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Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing
采用DNA梳理技术对芽殖酵母和人类细胞中DNA复制动力学进行单分子分析    

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Abstract

The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical and NGS-based methods, DNA combing provides unique information on cell-to-cell variations in DNA replication profiles, including initiation and elongation. Finally, this assay can be used to monitor the effect of DNA lesions on fork progression, arrest and restart.

Keywords: Replication(复制), Yeast(酵母), Human cells(人类细胞), Fork speed(分叉速度), Replication origin(复制起始), DNA stretching(DNA延伸)

Background

DNA synthesis is initiated at thousands of sites on eukaryotic chromosomes called replication origins. Origin activation follows a well-defined replication timing program that is controlled by checkpoint kinases and epigenetic modifications of chromatin (Prioleau and MacAlpine, 2016). Replication forks frequently stall during a normal S phase. Fork arrest is caused by multiple events, such as DNA lesions, tightly bound protein complexes, and transcription at highly expressed genes (Tourriere and Pasero 2007; Zeman and Cimprich, 2013). Eukaryotes have developed different strategies to deal with this replication stress, including repair mechanisms to restart arrested forks and activation of dormant replication origins to rescue terminally-arrested forks.

DNA combing is a method of choice to monitor different aspects of replication (fork speed, origin usage, fork restart, sister fork asymmetry). Unlike other DNA fiber methods such as DNA fiber spreading, the stretching, density and alignment of DNA molecules are highly reproducible and tightly controlled in the DNA combing method. Stretching is imposed by the force exerted by a receding air/water interface, independently of the length of DNA fibers (Bensimon et al., 1994; Michalet et al., 1997). Origin firing and progression of replication forks are followed after incorporation of thymidine analogs, such as 5-bromo-2’-deoxyuridine (BrdU), 5-iodo-2’-deoxyuridine (IdU) and 5-chloro-2’-deoxyuridine (CldU) in newly-synthesized DNA. This technique has been successfully used to monitor DNA replication dynamics in a variety of organisms, including bacteria, yeast, Drosophila, Xenopus and mammals.

Here, we provide detailed protocols to analyze newly synthesized DNA fibers in budding yeast and in human cells and to investigate various aspects of DNA replication in normal growth conditions and under replicative stress.

Materials and Reagents

  1. Common to human/yeast cells
    1. Tape
    2. 14 ml round-bottom polypropylene tubes (Corning, Falcon®, catalog number: 352059 )
    3. Tips 1 ml, 200 µl, 20 µl
    4. Silanized coverslips (Genomic Vision, catalog number: COV-001 ) purchased from Genomic Vision or prepared as described (Labit et al., 2008)
    5. 2 ml Teflon reservoir (Reservoir MCS Support [x 2]; from Genomic Vision)
    6. Whatman paper
    7. Microscope slides SuperFrost (VWR, catalog number: 630-1987 )
    8. Cyanoacrylate glue
    9. Diamond tip engraving pen (Sigma-Aldrich, catalog number: Z225568-1EA )
    10. Saran plastic film (Dominique Dutscher, catalog number: 090264 )
    11. Coplin Jar
    12. EDTA (Sigma-Aldrich, catalog number: E6758 )
    13. LMP agarose (Bio-Rad Laboratories, catalog number: 161-3111 )
    14. Plug mold (Bio-Rad Laboratories, catalog number: 170-3713 )
    15. Proteinase K (Sigma-Aldrich, catalog number: P6556 )
    16. 10x PBS (Sigma-Aldrich, catalog number: D1408 )
    17. YOYO-1 (Thermo Fisher Scientific, InvitrogenTM, catalog number: Y3601 )
    18. Sodium chloride (NaCl) (VWR, catalog number: 27810-295 )
    19. β-agarase (New England Biolabs, catalog number: M0392L )
    20. Sodium hydroxide (NaOH) (Merck, catalog number: 1.06462.1000 )
    21. BSA fraction V (Sigma-Aldrich, catalog number: A9647 )
    22. Prolong Gold Antifade reagent (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36930 )
    23. BrdU (Sigma-Aldrich, catalog number: B5002 )
    24. IdU (Sigma-Aldrich, catalog number: I7125 )
    25. CldU (MP Biomedicals, catalog number: 0 2105478 )
    26. DMSO (Sigma-Aldrich, catalog number: D2650 )
    27. Hydroxyurea (Sigma-Aldrich, catalog number: H8627 )
    28. N-laurylsarcosine sodium salt (Sigma-Aldrich, catalog number: L9150 )
    29. MES hydrate (Sigma-Aldrich, catalog number: M2933 )
    30. MES sodium salt (Sigma-Aldrich, catalog number: M5057 )
    31. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
    32. Mouse anti-BrdU clone B44 IgG1 (BD, BD Biosciences, catalog number: 347580 )
    33. Rat anti-BrdU clone BU1/75 (Bio-Rad Laboratories, catalog number: OBT0030 )
    34. Mouse anti ssDNA (poly dT) IgG2a (EMD Millipore, catalog number: MAB3034 or MAB3868 )
    35. Goat anti-Rat Alexa 488 (Thermo Fisher Scientific, Invitrogen, catalog number: A-11006 )
    36. Goat anti-Mouse Alexa 546 (Thermo Fisher Scientific, Invitrogen, catalog number: A-11030 )
    37. Goat anti-Mouse IgG2a Alexa 647 (Thermo Fisher Scientific, Invitrogen, catalog number: A-21241 )
    38. Goat anti-Mouse IgG1 Alexa 546 (Thermo Fisher Scientific, Invitrogen, catalog number: A-21123 )
    39. LMP agarose (see Recipes)
    40. TE50 (see Recipes)
    41. TE 1x (see Recipes)
    42. 10x MES buffer pH 6 (see Recipes)
    43. 1 N NaOH (see Recipes)
    44. PBS/T (see Recipes)
    45. Antibodies (dilution in PBS/T) (see Recipes)
    46. Detection of CldU/IdU/ssDNA (see Recipes)
    47. Detection of BrdU/ssDNA (see Recipes)

  2. S. cerevisiae specific reagents
    1. Yeast strain PP872 (genetic background: W303; genotype: MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL, psi+, RAD5, ura3::URA3-GPD-TK7) (Crabbe et al., 2010)
    2. Yeast strain HSV-TK + hENT1 (Viggiani and Aparicio, 2006)
    3. Bacto peptone (BD, BactoTM, catalog number: 211677 )
    4. Adenine (Sigma-Aldrich, catalog number: A8626 )
    5. Yeast extract (Sigma-Aldrich, catalog number: 70161 )
    6. Glucose (VWR, catalog number: 101175P )
    7. Alpha factor (custom peptide synthesis, Sequence: WHWLQLKPGQPMY)
    8. Pronase (EMD Millipore, catalog number: 53702-50KU )
    9. Sodium azide (NaN3) (Sigma-Aldrich, catalog number: 71289 )
    10. Tris-HCl (Sigma Aldrich, catalog number: RES3098T )
    11. Citric acid monohydrate (C6H8O7·H2O) (Sigma-Aldrich, catalog number: C1909 )
    12. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
    13. Monopotassium phosphate (KH2PO4) (VWR, catalog number: 26936.293 )
    14. Dibasic potassium phosphate (K2HPO4) (VWR, catalog number: 26930.293 )
    15. Enzyme powder Zymolyase 20T (MP Biomedicals, catalog number: 0 8320921 )
    16. YPAD (see recipes)
    17. Zymolyase buffer (see Recipes)
    18. 1 M potassium phosphate buffer pH 7.0 (see Recipes)
    19. 0.1 M citrate phosphate buffer pH 5.6 (see Recipes)
    20. BrdU stock solution (see Recipes)
    21. Proteinase K buffer (see Recipes)

  3. Human cells specific reagents
    1. 6 well plates (Dominique Dutscher, catalog number: 009206 )
    2. 1x PBS (Sigma-Aldrich, catalog number: D8537 )
    3. 0.05% trypsin-EDTA
    4. Regular recommended cell growth medium
    5. BrdU stock solution (see Recipes)
    6. IdU stock solution (see Recipes)
    7. CldU stock solution (see Recipes)
    8. Proteinase K buffer (see Recipes)

Equipment

  1. Centrifuge (Eppendorf, model: 5810 R )
  2. Microcentrifuge (Eppendorf, model: MiniSpin® )
  3. Thermoblock (thermomixer comfort Eppendorf with 2 ml; 15 ml and 50 ml block)
  4. Cell counter (CASY® Modell TT - Cell Counter, OLS for yeast cells or Malassez hemocytometer [VWR, catalog number: 631-0975 ] for human cells)
  5. Pasteur pipette Rubber bulb (Dominique Dutscher, catalog number: 042250 )
  6. Microwave
  7. Roller mixer (Cole-Parmer, Stuart, model: SRT9D )
  8. Leica DM6000B microscope (Leica Microsystems, model: DM6000B )
  9. CoolSNAP HQ CCD camera (Photometrics, model: CoolSNAP HQ CCD )
  10. P1000, P200, P20 pipetman
  11. DNA combing device (Genomic Vision, catalog number: MCS-001 ). Can also be assembled as described (Gallo et al., 2016; Kaykov et al., 2016; Norio and Schildkraut, 2001)
  12. Metal coverslip holder
  13. Humid chamber (StainTray slide staining system) (Sigma-Aldrich, catalog number: Z670146-1EA )
  14. Hybridization oven (Shake ‘n’ StackTM Hybridization Ovens) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 6240TS )
  15. Epifluorescence microscope Zeiss Axio Imager Z2 (Zeiss, model: Axio Imager Z2 ) with a camera Hamamatsu ORCA-Flash4.0 LT, Cmos sensor of 6.5 µm with an objective Zeiss 40x PL APO 1.4 oil and with the filters GFP HE: BP470/40 FT495 BP525/50; Texas Red: BP560/40 FT585 BP630/75; CY5: BP 640/30 FT660 BP690/50)
  16. Microscope (Nikon Instruments, model: YS100 )

Software

  1. MetaMorph (Molecular Devices) or open source software such as ImageJ (http://rsbweb.nih.gov/ij/) with bio-formats plugins
  2. IDeFIx Montpellier (IGMM, CNRS) or software developed by Genomic Vision
  3. Prism 7.0 (GraphPad) or open source software such as R (https://www.r-project.org/)

Procedure

  1. Saccharomyces cerevisiae
    S. cerevisiae cells are unable to incorporate BrdU into DNA because they lack the nucleotide salvage pathway that enables uptake of extracellular thymidine or its analogs. To circumvent this problem, strains for molecular combing have been engineered to ectopically express the Herpes simplex virus thymidine kinase (HSV-TK) under the control of the constitutive GPD promoter, inserted at the URA3 locus on chromosome V (strain PP872; genetic background: W303; genotype: MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, GAL, psi+, RAD5, ura3::URA3-GPD-TK7) (Lengronne et al., 2001; Viggiani and Aparicio, 2006). Cells bearing one integrated copy of the HSV-TK + hENT1 vector (human equilibrative nucleoside transporter 1) (Viggiani and Aparicio, 2006) show comparable levels of BrdU incorporation to cells with seven copies of HSV-TK alone (Bianco et al., 2012).
    Note: DNA molecular combing procedure doesn’t differ for fission yeast excepted for growth and labelling conditions (Kaykov et al., 2016).
    1. BrdU labeling of synchronized HU-arrested cells and preparation of DNA genomic plugs
      1. Grow cells overnight at 25 °C to a density of 5 x 106 cells/ml in 100 ml of YPAD.
      2. Add α-factor (2 μg/ml) at T0 in order to synchronized cells in G1. A second (4 μg/ml) and a third dose (2 μg/ml) of α-factor are added part way at respectively T1h and T2h to ensure that cells do not escape the G1 arrest.
        Note: The length of G1 synchronisation varies from 2.5 to 3 h depending on the doubling time of yeast strains. Make sure that cells are in G1 (unbudded cells) before the release into S phase (Figure 1).


        Figure 1. Yeast cell morphology in G1 and early S phase. A. G1 synchronised yeast cells; B. Yeast cells in S phase + HU after release from G1 for 90 min.

      3. Add (i) BrdU to a final concentration of 400 μg/ml (or 40 μg/ml if cells express the nucleotide transporter hENT1), and (ii) Hydroxyurea (HU, 200 mM) at least 15 min before releasing cells into S phase.
        Note: Elongation from early-firing origins is inhibited with the addition of HU in order to restrict the length of newly replicated regions to 10 kb.
      4. Release cells from α-factor arrest into S phase by addition of 50 μg/ml Pronase (degrades alpha factor peptide in medium). Adjust the pH of the medium to 7.0 with potassium phosphate buffer.
      5. After 90 min, collect cells by centrifugation at 4 °C; 1,096 x g in a 50 ml tube containing cold 0.1% NaN3 (sodium azide).
        Note: Sodium azide inhibits cytochrome oxidase (last enzyme in the respiratory electron transport chain of mitochondria or bacteria) by binding irreversibly to the heme cofactor and stops all cell metabolism, including DNA replication.
      6. Resuspend the cells in one volume of cold TE50, 0.1% NaN3 and keep them on ice. Check cells under a microscope (Nikon YS100). More than 90% of the cells should display small buds, which are indicative of entry into S phase (Figure 1).
    2. Preparation of genomic DNA plugs
      Genomic DNA is prepared in low melting point (LMP) agarose plugs to prevent mechanical shearing.
      1. Freshly prepare molten 2% LMP agarose in bi-distilled water and Zymolyase buffer. Keep at 42 °C in thermomixer until use.
      2. Determine cell concentration with a cell counter.
      3. Wash cells once with NZ buffer.
      4. Resuspend cells in prewarmed Zymolyase buffer (42 °C) to a final concentration of 4 x 108 cells/ml and carefully mixed with an equal volume of molten 2% LMP agarose (42 °C).
        Note: The Zymolyase will digest the cell wall.
      5. Transfer the cellular suspension into a plug mold sealed with tape to generate 90 μl plugs (Figure 2) containing 2 x 107 cells or approximately 350 ng of genomic DNA per plug at room temperature.
        Note: Prepare at least 4 plugs per sample.
      6. Cover molds with a plastic film (Figure 2) and incubate for 30 min at 37 °C.


        Figure 2. Pictures of a plug mold. A and B. Top and bottom views; C. Plug mold sealed with a tape and covered with a saran plastic film.

      7. Place agarose plugs at 4 °C for 10 min to let the agarose solidify.
      8. Transfer plugs into 14 ml round-bottom polypropylene tubes using a Pasteur pipette rubber bulb and incubate them at 37 °C in Proteinase K buffer (2 ml for 4 plugs) for 1 h under gentle agitation. Transfer plugs into fresh Proteinase K buffer and incubate for 24 h at 37 °C. Repeat once for an additional 24 h.
        Note: Incubation with PK buffer is critical to degrade proteins that perturb DNA fiber stretching.
      9. Wash plugs for two days in TE50, 100 mM NaCl on a roller mixer at room temperature. Change the buffer from time to time.
      10. Keep plugs at 4 °C in TE50 buffer until use (stable for months). Note that DNA plugs are translucent and fragile.

  2. Human cells
    Note: This protocol is available for any kind of mammalian cells.
    1. BrdU labelling and preparation of DNA genomic plugs
      Unlike budding yeast, human cells cannot be easily synchronized and experiments are usually performed with asynchronous cultures. Under these conditions, it is critical to use a combination of two modified nucleotides (IdU and CldU) to determine the polarity of replication forks and the position of initiation sites (Figure 3). (Conti et al., 2007)


      Figure 3. Schematic patterns obtained by DNA combing after IdU/CldU pulses in asynchronous mammalian cells. To determine fork velocity, green tracks adjacent to red tracks are measured in B, C, E. Stalled forks are represented in A and E and interorigin distances (IOD) in B and D. ORI: origin of replication. Arrows indicate fork direction. F. Schematic pattern obtained by DNA combing after BrdU pulse in synchronized budding yeast cells.
       
      1. Grow cells to a confluence of about 70% in 6-well plates. At the time of the experiment each well need to contain a minimum of 1 x 105 cells.
      2. Label cells by adding IdU (20 μM final) directly in 2 ml medium.
      3. Incubate cells at 37 °C for at least 10 min.
      4. Replace medium with 2 ml of prewarmed medium containing 200 μM CldU. Incubate cells at 37 °C for 20 min.
        Note: To avoid thermal shock and other perturbations, add nucleotides directly to growth medium (first pulse). Use prewarmed medium and a much higher concentration for the second pulse to avoid washes.
        The range of IdU/CldU staining time should be adjusted for each cell line from 10 to 30 min depending on S phase duration.
      5. Rinse cells with PBS and trypsinize cells as usual, adjust trypsinization time to cell type. For instance, incubate cells for 3-5 min in 0.05% trypsin, 1 mM EDTA.
      6. Collect cells, place them on ice and spin for 5 min at 1,000 x g at 4 °C. Keep the cells on ice until use (to stop DNA synthesis).
    2. Preparation of genomic DNA plugs
      1. Resuspend cells in 5 ml cold PBS, spin for 5 min at 1,000 x g at 4 °C and resuspend in 1 ml cold PBS.
      2. Count cells and resuspend at 8 x 105 to 2 x 106 cells/ml in cold PBS, depending on cell size.
      3. Prepare a solution of 2% LMP agarose in PBS and keep at 42 °C until use.
      4. Add an equal volume of 2% LMP agarose to cells. To prepare 4 plugs, add 200 μl of 2% LMP agarose solution to 200 μl of prewarmed cells solution.
      5. Mix very gently with a P1000 pipetman (optional: cut tip).
      6. Transfer 90 μl per plug into a plug mold sealed with tape.
      7. Let plugs solidify for 25 min at room temperature and for 10 min at 4 °C.
      8. Use a Pasteur pipette rubber bulb to eject plugs into 14 ml round-bottom tubes containing 0.5 ml of Proteinase K buffer per plug. Mix gently on roller mixer and change the PK buffer after 1 h.
      9. Incubate for 48 h at 37 °C. Change the PK solution twice more during this incubation. Mix gently on a roller mixer.
        Note: Incubation with PK buffer is critical to degrade proteins that perturb DNA fiber stretching.
      10. Gently remove the solution by blocking the agarose plug with a cell scraper. Plugs are transparent and difficult to manipulate after PK treatment.
      11. Wash plugs several times for two days in TE50, 100 mM NaCl on the roller mixer at room temperature.
      12. Store at 4 °C in TE50 until use. Genomic DNA is stable for months at 4 °C in TE50 buffer.

  3. Procedures common to yeast and human cells
    1. Melting of agarose plugs containing genomic DNA
      1. Transfer a plug in a 12 ml round-bottom tube. Plugs are washed 3 times for 1 h in 1x TE pH 7.5, 100 mM NaCl.
      2. After removing of the last wash, add 100 μl 1x TE and 1.5 μl YOYO-1 to stain genomic DNA. Keep in the dark for 30 min at room temperature.
        Note: YOYO staining is lost after DNA denaturation but is used to check combing before detection.
      3. Wash 4 times for 5 min with 10 ml 1x TE with gentle shaking.
      4. Wash once for 5 min with 2 ml of 50 mM MES pH 6, 100 mM NaCl.
      5. Replace with 3 ml of 50 mM MES pH 6, 100 mM NaCl.
        Note: Increase the volume of MES if fiber density is too high after combing. The pH of MES is critical to obtain a correct density and stretching of DNA fibers.
      6. Incubate for 15-30 min for human cells and 45 min for yeast cells at 67 °C in a heating block.
        Note: From now on, manipulate samples with extreme care to avoid DNA shearing.
      7. Check the DNA solution. If the shape of the plug is still visible, put it back at 67 °C until complete melting.
      8. Let the solution cool down to 42 °C before adding β-agarase (3 U per plug).
      9. Incubate overnight at 42 °C.
      10. Visually inspect the DNA solution. If you still see agarose aggregates; add β-agarase (1 U per plug) for 2 more hours.
      11. Incubate at 65 °C for 10 min and store at room temperature in dim light until use.
    2. DNA combing
      DNA combing is performed on silanized coverslips as described (Bensimon et al., 1994; Lengronne et al., 2001; Michalet et al., 1997). The quality of silanized coverslips is critical for the subsequent analysis of replication profiles. The whole procedure using the genomic vision device can be visualized on this video: https://youtu.be/LjjpYby3YIk
      1. Carefully pour the DNA solution into a 2 ml Teflon reservoir. Save the rest for further use.
        Note: Genomics Vision sells disposable 2 ml plastic reservoirs to melt plug directly in the reservoir and avoid manipulation of the DNA solution.
      2. Insert a silanized coverslip into the DNA solution and incubate for 5 min at room temperature.
      3. Using a DNA combing device, remove carefully the coverslip from the reservoir at a constant speed of 300 μm/sec. Repeat with another coverslip as many times as needed (2 slides per sample is usually sufficient).
      4. At this stage, DNA is stretched on both sides of the coverslip. Visualize DNA fibers on one side under the microscope (Nikon YS100) using a 40x objective and a FITC filter block. To this aim, fix with tape the coverslip to a metal coverslip holder, put a drop of immersion oil directly on the coverslip to visualize DNA fibers.
      5. Place the coverslip on a sheet of Whatman paper to soak up oil and bake for 2 h at 60 °C to crosslink DNA to coverslips.
      6. Stick the oiled side of coverslips on glass slides with cyanoacrylate glue. Label slides with a diamond tip engraving pen and store at -20 °C until use.
        Note: If needed, some steps presented in A2/B2 and C1/C2 sections can be modified to increase the length of stretched DNA molecules as described (Kaykov et al., 2016). If the density of fibers is too low, the DNA solution can be kept for a few days at 4 °C to increase DNA resuspension in MES buffer before combing.
    3. Immunodetection
      Specific combinations of primary and secondary antibodies are used to detect BrdU or CldU/IdU and DNA fibers simultaneously.
      1. Dehydrate slides for 5 min in successive baths of 70%, 90% and 100% ethanol in Coplin jar. Let air dry the slides.
      2. Incubate in Coplin jar for 25 min in freshly prepared 1 N NaOH to denature the DNA duplex.
        Note: Prolonged incubation in NaOH degrades the silane layer and should be avoided.
      3. Wash extensively with PBS pH 7.4 to neutralize NaOH (5 washes of 1 min each).
      4. Saturate slides for 15 min in PBS/T containing 1% BSA.
      5. Add 20 μl of PBS/T containing primary antibodies and cover with a coverslip. Incubate for 45 min at 37 °C in a humid chamber.
      6. Wash 5 min x 2 times with PBS/T in Coplin jar.
        Note: Dip slide in a Coplin jar containing PBS/T in order to remove the upper coverslip without damaging the DNA fibers.
      7. Detect with secondary antibodies (30 min at 37 °C in a humid chamber).
      8. Wash 5 min x 3 times with PBS/T in Coplin Jar.
      9. Air dry slides and mount by adding 5 μl Prolong Gold Antifade reagent on coverslip covered with a second coverslip. Let mounting reagent polymerize for at least 2 h at room temperature (preferably overnight and protect from light) before proceeding with microscopy. Mounted coverslips are stable for months at -20 °C.
    4. Image acquisition and data analysis
      1. Image acquisition is performed with a 40x objective on a motorized microscope equipped with a camera and controlled with Zeiss ZEN Pro 2 software or equivalent (Figure 4). The conversion factor (CF) from pixel to bp (CF = P/M x S) depends on the pixel size of the CCD camera (P in μm), on the magnification of the objective (M) and on the stretching of DNA fibers (S, 2 kb/µm for DNA combing). DNA molecules of known length, such as concatemers of bacteriophage lambda DNA, can be used as size standards.
      2. BrdU tracks and DNA fibers can be measured manually with MetaMorph (Molecular Devices) or open source software such as ImageJ (http://rsbweb.nih.gov/ij/). Data are transferred to an Excel spreadsheet for analysis. DNA fiber identification and length measurements can be automated with software like IDeFIx, developed by the DNA combing facility of Montpellier (IGMM, CNRS) or software developed by Genomic Vision. Critical issues regarding potential biases and limitations of DNA fiber analysis have been extensively discussed elsewhere (Techer et al., 2013; Tuduri et al., 2010).


        Figure 4. Representative examples of replication patterns observed by DNA combing. A and B. Yeast cells were released from G1 in the presence of 200 mM HU and labeled with BrdU for 90 min. DNA fibers were purified and stretched by DNA combing. BrdU and ssDNA were detected as indicated. A representative field of view is shown. Bar is 50 kb. C and D. Human cells were labeled for 15 min with IdU and 60 min with CldU. The combination of antibodies used to detect IdU, CldU and ssDNA is indicated. A representative field of view is shown. Bar is 50 kb. Image acquisition is performed with a motorized Leica DM6000B microscope equipped with a CoolSNAP HQ CCD camera (6.45 µm/pixel) and a 40x oil immersion objective. With this setup and a DNA stretching of 2 kb/µm, one pixel corresponds to (6.45/40) x 2 = 323 bp.

Data analysis

  1. Statistical analysis of BrdU track length and inter-origin distances is performed with Prism 7.0 (GraphPad) or R Statistical software. Since BrdU track lengths and inter-origin distances do not display a Gaussian distribution, statistical significance of differences between samples is tested using nonparametric tests such as the Mann-Whitney rank sum test.
  2. To determine fork speed, the median length of CldU tracks flanked with IdU tracks on one side (single fork) is divided by the duration of the second pulse (usually 20 min). CldU tracks alone and CldU tracks surrounded with two IdU tracks are not taken into account. CldU and IdU can also be used to measure inter-origin distances (IODs), global fork densities (Bialic et al., 2015) and to monitor replication fork arrest/restart (Techer et al., 2013; Tuduri et al., 2009) (Figure 3).
  3. IdU and CldU pulses can be used to visualize the progression of sister replication forks. In normal growth conditions, these signals are normally symmetrical as sister forks progress with the same speed. When asymmetrical patterns are detected this is indicative of increased replication fork pausing or stalling (Tuduri et al., 2009). This increased rate of fork stalling can be expressed as the ratio of the longest to the shortest CldU tracks, or represented graphically as a scatter plot of the distance covered by the two sister forks during the CldU pulse. Large ratio or dispersed scatter plots are indicative of increased fork arrest.
  4. Another useful application of IdU/CldU double labeling is the analysis of DNA replication recovery after a genotoxic insult (Sidorova et al., 2013; Tourriere et al., 2005). In this assay, cells are exposed to genotoxic agents or replication inhibitors such as hydroxyurea (HU), methyl methanesulfonate (MMS) or camptothecin (CPT) in the presence of IdU and are released in fresh medium in the presence of CldU. This approach can be used to monitor cells ability to restart stalled forks, to activate late or dormant origins and to complete DNA replication after release from the drug.
  5. It has been recently reported that stalled forks can be processed by nucleases. Resection of nascent DNA at forks arrested by genotoxic agents such as HU or CPT can be visualized after IdU and CldU pulses as described (Ray Chaudhuri et al., 2016; Schlacher et al., 2011).

Recipes

  1. Yeast cells
    1. YPAD
      1% yeast extract
      2% Bacto peptone
      0.005% adenine
      2% glucose
      Bi-distilled water
    2. BrdU
      10 mg/ml stock solution in Milli-Q H2O (freshly prepared)
    3. Zymolyase buffer
      50 mM citrate phosphate buffer, pH 5.6
      50 mM EDTA, pH 8.0
      1.2 M sorbitol
      1 mg/ml Zymolyase 20T (use enzyme powder)
    4. NZ (no Zymolyase) buffer
      50 mM citrate phosphate buffer, pH 5.6
      50 mM EDTA, pH 8.0
      1.2 M sorbitol
    5. 1 M potassium phosphate buffer pH 7.0; 100 ml
      61.5 ml KH2PO4, 2 M (M.W. = 136)
      38.5 ml K2HPO4, 2 M (M.W. = 174)
    6. 0.1 M citrate phosphate buffer pH 5.6; 100 ml
      58 ml of 0.2 M Na2HPO4 (dibasic; M.W. = 142)
      42 ml of 0.1 M citric acid (C6H8O7·H2O; M.W. = 210)
    7. Proteinase K buffer
      125 mM EDTA pH 9.5
      1% N-laurylsarcosine
      1 mg/ml Proteinase K

  2. Human cells
    1. BrdU: 25 mM stock solution in PBS, 10% DMSO. Store at -20 °C
    2. IdU: 25 mM stock solution in PBS, 10% DMSO. Store at -20 °C
    3. CldU: 200 mM stock solution in PBS, 10% DMSO. Store at -20 °C
    Note: Dilute directly in the culture medium during experiment.
    1. Proteinase K buffer
      10 mM Tris pH 7.5
      100 mM EDTA pH 8.0
      1% N-laurylsarcosine
      1 mg/ml Proteinase K

  3. Common to human/yeast cells
    1. LMP agarose, freshly prepared, check volume: 2% in water for yeast cells or in PBS for human cells. LMP can be melted in a microwave at a very low power; please make sure to avoid any boiling of the solution
    2. TE50
      10 mM Tris-HCl, pH 7.0
      50 mM EDTA, pH 8.0
    3. 1x TE
      10 mM Tris-HCl, pH 7.0
      1 mM EDTA, pH 8.0
    4. 10x MES buffer pH 6
      70 ml of 500 mM MES hydrate
      30 ml of 500 mM MES sodium salt
      Adjust to pH 6 with 500 mM MES sodium salt
    5. 1 N NaOH (freshly prepared)
    6. PBS/T
      PBS pH 7.4
      0.1% Triton X-100
    7. Antibodies (dilution in PBS/T)
      # 01 Mouse anti-BrdU clone B44 IgG1
      # 02 Rat anti-BrdU clone BU1/75
      # 03 Mouse anti ssDNA (poly dT) IgG2a
      # 04 Goat anti-Rat Alexa 488
      # 05 Goat anti-Mouse Alexa 546
      # 06 Goat anti-Mouse IgG2a Alexa 647
      # 07 Goat anti-Mouse IgG1 Alexa 546
    8. Detection of CldU/IdU/ssDNA
      Primary antibodies:
      CldU: #01 (1/20)
      IdU: #02 (1/20)
      ssDNA: #03 (1/50) 
      Secondary antibodies:
      #07 (1/50)
      #04 (1/50)
      #06 (1/50)
    9. Detection of BrdU/ssDNA
      Primary antibodies:
      BrdU: #02 (1/20)
      ssDNA: #03 (1/50)
      Secondary antibodies:
      #04 (1/50)
      #05 (1/50)

Acknowledgments

We thank the members of the Pasero laboratory for their contribution to the optimization of this protocol, which was adapted from protocols of several other groups, including the Bensimon, Schwob, Debatisse and Nurse laboratories. We thank the Montpellier RIO Imaging facility for support and the DNA combing facility of Montpellier for providing silanized coverslips. This work was supported by grants from Agence Nationale pour la Recherche (ANR), Institut National du Cancer (INCa), Ligue contre le Cancer (Equipe labellisée LIGUE, 2017) and the MSDAvenir fund.

References

  1. Bensimon, A., Simon, A., Chiffaudel, A., Croquette, V., Heslot, F. and Bensimon, D. (1994). Alignment and sensitive detection of DNA by a moving interface. Science 265(5181): 2096-2098.
  2. Bialic, M., Coulon, V., Drac, M., Gostan, T. and Schwob, E. (2015). Analyzing the dynamics of DNA replication in Mammalian cells using DNA combing. Methods Mol Biol 1300: 67-78.
  3. Bianco, J. N., Poli, J., Saksouk, J., Bacal, J., Silva, M. J., Yoshida, K., Lin, Y. L., Tourriere, H., Lengronne, A. and Pasero, P. (2012). Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing. Methods 57(2): 149-157.
  4. Conti, C., Sacca, B., Herrick, J., Lalou, C., Pommier, Y. and Bensimon, A. (2007). Replication fork velocities at adjacent replication origins are coordinately modified during DNA replication in human cells. Mol Biol Cell 18(8): 3059-3067.
  5. Crabbe, L., Thomas, A., Pantesco, V., De Vos, J., Pasero, P. and Lengronne, A. (2010). Analysis of replication profiles reveals key role of RFC-Ctf18 in yeast replication stress response. Nat Struct Mol Biol 17(11): 1391-1397.
  6. Gallo, D., Wang, G., Yip, C. M. and Brown, G. W. (2016). Single-molecule analysis of replicating yeast chromosomes. Cold Spring Harb Protoc 2016(2): pdb top077784.
  7. Kaykov, A., Taillefumier, T., Bensimon, A. and Nurse, P. (2016). Molecular combing of single DNA molecules on the 10 megabase scale. Sci Rep 6: 19636.
  8. Labit, H., Goldar, A., Guilbaud, G., Douarche, C., Hyrien, O. and Marheineke, K. (2008). A simple and optimized method of producing silanized surfaces for FISH and replication mapping on combed DNA fibers. Biotechniques 45(6): 649-652, 654, 656-648.
  9. Lengronne, A., Pasero, P., Bensimon, A. and Schwob, E. (2001). Monitoring S phase progression globally and locally using BrdU incorporation in TK+ yeast strains. Nucleic Acids Res 29(7): 1433-1442.
  10. Michalet, X., Ekong, R., Fougerousse, F., Rousseaux, S., Schurra, C., Hornigold, N., van Slegtenhorst, M., Wolfe, J., Povey, S., Beckmann, J. S. and Bensimon, A. (1997). Dynamic molecular combing: stretching the whole human genome for high-resolution studies. Science 277(5331): 1518-1523.
  11. Norio, P. and Schildkraut, C. L. (2001). Visualization of DNA replication on individual Epstein-Barr virus episomes. Science 294(5550): 2361-2364.
  12. Prioleau, M. N. and MacAlpine, D. M. (2016). DNA replication origins-where do we begin? Genes Dev 30(15): 1683-1697.
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  14. Schlacher, K., Christ, N., Siaud, N., Egashira, A., Wu, H. and Jasin, M. (2011). Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11. Cell 145(4): 529-542.
  15. Sidorova, J. M., Kehrli, K., Mao, F. and Monnat, R., Jr. (2013). Distinct functions of human RECQ helicases WRN and BLM in replication fork recovery and progression after hydroxyurea-induced stalling. DNA Repair (Amst) 12(2): 128-139.
  16. Techer, H., Koundrioukoff, S., Azar, D., Wilhelm, T., Carignon, S., Brison, O., Debatisse, M. and Le Tallec, B. (2013). Replication dynamics: biases and robustness of DNA fiber analysis. J Mol Biol 425(23): 4845-4855.
  17. Tourriere, H. and Pasero, P. (2007). Maintenance of fork integrity at damaged DNA and natural pause sites. DNA Repair (Amst) 6(7): 900-913.
  18. Tourriere, H., Versini, G., Cordon-Preciado, V., Alabert, C. and Pasero, P. (2005). Mrc1 and Tof1 promote replication fork progression and recovery independently of Rad53. Mol Cell 19(5): 699-706.
  19. Tuduri, S., Crabbe, L., Conti, C., Tourriere, H., Holtgreve-Grez, H., Jauch, A., Pantesco, V., De Vos, J., Thomas, A., Theillet, C., Pommier, Y., Tazi, J., Coquelle, A. and Pasero, P. (2009). Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription. Nat Cell Biol 11(11): 1315-1324.
  20. Tuduri, S., Tourriere, H. and Pasero, P. (2010). Defining replication origin efficiency using DNA fiber assays. Chromosome Res 18(1): 91-102.
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  22. Zeman, M. K. and Cimprich, K. A. (2013). Causes and consequences of replication stress. Nat Cell Biol 16: 2-9.

简介

DNA梳理方法允许在沿着硅烷涂覆的玻璃盖玻片拉伸的单个DNA分子的水平上分析DNA复制。在DNA提取前,进行的DNA合成用胸苷的卤化类似物标记。使用特异性抗体通过免疫荧光可视化复制轨迹。与生物化学和基于NGS的方法不同,DNA梳理提供了DNA复制谱中细胞间细胞变化的独特信息,包括引发和延长。最后,该测定可用于监测DNA损伤对叉进展,停止和重新启动的影响。

背景 在称为复制起点的真核染色体上的数千个位点处启动DNA合成。原始激活遵循由检查点激酶和染色质的表观遗传修饰(Prioleau和MacAlpine,2016)控制的定义良好的复制计时程序。复制叉在正常S阶段经常停顿。叉停止是由多个事件引起的,例如DNA损伤,紧密结合的蛋白质复合物和高表达基因的转录(Tourriere和Pasero 2007; Zeman and Cimprich,2013)。真核生物已经制定了不同的策略来应对这种复制压力,包括修复机制来重新启动捕获的叉子和激活休眠复制起源以抢救终末抓捕的叉。
  DNA梳理是一种监控复制方式的选择方法(叉速度,原点使用,叉重启,姐妹叉不对称)。与其他DNA纤维方法如DNA纤维扩散不同,DNA分子的拉伸,密度和取向在DNA梳理方法中是高度可重复的和严格控制的。拉伸是由后退的空气/水界面施加的力,与DNA纤维的长度无关(Bensimon等人,1994; Michalet等人,1997年) )。引入胸苷类似物如5-溴-2'-脱氧尿苷(BrdU),5-碘-2'-脱氧尿苷(IdU)和5-氯-2'-脱氧尿苷( CldU)。这种技术已成功地用于监测各种生物体中的DNA复制动力学,包括细菌,酵母,果蝇,非洲爪蟾和哺乳动物。
 这里,我们提供详细的协议来分析芽殖酵母和人类细胞中新合成的DNA纤维,并研究正常生长条件和复制应激下DNA复制的各个方面。

关键字:复制, 酵母, 人类细胞, 分叉速度, 复制起始, DNA延伸

材料和试剂

  1. 人/酵母细胞共有
    1. 磁带
    2. 14ml圆底聚丙烯管(Corning,Falcon ®,目录号:352059)
    3. 提示1 ml,200μl,20μl
    4. 从Genomic Vision购买或按照描述(Labit等人,2008)
      购买的硅化盖玻片(Genomic Vision,目录号:COV-001)
    5. 2 ml特氟隆水库(Reservoir MCS Support [x 2]; from Genomic Vision)
    6. Whatman纸
    7. 显微镜幻灯片SuperFrost(VWR,目录号:630-1987)
    8. 氰基丙烯酸酯胶
    9. 金刚石刻笔(Sigma-Aldrich,目录号:Z225568-1EA)
    10. Saran塑料薄膜(Dominique Dutscher,目录号:090264)
    11. Coplin Jar
    12. EDTA(Sigma-Aldrich,目录号:E6758)
    13. LMP琼脂糖(Bio-Rad Laboratories,目录号:161-3111)
    14. 插头模具(Bio-Rad Laboratories,目录号:170-3713)
    15. 蛋白酶K(Sigma-Aldrich,目录号:P6556)
    16. 10x PBS(Sigma-Aldrich,目录号:D1408)
    17. YOYO-1(Thermo Fisher Scientific,Invitrogen TM,目录号:Y3601)
    18. 氯化钠(NaCl)(VWR,目录号:27810-295)
    19. β-琼脂糖酶(New England Biolabs,目录号:M0392L)
    20. 氢氧化钠(NaOH)(Merck,目录号:1.06462.1000)
    21. BSA级分V(Sigma-Aldrich,目录号:A9647)
    22. ProLong Gold Antifade试剂(Thermo Fisher Scientific,Invitrogen TM,目录号:P36930)
    23. BrdU(Sigma-Aldrich,目录号:B5002)
    24. IdU(Sigma-Aldrich,目录号:I7125)
    25. CldU(MP Biomedicals,目录号:02105478)
    26. DMSO(Sigma-Aldrich,目录号:D2650)
    27. 羟基脲(Sigma-Aldrich,目录号:H8627)
    28. 月桂基肌氨酸钠盐(Sigma-Aldrich,目录号:L9150)
    29. MES水合物(Sigma-Aldrich,目录号:M2933)
    30. MES钠盐(Sigma-Aldrich,目录号:M5057)
    31. Triton X-100(Sigma-Aldrich,目录号:T8787)
    32. 小鼠抗BrdU克隆B44 IgG1(BD,BD Biosciences,目录号:347580)
    33. 大鼠抗BrdU克隆BU1 / 75(Bio-Rad Laboratories,目录号:OBT0030)
    34. 小鼠抗ssDNA(poly dT)IgG2a(EMD Millipore,目录号:MAB3034或MAB3868)
    35. 山羊抗大鼠Alexa 488(Thermo Fisher Scientific,Invitrogen,目录号:A-11006)
    36. 山羊抗小鼠Alexa 546(Thermo Fisher Scientific,Invitrogen,目录号:A-11030)
    37. 山羊抗小鼠IgG2a Alexa 647(Thermo Fisher Scientific,Invitrogen,目录号:A-21241)
    38. 山羊抗小鼠IgG1 Alexa 546(Thermo Fisher Scientific,Invitrogen,目录号:A-21123)
    39. LMP琼脂糖(见食谱)
    40. TE 50 (见配方)
    41. TE 1x(见食谱)
    42. 10倍MES缓冲液pH 6(参见食谱)
    43. 1 N NaOH(参见食谱)
    44. PBS / T(见食谱)
    45. 抗体(PBS / T稀释)(见配方)
    46. 检测CldU / IdU / ssDNA(参见食谱)
    47. 检测BrdU / ssDNA(见配方)

  2. S上。啤酒酵母特异性试剂
    1. 酵母菌株PP872(遗传背景:W303;基因型:MATa,ade2-1,trp1-1,can1-100,leu2-3,112,his3-11,15,ura3,GAL,psi +,RAD5,ura3 :: URA3 -GPD-TK 7 )(Crabbe等人,2010)
    2. 酵母菌株HSV-TK + hENT1(Viggiani和Aparicio,2006)
    3. 细菌蛋白胨(BD,Bacto TM ,目录号:211677)
    4. 腺嘌呤(Sigma-Aldrich,目录号:A8626)
    5. 酵母提取物(Sigma-Aldrich,目录号:70161)
    6. 葡萄糖(VWR,目录号:101175P)
    7. α因子(定制肽合成,序列:WHWLQLKPGQPMY)
    8. Pronase(EMD Millipore,目录号:53702-50KU)
    9. 叠氮化钠(NaN 3 3)(Sigma-Aldrich,目录号:71289)
    10. Tris-HCl(Sigma Aldrich,目录号:RES3098T)
    11. 柠檬酸一水合物(C 6 H 8 O 7 H 2 O)(Sigma-Aldrich,目录号:C1909)
    12. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S3264)
    13. 磷酸二氢钾(KH 2 PO 4)(VWR,目录号:26936.293)
    14. 磷酸二氢钾(K 2 HPO 4)(VWR,目录号:26930.293)
    15. 酶粉末酵解酶20T(MP Biomedicals,目录号:08320921)
    16. YPAD(见食谱)
    17. 酵解酶缓冲液(参见食谱)
    18. 1M磷酸钾缓冲液pH 7.0(参见食谱)
    19. 0.1 M柠檬酸磷酸盐缓冲液pH 5.6(参见食谱)
    20. BrdU库存解决方案(见配方)
    21. 蛋白酶K缓冲液(参见食谱)

  3. 人细胞特异性试剂
    1. 6孔板(Dominique Dutscher,目录号:009206)
    2. 1x PBS(Sigma-Aldrich,目录号:D8537)
    3. 0.05%胰蛋白酶-EDTA
    4. 常规推荐的细胞生长培养基
    5. BrdU库存解决方案(见配方)
    6. IdU库存解决方案(见配方)
    7. CldU库存解决方案(请参阅食谱)
    8. 蛋白酶K缓冲液(参见食谱)

设备

  1. 离心机(Eppendorf,型号:5810 R)
  2. 微量离心机(Eppendorf,型号:MiniSpin ®
  3. Thermoblock(温热混合器舒适Eppendorf 2毫升; 15毫升和50毫升块)
  4. 细胞计数器(CASY ® Modell TT-细胞计数器,用于酵母细胞的OLS或用于人细胞的Malassez血细胞计数器[VWR,目录号:631-0975])
  5. 巴斯德吸管橡胶球(Dominique Dutscher,目录号:042250)
  6. 微波
  7. 辊式搅拌机(Cole-Parmer,Stuart,型号:SRT9D)
  8. 徕卡DM6000B显微镜(Leica Microsystems,型号:DM6000B)
  9. CoolSNAP HQ CCD相机(Photometrics,型号:CoolSNAP HQ CCD)
  10. P1000,P200,P20移液器
  11. DNA精梳装置(Genomic Vision,目录号:MCS-001)。也可以按照描述进行组装(Gallo等人,2016; Kaykov等人,2016; Norio和Schildkraut,2001)
  12. 金属盖玻片架
  13. 潮湿室(StainTray幻灯片染色系统)(Sigma-Aldrich,目录号:Z670146-1EA)
  14. 杂交炉(Shake'n'Stack TM Hybridization Ovens)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:6240TS)
  15. 荧光显微镜蔡司Axio Imager Z2(Zeiss,型号:Axio Imager Z2),带有相机浜松ORCA-Flash4.0 LT,具有目标蔡司40x PL APO 1.4油和滤光片GFP HE:BP470 / 40的6.5μmCmos传感器FT495 BP525 / 50;得克萨斯红:BP560 / 40 FT585 BP630 / 75; CY5:BP 640/30 FT660 BP690 / 50)
  16. 显微镜(Nikon Instruments,型号:YS100)

软件

  1. MetaMorph(Molecular Devices)或开源软件,如ImageJ( http:// rsbweb。 nih.gov/ij/ )使用生物格式插件
  2. IDeFIx蒙彼利埃(IGMM,CNRS)或由Genomic Vision开发的软件
  3. Prism 7.0(GraphPad)或开源软件,如R( https:// www。 r-project.org/

程序

  1. 酿酒酵母
    酿酒酵母
    S上。酿酒酵母细胞不能将BrdU并入DNA中,因为它们缺乏能够吸收细胞外胸苷或其类似物的核苷酸补救途径。为了克服这个问题,分子组合的菌株已经被设计成异位表达在组成型GPD启动子控制下的单纯疱疹病毒胸苷激酶(HSV-TK),插入在染色体V上的URA3位点(菌株PP872;遗传背景: W303;基因型:MATa,ade2-1,trp1-1,can1-100,leu2-3,112,his3-11,15,ura3,GAL,psi +,RAD5,ura3 :: URA3-GPD-TK7 )(Lengronne等人,2001; Viggiani和Aparicio,2006)。携带HSV-TK + hENT1载体(人平衡核苷转运蛋白1)(Viggiani和Aparicio,2006)的一个整合拷贝的细胞显示与单独的HSV-TK七拷贝(Bianco等)的BrdU掺入细胞的可比水平。,2012)。
    注意:除了生长和标记条件以外的裂变酵母,DNA分子组合程序没有差异(Kaykov et al。,2016)。
    1. BrdU标记同步HU阻滞细胞和DNA基因组插入物的制备
      1. 在100ml YPAD中,在25℃下将细胞生长至5×10 6细胞/ ml的密度。
      2. 在T 0 0加入α因子(2μg/ ml),以使G1中的细胞同步。分别在T 1h和T 2h 分别加入第二(4μg/ ml)和第三剂量(2μg/ ml)的α因子,以确保细胞不能逃避G 1 逮捕。
        注意:根据酵母菌株的倍增时间,G <1>同步的长度在2.5至3小时之间变化。确保在释放到S期之前,单元格处于G <1>(unbudded单元格)(图1)。


        图1. G 1和早期S期的酵母细胞形态。A. G1同步酵母细胞; B.从G1释放90分钟后,S期+ HU的酵母细胞
      3. 将(i)BrdU添加至终浓度为400μg/ ml(如果细胞表达核苷酸转运蛋白hENT1,则为40μg/ ml),和(ii)羟基脲(HU,200mM)至少15分钟,然后将细胞释放至S期。
        注意:通过添加HU来抑制早期起始起始的延长,以便将新复制的区域的长度限制在10kb。
      4. 通过加入50μg/ ml雌激素酶(介质中降解α因子肽)将细胞从α因子停滞释放到S期。用磷酸钾缓冲液调节培养基的pH至7.0
      5. 90分钟后,在4℃下离心收集细胞;在含有冷的0.1%NaN 3 N(叠氮化钠)的50ml管中,将1096×g
        注意:叠氮化钠通过不可逆地结合血红素辅因子来抑制细胞色素氧化酶(线粒体或细菌的呼吸电子传递链中的最后一种酶),并停止所有细胞代谢,包括DNA复制。
      6. 将细胞重悬在一个体积的冷TE 50,0.1%NaN 3 3中,并保持在冰上。在显微镜下检查细胞(Nikon YS100)。超过90%的细胞应显示小芽,这表明进入S期(图1)。
    2. 基因组DNA插入物的制备
      在低熔点(LMP)琼脂糖塞中制备基因组DNA以防止机械剪切。
      1. 新鲜制备熔融的2%LMP琼脂糖在双蒸水和酵解酶缓冲液中。在温热混合器中保持在42°C,直到使用。
      2. 用细胞计数器确定细胞浓度。
      3. 用NZ缓冲液洗涤细胞一次。
      4. 将细胞重新悬浮在预热的酵解酶缓冲液(42℃)中,终浓度为4×10 8细胞/ ml,并仔细与等体积的熔融2%LMP琼脂糖(42℃)混合。
        注意:酵解酶会消化细胞壁。
      5. 将细胞悬浮液转移到用胶带密封的塞子模具中,以在室温下产生含有2×10 7个细胞的约90μl塞子(图2)或每个塞子约350ng的基因组DNA。
        注意:每个样品至少准备4个插头。
      6. 用塑料薄膜覆盖模具(图2),并在37℃下孵育30分钟。


        图2.插头模具的图片。 A和B.顶部和底部视图; C.将模具用胶带密封并用萨兰胶膜覆盖。

      7. 将琼脂糖凝胶插入4℃10分钟使琼脂糖凝固。
      8. 使用巴斯德吸管橡胶球将塞子转移到14ml圆底聚丙烯管中,并在37℃下在蛋白酶K缓冲液(2ml,4个塞子)中孵育1小时并在温和搅拌下孵育。将塞子插入新鲜的蛋白酶K缓冲液中,37℃孵育24小时。重复一次24小时。
        注意:PK缓冲液的孵育对于降解干扰DNA纤维伸展的蛋白质至关重要。
      9. 在TE 50℃,100mM NaCl中,在辊式混合器中室温下将塞子洗涤两天。不时更改缓冲区。
      10. 在4℃保存在TE 50微量缓冲液中直到使用(稳定数月)。请注意,DNA插头是半透明和脆弱的。

  2. 人类细胞
    注意:该协议适用于任何种类的哺乳动物细胞。
    1. BrdU标记和DNA基因组插入物的制备
      不像发芽酵母,人类细胞不能轻易同步,而且通常用异步培养进行实验。在这些条件下,使用两个修饰的核苷酸(IdU和CldU)的组合来确定复制叉的极性和起始位点的位置是至关重要的(图3)。 (Conti等人,2007)


      图3.通过在异步哺乳动物细胞中的IdU / CldU脉冲之后的DNA梳理获得的示意图。为了确定叉速度,在B,C,E中测量与红色轨迹相邻的绿色轨迹。 A和E以及B和D中的interorigin距离(IOD)。ORI:复制起点。箭头表示叉方向。 F.通过在同步芽殖酵母细胞中BrdU脉冲后DNA合成获得的示意图 &nbsp;
      1. 在6孔板中生长细胞至约70%的汇合物。在实验时,每个孔需要至少包含1×10 5个细胞。
      2. 通过直接在2ml培养基中添加IdU(最终20μM)来标记细胞。
      3. 在37℃孵育细胞至少10分钟
      4. 用2 ml含有200μMCldU的预热培养基更换培养基。在37℃孵育细胞20分钟 注意:为了避免热休克和其他扰动,将核苷酸直接添加到生长培养基(第一脉冲)。使用预热的介质和更高的浓度用于第二脉冲以避免洗涤。
        应根据S期持续时间调整10至30分钟的每个细胞系的IdU / CldU染色时间范围。
      5. 用PBS冲洗细胞,像往常一样对细胞进行胰蛋白酶消化,将胰蛋白酶化时间调整为细胞类型。例如,在0.05%胰蛋白酶,1mM EDTA中孵育细胞3-5分钟
      6. 收集细胞,将其置于冰上,并在4℃下以1,000×g旋转5分钟。保持细胞在冰上直到使用(停止DNA合成)。
    2. 基因组DNA插入物的制备
      1. 将细胞重悬于5ml冷PBS中,在4℃以1000×g旋转5分钟并重悬于1ml冷PBS中。
      2. 根据细胞大小,在冷PBS中计数细胞并在8×10 5个重复悬浮至2×10 6个细胞/ ml。
      3. 在PBS中制备2%LMP琼脂糖溶液,并保持在42°C直至使用
      4. 向细胞中加入等体积的2%LMP琼脂糖。为了制备4个插头,向200μl预热的细胞溶液中加入200μl的2%LMP琼脂糖溶液。
      5. 用P1000移液器轻轻混合(可选:切割头)。
      6. 将每个插头90μl转移到用胶带密封的插头模具中。
      7. 让塞子在室温下固化25分钟,在4℃下固化10分钟
      8. 使用巴斯德吸管橡胶球将插头插入14毫升圆底管中,每个插头含有0.5毫升蛋白酶K缓冲液。在滚筒搅拌机上轻轻混合,1小时后更换PK缓冲液
      9. 在37℃孵育48小时。在孵化过程中更换PK溶液两次。在滚筒搅拌机上轻轻混合。
        注意:PK缓冲液的孵育对于降解干扰DNA纤维伸展的蛋白质至关重要。
      10. 通过用细胞刮刀堵塞琼脂糖插塞轻轻取出溶液。 PK治疗后,插头是透明的,难以操作。
      11. 在TE 50℃,100mM NaCl中,在辊式混合器中室温下将塞子洗涤两次,持续两天。
      12. 在4℃保存在TE 50中直到使用。基因组DNA在TE 50%缓冲液中在4℃下稳定数月。

  3. 酵母和人类细胞常见的程序
    1. 熔化含有基因组DNA的琼脂糖插塞
      1. 将塞子插入12毫升圆底管中。将塞子在1x TE pH 7.5,100mM NaCl中洗涤3次1小时。
      2. 去除最后一次洗涤后,加入100μl1x TE和1.5μlYOYO-1染色基因组DNA。在室温下保持在黑暗中30分钟。
        注意:DNA变性后YOYO染色失去,但用于检测前的梳理。
      3. 用10毫升1x TE轻轻晃动洗涤4次5分钟。
      4. 用2ml 50mM MES pH6,100mM NaCl洗涤5分钟。
      5. 用3ml 50mM MES pH6,100mM NaCl代替 注意:如果梳理后纤维密度过高,则增加MES的体积。 MES的pH对于获得正确的密度和DNA纤维的拉伸是至关重要的。
      6. 人体细胞孵育15-30分钟,酵母细胞在加热块中孵育45分钟。
        注意:从现在开始,非常小心操作样品,以避免DNA剪切。
      7. 检查DNA溶液。如果插头的形状仍然可见,请将其放回67°C直到完全熔化。
      8. 让溶液冷却至42℃,然后加入β-琼脂糖(每插塞3 U)
      9. 在42°C孵育过夜。
      10. 目视检查DNA溶液。如果您仍然看到琼脂糖聚集体;加入β-琼脂糖(每插头1 U)2个小时。
      11. 在65°C孵育10分钟,并在室温下暗淡存放,直至使用。
    2. DNA精梳
      如(Bensimon等人,1994; Lengronne等人,2001; Michalet等人)所述,在硅烷化盖玻片上进行DNA梳理, 1997年)。硅烷化盖玻片的质量对于随后的复制轮廓分析至关重要。使用基因组视觉设备的整个过程可以在此视频上显示: https://youtu.be / LjjpYby3YIk 。&nbsp;
      1. 小心地将DNA溶液倒入2ml特氟隆储存器中。节省其余的进一步使用。
        注意:Genomics Vision销售一次性2ml塑料储存器,将塞子直接熔化在储存器中,避免DNA溶液的操作。
      2. 将硅烷化的盖玻片插入DNA溶液中,并在室温下孵育5分钟。
      3. 使用DNA精梳装置,以300微秒/秒的恒定速度从容器中小心地取出盖玻片。根据需要重复另一个盖玻片多次(每个样品2个幻灯片通常就足够了)。
      4. 在这个阶段,DNA在盖玻片的两侧被拉伸。在显微镜(Nikon YS100)的一侧可视化DNA纤维,使用40x物镜和FITC滤光片。为此目的,用胶带将盖玻片固定到金属盖玻片支架上,将一滴浸油直接放在盖玻片上以显现DNA纤维。
      5. 将盖玻片放在Whatman纸上,浸泡油并在60℃下烘烤2小时以将DNA与盖玻片交联。
      6. 用氰基丙烯酸酯胶将玻片盖的油面贴在玻璃片上。用金刚石尖端雕刻笔标记幻灯片,并保存在-20°C直到使用。
        注意:如果需要,可以修改A2 / B2和C1 / C2部分中提出的一些步骤,以增加拉伸的DNA分子的长度(Kaykov等,2016)。如果纤维的密度太低,则可以在4℃下将DNA溶液保持几天,以在梳理之前在MES缓冲液中增加DNA重悬。
    3. 免疫检测
      使用一级和二级抗体的特异性组合同时检测BrdU或CldU / IdU和DNA纤维
      1. 在Coplin罐中,在70%,90%和100%乙醇的连续浴中脱水5分钟。让空气干燥幻灯片。
      2. 在新鲜制备的1N NaOH中,在Coplin罐中孵育25分钟使DNA双链体变性。
        注意:在NaOH中延长的温育会降解硅烷层,应避免使用。
      3. 用PBS pH 7.4大量洗涤以中和NaOH(5次洗涤1分钟)
      4. 含有1%BSA的PBS / T中饱和载玻片15分钟
      5. 加入20μl含有一抗的PBS / T,盖上盖玻片。在潮湿室中37℃孵育45分钟。
      6. 在Coplin罐中用PBS / T洗5分钟×2次。
        注意:在含有PBS / T的Coplin罐中浸泡,以去除上盖玻片而不损坏DNA纤维。
      7. 用二次抗体检测(37℃,潮湿室30分钟)
      8. 在Coplin罐中用PBS / T洗涤5分钟×3次。
      9. 通过在覆盖有第二盖玻片的盖玻片上加入5μlProlong Gold Antifade试剂,将其悬空。在进行显微镜检查之前,让试剂在室温下聚合至少2小时(优选过夜并防止光照)。安装的盖玻片在-20°C下稳定数月。
    4. 图像采集和数据分析
      1. 在装有相机的电动显微镜上使用40x物镜执行图像采集,并使用Zeiss ZEN Pro 2软件或等效物进行控制(图4)。从像素到bp(CF = P / M×S)的转换因子(CF)取决于CCD照相机的像素尺寸(P inμm),物镜(M)的放大倍数和DNA纤维的拉伸(S,2kb /μm,用于DNA精梳)。可以使用已知长度的DNA分子,例如噬菌体λDNA的并列物质作为尺寸标准。
      2. 可以使用MetaMorph(Molecular Devices)或开源软件如ImageJ( http://rsbweb.nih.gov/ij/ )。数据将传输到Excel电子表格进行分析。 DNA光纤识别和长度测量可以通过由蒙彼利埃(IGMM,CNRS)的DNA精梳设备开发的IDeFIx软件或由Genomic Vision开发的软件自动进行。关于DNA纤维分析的潜在偏差和限制的关键问题已在其他地方进行了广泛讨论(Techer等人,2013; Tuduri等人,2010)。


        图4.通过DNA合成观察到的复制模式的代表性实例.A和B.在200mM HU存在下从G1释放酵母细胞,并用BrdU标记90分钟。纯化DNA纤维并通过DNA梳理进行拉伸。如所示检测BrdU和ssDNA。显示了代表性的视野。酒吧是50 kb。 C和D。用IdU标记人细胞15分钟,用CldU标记60分钟。表明用于检测IdU,CldU和ssDNA的抗体的组合。显示了代表性的视野。酒吧是50 kb。采用配备CoolSNAP HQ CCD相机(6.45μm/像素)和40x油浸物镜的电动徕卡DM6000B显微镜进行图像采集。通过该设置和2kb /μm的DNA延伸,一个像素对应于(6.45 / 40)×2 = 323bp。

数据分析

  1. 使用Prism 7.0(GraphPad)或R Statistical软件进行BrdU轨迹长度和原点间距离的统计分析。由于BrdU轨道长度和原点间距离不显示高斯分布,因此使用非参数检验(如曼 - 惠尼等级和)测试样本之间差异的统计学显着性。
  2. 为了确定叉速度,将一侧(单叉)上的IdU轨迹的CldU轨迹的中间长度除以第二脉冲的持续时间(通常为20分钟)。 CldU单独跟踪,并且不考虑用两个IdU轨道包围的CldU轨道。 CldU和IdU也可用于测量起源距离(IOD),全球叉密度(Bialic等人,2015),并监控复制叉停止/重启(Techer等人,2013; Tuduri等人,2009)(图3)。
  3. IdU和CldU脉冲可用于可视化姐妹复制叉的进展。在正常生长条件下,这些信号通常对称,因为姐妹叉以相同的速度进行。当检测到非对称模式时,这表明增加复制叉暂停或停滞(Tuduri等人,2009)。叉停止速率的增加可以表示为最长与最短CldU轨迹的比率,或者以CARYU脉冲中两个姐妹叉所覆盖的距离的散点图形式表示。大比例或分散的散点图指示叉停止增加。
  4. IdU / CldU双标记的另一个有用的应用是基因毒性侮辱后的DNA复制恢复的分析(Sidorova等人,2013; Tourriere等人,2005)。在该测定中,在IdU存在下,将细胞暴露于基因毒性试剂或复制抑制剂如羟基脲(HU),甲磺酸甲酯(MMS)或喜树碱(CPT))中,并在CldU存在下释放到新鲜培养基中。这种方法可用于监测细胞重新启动停滞的叉的能力,激活晚期或休眠的起源,并在药物释放后完成DNA复制。
  5. 最近已经报道,停滞的叉可以被核酸酶加工。可以在IdU和CldU脉冲之后显现由诸如HU或CPT等基因毒素药物捕获的叉的新生DNA的切除(Ray Chaudhuri等,2016; Schlacher等人, >。,2011)。

食谱

  1. 酵母细胞
    1. YPAD
      1%酵母提取物
      2%Bacto蛋白胨
      0.005%腺嘌呤
      2%葡萄糖
      双蒸水
    2. BrdU
      在Milli-Q H 2 O(新鲜制备)中的10mg / ml储备溶液
    3. 酵解酶缓冲液
      50mM柠檬酸磷酸盐缓冲液,pH 5.6
      50mM EDTA,pH 8.0
      1.2 M山梨醇
      1 mg / ml酵解酶20T(使用酶粉)
    4. NZ(无酵解酶)缓冲液
      50mM柠檬酸磷酸盐缓冲液,pH 5.6
      50mM EDTA,pH 8.0
      1.2 M山梨醇
    5. 1M磷酸钾缓冲液pH7.0; 100 ml
      61.5ml KH 2 PO 4,2M(M.W. = 136)
      38.5ml K 2 HPO 4,2M(M.W. = 174)
    6. 0.1M柠檬酸磷酸盐缓冲液pH 5.6; 100 ml
      58ml 0.2M Na 2 HPO 4(二元; M.W = 142)
      42ml 0.1M柠檬酸(C 6 H 8 O 7 H 2 O 2 O; MW = 210 )
    7. 蛋白酶K缓冲液
      125 mM EDTA pH 9.5
      1% - 月桂基肌氨酸 1 mg / ml蛋白酶K

  2. 人类细胞
    1. BrdU:25mM储备液,PBS,10%DMSO。储存于-20°C
    2. IdU:PBS中的25mM储备溶液,10%DMSO。储存于-20°C
    3. CldU:200mM PBS溶液,10%DMSO。储存于-20°C
    注意:在实验过程中直接在培养基中稀释。
    1. 蛋白酶K缓冲液
      10 mM Tris pH 7.5
      100 mM EDTA pH 8.0
      1% - 月桂基肌氨酸 1 mg / ml蛋白酶K

  3. 人/酵母细胞共有
    1. LMP琼脂糖,新鲜制备,检查体积:酵母细胞水中的2%或人类细胞的PBS。 LMP可以在微波炉中以非常低的功率熔化;请确保避免溶液沸腾的问题
    2. TE 50
      10mM Tris-HCl,pH7.0
      50mM EDTA,pH 8.0
    3. 1x TE
      10mM Tris-HCl,pH7.0
      1mM EDTA,pH 8.0
    4. 10倍MES缓冲液pH 6
      70 ml 500 mM MES水合物
      30 ml 500 mM MES钠盐
      用500 mM MES钠盐调节至pH 6
    5. 1 N NaOH(新鲜制备)
    6. PBS / T
      PBS pH 7.4
      0.1%Triton X-100
    7. 抗体(PBS / T稀释)
      #01小鼠抗BrdU克隆B44 IgG1
      #02大鼠抗BrdU克隆BU1 / 75
      #03小鼠抗ssDNA(poly dT)IgG2a
      #04山羊抗鼠Alexa 488
      #05山羊抗鼠Alexa 546
      #06山羊抗小鼠IgG2a Alexa 647
      #07山羊抗小鼠IgG1 Alexa 546
    8. 检测CldU / IdU / ssDNA
      一抗:
      CldU:#01(1/20)
      IdU:#02(1/20)
      ssDNA:#03(1/50)&nbsp;
      二抗:
      #07(1/50)
      #04(1/50)
      #06(1/50)
    9. 检测BrdU / ssDNA
      一抗:
      BrdU:#02(1/20)
      ssDNA:#03(1/50)
      二抗:
      #04(1/50)
      #05(1/50)

致谢

我们感谢Pasero实验室的成员对本协议的优化做出了贡献,该方案根据其他几个组织的协议(包括Bensimon,Schwob,Debatisse和Nurse实验室)进行了改进。我们感谢蒙彼利埃RIO成像设施的支持和蒙彼利埃的DNA精梳设施,提供硅藻土盖玻片。这项工作得到了Agence Nationale pour la Recherche(ANR),国家癌症研究所(INCa),Ligue contre le Cancer(EquipelabelliséeLIGUE,2017)和MSDAvenir基金的资助。

参考

  1. Bensimon,A.,Simon,A.,Chiffaudel,A.,Croquette,V.,Heslot,F.and Bensimon,D.(1994)。通过移动界面对DNA的对准和敏感检测。 科学 265(5181):2096 -2098。
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引用:Tourrière, H., Saksouk, J., Lengronne, A. and Pasero, P. (2017). Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing. Bio-protocol 7(11): e2305. DOI: 10.21769/BioProtoc.2305.
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