶液。储备溶液可以在4 °C下保存一年以上。从APS储备液中准备15 ml的有效APS(167μM )。
从优质云母片上切下1 × 3厘米的云母带。对角放置在比色杯中时,检查其是否合适。A S电气原理的过程以制备APS的官能云母用于AFM成像在所示Stumme-DIERS等。(2019)。用刀片将云母层劈开,直到两面都被新劈开为止,并且切片应该很薄(〜0.1毫米)。立即将云母片放入APS填充的比色杯中,孵育30分钟。
在运行的DDI H 2 O oplet或慢速液体下冲洗云母片约10 s 。在柔和的氩气气流下,将APS云母带的两面完全干燥。
注:ü瑟锁钳取云母片。非织造的纤维素和聚酯擦拭布(推荐的擦拭材料在材料中详细介绍)可用于在干燥时帮助从云母边缘吸水。使云母片垂直于抹布,以免损坏功能化的云母表面。


APS功能化的云母已准备就绪。为了存储,将干燥的云母带放入干净的比色杯中,并将其保存在真空室内。功能化的云母可以在真空室或氩气气氛中保存至少一周。


准备蛋白质-DNA复合物
从制备的结合溶液中的10 ×宾迪纳克缓冲液(见配方)。取1μl的10 ×结合缓冲液,并用DDI H 2 O补足至10μl 。结合溶液包含10 mM Tris-HCl (pH 7.5),50 mM NaCl ,5 mM MgCl 2和1 mM DTT 。
准备蛋白质-DNA混合物
准备PriA -DNA复合体。混合3.6微升的的PRIA monom ER(摩尔浓度:100纳米)以1微升:DNA底物(45摩尔浓度nM的在8:1的摩尔比):1。添加5.4微升的结合缓冲液在室温下(〜20的混合物,并培育℃)10分钟。
准备PriA -SSB-DNA复合体。将10μl的SSB四聚体(摩尔浓度:50 nM )与10μl的PriA单体混合,并在使用前将混合物在冰上放置30分钟。取3.6μl的蛋白质混合物,以1:2:4(DNA底物:SSB:PriA )的摩尔比将其添加到叉子DNA底物中。用结合缓冲液补足体积至10 µl,并将混合物在室温(〜20 °C)下孵育10分钟。
孵育后,稀释复合物溶液以降低DNA浓度(〜2 nM ),可将其沉积到APS功能化的云母上。
注意:在执行此步骤之前,请安排实验以准备使用APS-mica。混合物稀释后,应立即沉积在云母上。


在APS云母上制备蛋白质DNA样品
将双面胶带粘贴到多个磁性圆盘上,然后将其放在一边。
将APS云母基板切成1 × 1 cm的正方形。将这些碎片在一个干净的培养皿中,并保持它们覆盖。
使用结合缓冲液制备稀释的组装蛋白-DNA复合物(使最终DNA浓度保持在1.0至2.0 nM )。 
沉积10微升经稀释的蛋白质的DNA样品在APS-云母片孵育两分钟的中心。用DDI H 2 O小滴轻轻冲洗样品约10 s,以除去所有缓冲液成分。
在干净的氩气的作用下,借助干净的抹布将沉积的样品干燥。将样品附着到磁性圆盘上,并在成像前将其保存在装有氩气的真空柜中至少3 h。
注意:乙ë小心不要样品沉积后接触到云母表面。


原子力显微镜成像
使用MultiMode 8,Nanoscope V系统(布鲁克,加利福尼亚州圣巴巴拉)在同一供应商的TESPA探头(标称频率为320 kHz,弹簧常数为42 N / m)上以轻敲模式在空中操作获取图像。


将探头安装到悬臂支架中。确保它与凹槽的末端牢固接触。将悬臂支架安装到扫描仪头的末端。
将样品安装在AFM载物台上。
注:乙Ë小心不要接触样品表面。


调整激光,直到总和最大,使其位于悬臂上。调整光电探测器并将垂直和水平偏转值设置为接近零。
调整悬臂。单击“自动调谐”以找到悬臂的自由空气共振频率,并将峰值偏移调整为〜3%。
设置初始扫描参数。将初始扫描大小设置为1 µm,X和Y偏移设置为0,扫描角度设置为0。将积分增益设置为1.0,将比例增益设置为5,将扫描速率设置为1 Hz。单击“参与”按钮开始该方法。
接近后,逐渐优化振幅设定点,直到清晰可见样品表面为止。优化扫描参数后,将分辨率提高到1024 × 1024像素。检查在扫描过程中Trace和Retrace是否互相跟踪良好。单击捕获按钮,然后单击接合按钮以开始图像获取。
展平图像。氯ICK“拼合”在工具面板,并保存在同一文件夹中的副本。


注意:


使用制造商提供的校准参考和说明手册(NanoScope软件版本5,004-210-000)可以完成AFM校准。根据此协议,请使用铂金涂层校准栅(PG),周期为1 µm × 1 µm [深度100 nm( ± 10%)]作为校准参考,并按照仪器手册中的说明进行操作。对于Z轴校准,通过使用同一软件的深度分析测量图像的垂直特征来校正Z灵敏度参数。为此,将特征的实际深度(PG为100 nm)除以测得的深度(深度分析中用“峰峰值”值表示),然后将该商乘以“ Z校准”对话框中的Z灵敏度值,并用新结果替换以前的Z灵敏度值。
在CA的自动调谐ntilever ,所述索纳Ñ CE频率和驱动振幅被自动选择为最大的小号我GN人到NOI小号É比率。在我们的AFM成像系统(MultiMode 8,Nanoscope V系统)中,驱动幅度通常为5-15 mV,并产生200-250 mV的自由幅度。如果需要,自由振幅(在伏)可以是CONVER泰德到悬臂(以nm)的偏转与所述校准弹簧常数和逆光杠杆灵敏度悬臂的,由生产商提供的详细指令之后(的NanoScope软件版本5,004-210-000)。对于TESPA探针,所述标称弹簧常数是37 Ñ ñ / Ñ米和标称灵敏度为60nm / V ,因此名义上,自由振幅(200-250毫伏)是〜12-15纳米。
的设定点值进行手动扫描,以保持所述高分辨率图像,并尽量减少尖端清扫效果期间调整到最大的稳定值。操作幅度在不同的仪器和/或吸头上会有所不同。在我们的原子力显微镜成像系统(多模式8,的Nanoscope V系),振幅设定值是150毫伏,对应于〜8 0 %的悬臂的自由振幅的。




数据分析


使用FemtoScan Online软件包(俄罗斯莫斯科高级技术中心)对AFM图像进行了分析。图是由Origin软件(美国马萨诸塞州北安普敦的OriginLab公司)制作的。


测量轮廓长度。在FemtoScan中打开展平的图像,测量并记录游离DNA从一端到另一端的轮廓长度。对于内部长度校准,请使用测量值生成直方图,并将其与正态(高斯)分布拟合。为了获得校准因子,将峰中心(Xc )除以底物长度分成碱基对。
注:牛逼他标定系数应为0.34〜。


测量蛋白质的位置(图2)。从DNA底物上较短臂的末端开始,朝着蛋白质的中心开始,以获取蛋白质的位置(臂长1,如图2所示)。保持从蛋白质中心向DNA底物另一端的记录,以获取DNA的轮廓长度(臂长1和臂长2在一起,如图2所示)。将每个臂长除以计算出的校准因子,即可获得DNA碱基对中的臂长。可以从臂长1的数据集中制作蛋白质分布的直方图(图2C)。






图2.蛋白质位置的测量。A. ř具有代表性AFM图像(0.5 ×与F5为0.5μm)PRIA 。Z标度为3 nm。箭头指向F5 DNA底物上结合的PriA 。B.放大图像(0.25 × 0.25 µm),虚线显示轮廓长度测量值。从较短的臂的末端到蛋白质的中心(绿色虚线)测量每种蛋白质的位置,并从蛋白质的中心一直到蛋白质的末端连续测量与蛋白质结合的DNA底物的总长度。另一只手臂(蓝色虚线)。C.使用短臂长度的数据的PriA在F5 DNA上的位置的直方图。直方图由高斯拟合,其中心位于254±23 bp (SD)的单个峰,bin大小为20 bp。


对于SSB-PriA -DNA结果中的双重特征复合物,请使用软件的横截面特征按如下所述测量每个特征的高度,然后将较高的一个分配给SSB。对于长度测量,请从更靠近SSB的一端开始,连续测量并向SSB和PriA的中心记录,继续记录直到DNA底物另一侧的末端。通过将DNA长度设置为聚簇柱并将蛋白质位置设置为散点图,将这些值绘制为图表(图3)。






图3.含有PriA和SSB蛋白的复合物的蛋白质分布测量。A. PriA + SSB +叉子DNA(0.5 × 0.5 µm)的代表性AFM图像。Z标度为3 nm。B.双蛋白复合物的放大图像(0.25 × 0.25 µm),虚线表示轮廓长度测量值。绿色箭头指向综合体中的PriA ,而SSB位置则由蓝色箭头指示。C. S3位置对应于零值的F3 DNA底物上的蛋白质图。蓝色方块表示SSB的位置,红色点表示PriA的位置。


测量蛋白质的体积。对于每种蛋白质,从正交截面测量(图4 A )中收集两组高度(H)和半峰全宽(D)的全宽。应用测量结果,以式中吉尔摩描述等。(2009):V = 3.14 × H / 6 × (0.75×D 1 ×D 2 + H 2 )。将这些值绘制为直方图,并以正态(高斯)分布拟合峰,从而获得蛋白质群体的体积。
注意:在我们的AFM成像系统(MultiMode 8,Nanoscope V系统,E扫描仪)中,垂直测量的精度为亚纳米级(Lyubchenko ,2011年)。如我们实验室和其他实验室所进行的那样,空气中DNA的测量高度通常约为0.6至0.8 nm(Thomson等,1996; Hansma等,1996; Maeda等,1999; Pietrasanta等,1999 ;Ye等人,2000 ;Kato等人,2002;Shlyakhtenko等人,2003 ),并且可以将其用作蛋白质-DNA复合物的高度测量的对照。此外,负载的磷脂双层的测量厚度为4-5 nm(Lv等人,2018 ),这与通过其他方法获得的双层测量值完全吻合。






图4.蛋白质大小的测量。A.双蛋白复合物(0.2 × 0.2 µm)的代表性AFM图像。蛋白质的横截面轮廓(绿线和蓝线)产生(BE)中所示的高度分布曲线:(B)和(C)是SSB的图,而(D)和(E)显示了SSB的高度分布。PRIA 。根据这些曲线,将高度(H)和宽度(D)值用于计算蛋白质体积。


菜谱


10 ×结合缓冲液
100 mM的Tris-HCl (pH 7.5)


500毫米氯化钠


50毫米MgCl 2


10毫米DTT


致谢


这项工作得到了美国国立卫生研究院(NIH)的资助,其中R01 GM118006授予YLL,R01 GM100156授予PRB和YLL。


利益争夺


没有要宣布的利益冲突。






参考


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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Wang, Y., Sun, Z., Bianco, P. R. and Lyubchenko, Y. L. (2021). Characterize the Interaction of the DNA Helicase PriA with the Stalled DNA Replication Fork Using Atomic Force Microscopy. Bio-protocol 11(5): e3940. DOI: 10.21769/BioProtoc.3940.
  2. Wang, Y., Sun, Z., Bianco, P.R. and Lyubchenko, Y.L. (2020). Atomic force microscopy–based characterization of the interaction of PriA helicase with stalled DNA replication forks. J Biol Chem 295(18): 6043-6052.
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