Materials and Reagents

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, Invitrogen^TM, 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, Invitrogen^TM, 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)
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, Bacto^TM, 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)
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

Centrifuge (Eppendorf, model: 5810 R )
Microcentrifuge (Eppendorf, model: MiniSpin^® )
Thermoblock (thermomixer comfort Eppendorf with 2 ml; 15 ml and 50 ml block)
Cell counter (CASY^® Modell TT - Cell Counter, OLS for yeast cells or Malassez hemocytometer [VWR, catalog number: 631-0975 ] for human cells)
Pasteur pipette Rubber bulb (Dominique Dutscher, catalog number: 042250 )
Microwave
Roller mixer (Cole-Parmer, Stuart, model: SRT9D )
Leica DM6000B microscope (Leica Microsystems, model: DM6000B )
CoolSNAP HQ CCD camera (Photometrics, model: CoolSNAP HQ CCD )
P1000, P200, P20 pipetman
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)
Metal coverslip holder
Humid chamber (StainTray slide staining system) (Sigma-Aldrich, catalog number: Z670146-1EA )
Hybridization oven (Shake ‘n’ Stack^TM Hybridization Ovens) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 6240TS )
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)
Microscope (Nikon Instruments, model: YS100 )

Software

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

Procedure

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 T_1h and T_2h 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.
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
Procedures common to yeast and human cells
1. Melting of agarose plugs containing genomic DNA
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.
4. Image acquisition and data analysis

Data analysis

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.
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).
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.
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.
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

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
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
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

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