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In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication
真核先导链/后随链DNA复制的体外分析   

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Abstract

The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other proteins. We have previously established the mechanisms by which the polymerases ε and δ are targeted to their ‘correct’ strands, as well as quality control mechanisms that evict polymerases when they associate with an ‘incorrect’ strand. Here, we provide a practical guide to differentially assay leading and lagging strand replication in vitro using pure proteins.

Keywords: Eukaryotic DNA replication(真核DNA复制), Replisome assay(复制体测定), CMG helicase(CMG解旋酶), DNA polymerase(DNA聚合酶), RFC clamp loader(RFC钳装载器), PCNA sliding clamp(PCNA滑动钳), Leading strand(先导链), Lagging strand(后随链)

Background

Using pure proteins from Saccharomyces cerevisiae, our lab was the first to reconstitute a functional eukaryotic DNA replisome, a ~2 MDa complex that includes the 11-subunit CMG helicase (complex of Cdc45, Mcm2-7, GINS heterotetramer), the 4-subunit DNA polymerase (Pol) ε, the 4-subunit Pol α-primase, the PCNA (Proliferating Cell Nuclear Antigen) clamp homotrimer ring shaped processivity factor that encircles duplex DNA, the 5-subunit clamp loader RFC (Replication Factor C) that uses ATP to open and close the PCNA sliding clamp ring onto primed sites for polymerase processivity, and the RPA (Replication Protein A) heterotrimeric single-strand DNA binding protein that removes secondary structure obstacles to DNA polymerase progression. In our initial studies we discovered that Pol ε is targeted to CMG on the leading strand after priming by Pol α-primase, while Pol δ is targeted to PCNA clamps on the lagging strand primed sites (Georgescu et al., 2014; Langston et al., 2014). We next reconstituted a functional coupled leading/lagging strand replisome which included the 4-subunit Pol α-primase and 3-subunit Pol δ, in which we demonstrated that Pol ε is inactive on the lagging strand and Pol ε is inactive on the leading strand (Georgescu et al., 2015). Interestingly, the Pol α-primase, which lacks proofreading activity, was active with CMG on both strands, but when either Pol ε or Pol δ are present, which both contain a proofreading 3’-5’ exonuclease for high fidelity synthesis, they take over from the low fidelity Pol α-primase on either strand. However, Pol ε and Pol δ only performed optimal synthesis when on their respective correct strands (Georgescu et al., 2015). In a subsequent study we characterized the unprecedented quality control mechanisms that exclude these polymerases from incorrect strands, a job that bacterial replisomes do not need to do because they utilize identical polymerases for both strands (Schauer et al., 2017). We found that on the lagging strand, Pol ε is excluded from primed sites by competition with the RFC clamp loader for the primer terminus, while CMG binds and protects Pol ε from RFC inhibition on the leading strand. In contrast Pol δ is preferentially targeted to PCNA on lagging strand primed sites through a tight binding affinity to PCNA clamps that is over 20-fold greater than the PCNA affinity to Pol ε and is unaffected by competition by the RFC clamp loader (Schauer et al., 2017). Interestingly, no stabilizing interaction with CMG exists for Pol δ (Schauer et al., 2017). Furthermore, Pol δ is less stable on a completed DNA than when idling at a primer terminus or extending a primer. Specifically, Pol δ is known to be stable for over a half hour with PCNA, consistent with its high processivity, but upon completing replication of a section of DNA, and bumping into a completed dsDNA region, it dissociates rapidly (i.e., < 1 min) from PCNA-DNA in a process referred to as collision release (Langston and O’Donnell, 2008; Langston et al., 2014).This inherent instability of Pol δ-PCNA upon completing replication may serve as a quality control to destabilize Pol δ-PCNA on the leading strand because Pol δ-PCNA is much faster than CMG unwinding and will be in a constant state of having completed DNA and collision with CMG (Schauer et al., 2017). Destabilization of Pol δ-PCNA when there is no more DNA to be extended should not be taken to mean that Pol δ instantly ejects from PCNA. For example, Pol δ-PCNA remains on DNA for a few seconds to fill-in short gaps upon RNA removal at 5’ ends of Okazaki fragments (Stodola and Burgers, 2016).

In interrogating these various activities, we observed that CMG does not load onto small (100-200 bp) rolling circle replication substrates, which are often used to study replisome behavior in bacterial systems. Thus, we turned to linear DNA fork assays as an alternative to address biochemical mechanisms in eukaryotic replication. These assays enable one to easily separate leading from lagging strand replication activity by synthesis of a long linear DNA that has no dC in one strand, and thus no dG in the other strand. By doing so, one can specifically monitor leading or lagging strand synthesis depending on the radioactive deoxyribonucleoside triphosphate (dNTP) used in the assay.

Materials and Reagents

  1. Razor blade
  2. 1.57 mm OD polyethylene tubing (e.g., Clay Adams® Intramedic®, BD, catalog number: 427431 )
  3. Sephadex microcentrifuge columns (Illustra Microspin G-25) (GE Healthcare, catalog number: 27-5325-01 )
  4. Plastic wrap (e.g., Fisherbrand Clear Plastic Wrap, Fisher Scientific, catalog number: 22-305654 )
  5. C-fold paper towels (e.g., Scott paper towels, KCWW, Kimberly-Clark, catalog number: 01510 )
  6. Positively charged nylon DNA blotting membrane (Hybond-N+, 30.0 x 50.0 cm) (GE Healthcare, catalog number: RPN3050B )
  7. Chromatography transfer paper (Whatman 3MM, 46.0 x 57.0 cm) (GE Healthcare, catalog number: 3030-917 )
  8. Syringe tip (e.g., B-D 18 G 1 ½ PrecisionGlide® Needle) (BD, catalog number: 305196 )
  9. phiX174 virion DNA, 1 mg/ml (New England Biolabs, catalog number: N3023L )
  10. Phi29 DNA polymerase (New England Biolabs, catalog number: M0269S )
  11. 100 mM dNTP (deoxynucleotide triphosphate) set (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0181 )
  12. 1 μM CMG (Cdc45 Mcm2-7 Gins) helicase (see Georgescu et al. [2014] for purification details)
  13. pUC19, 1 mg/ml (New England Biolabs, catalog number: N3041L )
  14. BsaI-HF with CutSmart buffer (New England Biolabs, catalog number: R3535L )
  15. ‘blockLd’ oligo*
  16. ‘blockLg’ oligo*
  17. ‘Pr1B’ oligo*
  18. ‘160Ld’ oligo*
  19. ‘91Lg’ oligo*
  20. ‘Fork primer’ oligo*
  21. Nucleotide-biased template (synthesized by Biomatik, Wilmington DE)*

*Note: See Supplementary file 1.

  1. T4 ligase, including 10x ligase buffer (New England Biolabs, catalog number: M0202M )
  2. 100 mM ATP (GE Healthcare, catalog number: 27-2056-01 )
  3. 0.5 M EDTA, disodium salt (Sigma-Aldrich, catalog number: E5134 )
  4. 5 M NaCl (Sigma-Aldrich, catalog number: S9888 )
  5. Sepharose 4B size exclusion chromatography resin (GE Healthcare, catalog number: 17012001 )
  6. 1 kb MW marker (New England Biolabs, catalog number: N3232L )
  7. Ethidium bromide (EthBr, 10 mg/ml) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15585011 )
  8. T4 kinase and 10x T4 kinase buffer (New England Biolabs, catalog number: M0201L )
  9. 32P-γ-ATP, 3,000 Ci/mmol, 3.3 μM (PerkinElmer, catalog number: BLU002A )
  10. Type XI low-melt agarose (Sigma-Aldrich, catalog number: A3038 )
    Note: This product has been discontinued.
  11. BtsCI (New England Biolabs, catalog number: R0647L )
  12. β-Agarase I (New England Biolabs, catalog number: M0392L )
  13. 3 M sodium acetate (CH3COONa), pH 5.2 (Sigma-Aldrich, catalog number: S2889 )
  14. Isopropanol (Sigma-Aldrich, catalog number: 190764 )
  15. Glycogen, molecular biology grade (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0561 )
  16. Ethanol (Sigma-Aldrich, catalog number: E7023 )
  17. 1 μM RFC (Replication Factor C; see Georgescu et al. [2014] for purification details)
  18. 5 μM PCNA (Proliferating Cellular Nuclear Antigen; see Georgescu et al. [2014] for purification details)
  19. 2 μM Pol ε (see Georgescu et al. [2014] for purification details)
  20. 2 μM Pol δ (see Georgescu et al. [2014] for purification details)
  21. 2 μM Pol α (see Georgescu et al. [2014] for purification details)
  22. 20 μM RPA (Replication Protein A; see Georgescu et al. [2014] for purification details)
  23. 32P-α-dCTP, 3,000 Ci/mmol, 3.3 μM (PerkinElmer, catalog number: BLU013H )
  24. 32P-α-dGTP, 3,000 Ci/mmol, 3.3 μM (PerkinElmer, catalog number: BLU514H )
  25. LE agarose (BioExpress, GeneMate, catalog number: E-3120-500 )
  26. 10 N sodium hydroxide (NaOH) (Fisher Scientific, catalog number: SS255 )
  27. Glycerol
  28. Xylene cylanol
  29. Tris-HCl, pH 8.0
  30. Tris base (RPI, catalog number: T60040-500.0 )
  31. Boric acid (RPI, catalog number: B32050-5000.0 )
  32. Sodium citrate
  33. 1-Butanol
  34. Tris-acetate, pH 7.5
  35. Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S )
  36. Tris(2-carboxyethyl)phosphine (TCEP) pH 7.5
  37. 100 mM dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0861 )
  38. Potassium glutamate
  39. Magnesium acetate
  40. 1% SDS
  41. 6x gel loading dye (see Recipes)
  42. TE buffer, pH 8.0 (see Recipes)
  43. 10x TBE (Tris/Borate/EDTA; see Recipes)
  44. DNA elution buffer (see Recipes)
  45. 20x SSC (see Recipes)
  46. 1-Butanol saturated water (see Recipes)
  47. 5x TDBG (see Recipes)
  48. 10x MK (see Recipes)
  49. dA/dC mix (see Recipes)
  50. dT/dG mix (see Recipes)
  51. T/G/C mix (see Recipes)
  52. Stop solution (see Recipes)
  53. Alkaline running buffer (see Recipes)

Equipment

  1. Heating block (e.g., VWR, catalog number: 12621-084 )
  2. Fraction collector (e.g., Gilson, model: F203B )
  3. Variable mode gel imager (e.g., GE Typhoon)
  4. UV-vis spectrophotometer (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )
  5. Vacuum dessicator (e.g., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5309-0250 )
  6. UV light box
  7. UV blocking face shield (e.g., Sigma-Aldrich, catalog number: F8142 )
    Note: This product has been discontinued.
  8. Microcentrifuge
  9. Conductivity meter (e.g., Radiometer Medical, model: CDM 80 )
  10. Temperature controlled water bath with microcentrifuge tray (e.g., LabX, model: Lauda E100 and Brinkman 30x x 1.5 ml)
    Manufacturer: LAUDA-Brinkmann, model: E100 .
  11. Phosphorimaging screen (GE Healthcare)
  12. Phosphor imager (e.g., GE Typhoon)
  13. A heavy weight
    Note: We use giant lead blocks that we found; a ~50 lb dumbell would work.
  14. Computer with ImageJ and spreadsheet software (e.g., Apache Open Office) installed
  15. 1 x 30 cm glass column (e.g., glass Econo-Columns®) (Bio-Rad Laboratories, catalog number: 7371032 )
  16. 100 ml agarose gel electrophoresis apparatus
  17. Styrofoam box large enough to fit 100 ml agarose gel electrophoresis apparatus
  18. Electrophoresis power supply (e.g., Pharmacia Biotech, catalog number: EPS 3500 XL )
  19. Analytical balance
  20. Protective plexiglass samples shield
  21. 20 x 14 cm horizontal agarose gel electrophoresis apparatus (C.B.S. Scientific, catalog number: SGU-014T-02 )

Software

  1. ImageJ (https://imagej.nih.gov/ij/docs/menus/analyze.html#gels)

Procedure

  1. Linear forked DNA template construction
    We primarily use two types of linear forked DNA substrates to assay leading/lagging strand replication: a linear pUC based natural sequence substrate (referred to below as 3kbf, for 3 kb fork), and a 3.2 kb nucleotide-biased, leading/lagging strand substrate that is generated synthetically such that one strand has no dC and the other strand has no dG (referred to as 3nbf, for 3 kb nucleotide biased fork). The 3nbf substrate can be used in leading and lagging strand assays to monitor each strand independent of the other (see Figure 1). We sometimes prefer to use a 5’-32P-labeled primer to quantify leading strand replication as it is a direct reporter of primer elongation compared to use of 32P-dNTPs because it lacks potential end-labeling artifacts. Though both substrates can accommodate a primer, we typically only use the 3kbf substrate for this purpose because it is much simpler to prepare (i.e., does not require purification of the 3 kb synthetic nucleotide biased section from a low melt gel). Although the template length and composition differs between these substrates, we use the same small synthetic fork construct that is ligated onto either of these substrates.
    The fork is composed of a 160 mer for the leading strand and a 91 mer for the lagging strand. The leading strand contains a 3’ (dT)34 tail for CMG loading, a 37 nucleotide primer annealing site followed by a 4 nucleotide region lacking dC and dA residues and a 10 nucleotide region lacking dC and dT residues (see Recipes for oligonucleotide composition). The 4 nucleotide region upstream of the primer allows ‘idling’ of Pol ε on the leading strand by only including dATP and dCTP (i.e., to prevent the 3’-5’ exonuclease activity of Pol ε from degrading the primer). The 10 nucleotide region prevents ‘runaway’ Pol ε after potential misincorporation (i.e., even with dTTP withheld). Following these sequences, we include a (dT)40 spacer to the fork junction designed to accommodate the footprint of CMG (~10 nm). The lagging strand contains a (dT)60 5’-tail and 35 bp complementary to the 5’ end of the leading strand. The leading strand fork template oligo typically contains four 3’ phosphorothioate bonds to protect against exonuclease activity inherent in DNA polymerases by substituting a sulfur atom for a non-bridging phosphate oxygen atom in the oligonucleotide backbone (available as a modification from DNA synthesis companies, such as IDT). See Figure 1 for a schematic of the nucleotide biased substrate.


    Figure 1. Schematic of nucleotide biased substrate. The “3nbf” substrate, including the oligonucleotides described in the text, is shown with the relevant features highlighted. The bias is designed such that 32P-α-dCTP will only be incorporated during leading strand synthesis, while 32P-α-dGTP will only be incorporated during lagging strand synthesis. Dotted lines indicate restriction enzyme cut sites discussed in the text. Note that the “3kbf” substrate, which is constructed using the same synthetic fork but using a 2,686 bp template with natural (unbiased) sequence, is not shown. 

    3kbf substrate construction (typically used for leading strand reactions using a 32P-primer)
    1. Resuspend ‘160Ld’ to 20 μM in TE buffer (see Recipes)
    2. Resuspend ‘91Lg’ to 500 μM in TE buffer.
    3. Digest 35 pmol pUC19 with 4 μl BsaI-HF (20 U/μl) in 1x CutSmart buffer in 40 μl total at 37 °C for 4 h.
    4. Heat inactivate BsaI-HF at 75 °C for 30 sec.
    5. Add 17.5 μl (350 pmol) 160Ld and 4.2 μl (2,100 pmol) 91Lg in TE, and add 238.3 μl TE to 400 μl.
      Note: The 10:1 excess of fork to template is required to prevent concatamerization of linearized pUC19, and the 6:1 excess of 91Lg to 160Ld is to ensure all ligated 160Ld is annealed to 91Lg.
    6. Anneal fork to template by heating to 95 °C for 5 sec and let heat block cool to RT on benchtop.
    7. Ligate fork onto linear pUC19 by adding 15 μl T4 ligase, 36 μl 10x CutSmart buffer, 4 μl 100 mM ATP (1 mM final), adding TE to 400 μl volume. Incubate at 16 °C for ≥ 16 h.
    8. Add 10 μl 0.5 M EDTA to chelate Mg2+.
    9. Heat inactivate T4 ligase at 65 °C for 20 sec.
    10. Add 10 μl 5 M NaCl to bring [NaCl] to > 100 mM (necessary to prevent nonspecific binding to Sepharose 4B column).
    11. Pour a ~20 ml Sepharose 4B column and equilibrate with > 2 column volumes (CV) of cold DNA elution buffer (see Recipes).
    12. Adapt syringe tip to end of the column. Adapt polyethylene tubing to syringe tip and couple to fraction collector with the minimum length required for the device to reach all tubes.
    13. Add 420 μl reaction to 20 ml Sepharose 4B column, ensuring that buffer has completely seeped into the column bed before loading the reaction volume.
    14. After the sample has completely penetrated the column bed, add and elute with cold DNA elution buffer.
    15. Collect ~227 μl fractions (10 drops/fxn for the tubing we use). Under these conditions, the template should elute around fraction 20.
    16. Run 7 μl of each fraction on a 1% TBE agarose gel alongside a 1 kb MW marker (with gel loading buffer) at 100 V until the bromophenol blue marker reaches 2/3 the way down.
    17. Stain with 2-5 μl EthBr in 1x TBE and visualize on a gel imager.
      Notes:
      1. The gel shift of the linearized plasmid from ligated fork DNA is very small, so do not expect to see a difference. However the ligated fork DNA will elute much earlier than the excess of free synthetic forked DNA, which can be visualized on a properly stained/destained gel.
      2. We have quantified fork ligation with radiolabeled fork, and it is highly (> 95%) efficient, thus the 3 kb template is attached to the DNA fork under the conditions described above.
      3. Nonetheless, we encourage users to actually monitor their ligation efficiency using 32P-labelled fork DNA to be sure of ligation, and use of phi29 DNA polymerase in the extension of a 32P-labeled primer (i.e., see below) to further ensure ligation efficiency.
    18. Save fractions containing the ~2.7 kb band (this will be the sole visible band amongst all the fractions) and measure DNA concentration via A260 using a UV/Vis spectrophotometer.
    19. Follow manufacturer’s protocols to 5’-end-label the primer with T4 kinase and 32P-γ-ATP. We typically use a 25 μl reaction with a final primer concentration of 400 nM. Purify away free 32P-γ-ATP with a G-25 column that has been equilibrated with TE, according to the manufacturer’s protocol.
    20. To prime the 3kbf substrate with 32P-primer, add the required amount of 3kbf substrate with an equimolar amount of 32P-primer. Minimize the total volume, add 20x SSC (see Recipes) to a final 1x concentration, heat to 95 °C and cool to RT on the benchtop.

    3nbf substrate construction
    1. Resuspend ‘160Ld’ to 20 μM in TE buffer.
    2. Resuspend ‘91Lg’ to 500 μM in TE buffer.
    3. Digest ~400 μg plasmid containing 3.2 kb nucleotide-biased template (see Supplementary file 1 for sequence) with 12 μl (20 U/μl) BsaI-HF in 1x CutSmart buffer in 400 μl total at 37 °C for 7 h.
    4. Before the previous step is finished, run 0.5 μl on a 0.8% TBE agarose gel at 100 V until the bromophenol blue reaches 2/3 the way down to ensure complete cutting. If cutting is not complete, add more BsaI-HF and digest more; repeat until cutting is complete.
    5. Add 12 μl BtsCI (20 U/μl) plus enough 10x CutSmart buffer and to compensate for added volume. Incubate at 50 °C for 6 h.
    6. Before digest is finished, run 0.5 μl on gel as in step 4 (Procedure A, 3nbf substrate construction) to ensure complete digest.
    7. Add 21.5 μl 0.5 M EDTA.
    8. Place sample in vacuum desiccator for 6-8 h to reduce volume by roughly 1/3.
    9. Run entire sample on 1.2% TBE low-melt agarose gel (well is 8 of 15 teeth of comb taped together). Add 2 μl 10 mg/ml EthBr to 100 ml gel. Pre-chill TBE, adding 2 μl 10 mg/ml EthBr for every 100 ml running buffer used. Run at 40-50 V with the apparatus sitting in a Styrofoam box full of ice-water (a cold room is not sufficient) until the bromophenol blue reaches the bottom third of the gel. Donning UV-protective face shield, visualize on a UV light box and cut out the largest (3,260 bp) band out with a razor blade. Weigh the gel slice for the next steps.
    10. Melt gel slices at 65 °C.
    11. Weigh melted slices and add 1/9 V Tris-HCl, pH 6.8 and 1/224 V 0.5 M EDTA, under the rough approximation that the density of the gel is 1 g/ml.
    12. Cool to 42 °C.
    13. Add ~5 μl β-agarase I per gram of gel. Incubate at 42 °C for 5-7 h.
    14. Repeat step 13 (Procedure A, 3nbf substrate construction).
    15. Isopropanol precipitate the DNA fragment.
      Add 1/10 the volume of 3 M sodium acetate, pH 5.2, followed by an equal volume of isopropanol. Incubate for 1 h at -20 °C. Spin in a microcentrifuge at ≥ 15,000 x g for 30 min.
      Note: If too much agar has not been digested sufficiently to small oligosaccharides, you will see a lot of white precipitate. You will need to finish the precipitation, re-suspend in aqueous solution, and go back to step 13 (Procedure A, 3nbf substrate construction) to perform a further β-agarase I digestion.
    16. Decant supernatant and resuspend pellet in TE pH 8.0.
    17. Remove EthBr by extraction with an equal volume of 1-butanol saturated water (see Recipes). Gently vortex to mix. Spin at 1,500 x g, saving the bottom solution. Continue extracting until there is no visible pink color. Extract 3 more times.
    18. Ethanol precipitate
      Add 1 μl glycogen (20 mg/ml), 1/10 volume of 3 M sodium acetate pH 5.2, followed by an equal volume of cold ethanol. Incubate for 1 h at -20 °C. Spin in a microcentrifuge at ≥ 15,000 x g for 30 min.
    19. Resuspend DNA in 150 μl TE pH 8.0.
    20. Run 2 μl, 4 μl, and 8 μl of 1:10 and 1:100 dilutions on 0.8% TBE agarose gel to estimate concentration.
    21. Use 35 pmol of this material for the subsequent steps.
    22. Add 350 pmol 160Ld and 2,100 pmol 91Lg in TE, and add TE to 300 μl.
      Note: The 10:1 excess of fork to template is required to prevent concatamerization, and the 6:1 excess of 91Lg to 160Ld is to ensure all ligated 160Ld is annealed to 91Lg.
    23. Anneal fork to template by heating to 95 °C for 5 sec and let heat block cool to RT on benchtop.
    24. Ligate fork onto linear pUC19 by adding 15 μl T4 ligase, 36 μl 10x CutSmart buffer, 4 μl 100 mM ATP (1 mM final), adding TE to 400 μl volume. Incubate at 16 °C for at least 16 h.
    25. Add 10 μl 0.5 M EDTA to chelate Mg2+.
    26. Heat inactivate T4 ligase at 65 °C for 20 sec.
    27. Add 10 μl 5 M NaCl to bring [NaCl] to >100 mM (necessary to prevent nonspecific binding to Sepharose 4B column).
    28. Adapt syringe tip to end of column. Adapt polyethylene tubing to syringe tip and couple to fraction collector with the minimum length required for the device to reach all tubes.
    29. Pour a ~20 ml Sepharose 4B column and equilibrate with > 2 column volumes (CV) of cold DNA elution buffer.
    30. Add 420 μl reaction to 20 ml Sepharose 4B column, ensuring that buffer has completely seeped into the column bed before adding.
    31. After the sample has completely seeped into the column bed, elute with cold DNA elution buffer.
    32. Collect ~227 μl fractions (10 drops/fraction).
    33. Run 7 μl of each fraction on a 1% TBE agarose gel alongside a 1 kb MW marker (with gel loading buffer) at 100 V until the bromophenol blue marker reaches about 2/3 the way down.
    34. Stain with 2-5 μl ethidium bromide in 1x TBE and visualize on a gel imager.
      Note: The gel shift of the linearized plasmid from fork tethering is very small, so do not expect to see a difference. It is also difficult to visualize free fork on this gel. We have quantified fork ligation with radiolabeled fork, and it is extremely (> 95%) efficient, thus the template should have fork attached.
    35. Save fractions containing the ~3.2 kb band (this should be the only visible band) and measure concentration via A260 on the spectrophotometer.
    36. Blunt the non-fork end of the forked template with blocking oligos. Resuspend ‘blockLd’ and ‘blockLg’ to 10 μM in TE pH 8.0. Add each oligo at a 2:1 oligo: template molar ratio. Heat to 85 °C for 5 sec, cool to RT on benchtop.
      Note: This step is required to prevent end-labeling of the BtsCI cut site during 32P-α-dNTP incorporation (i.e., Pol could use the non-ligated 5’ overhang as a template), however, it may be more practical to order the nucleotide-biased sequence that is removed by a blunt-cut site at this region instead.
    37. Ligate block oligos by adding 10 μl T4 ligase. Add 1/10 volume 10x T4 ligase buffer and supplement with 1 mM ATP. Incubate at 16 °C for at least 16 h.
    38. Heat inactivate T4 ligase at 65 °C for 20 sec.

    Primed phiX174 (model lagging strand) construction
    In addition to CMG-directed leading/lagging strand synthesis, we also find it useful to use a primed, RPA-coated circular 5.4 kb ssDNA (phiX174 virion circular ssDNA) as a model lagging strand in order to isolate potential lagging strand activity in the absence of the full replisome context.
    1. Resuspend Pr1B to 5 μM in TE pH 8.0 (see Recipes).
    2. Add 12 pmol Pr1B to 10 pmol phiX174 DNA (a 1.2:1 primer: template molar excess). Add buffer TE pH 8.0 to 57 μl, and 3 μl 20x SSC. Heat to 95 °C for 5 sec, cool to RT on benchtop.

  2. Replication assays
    We assume the reader has purified the full repertoire of proteins required to reconstitute the eukaryotic replisome, including at a minimum CMG helicase, Pols ε, δ and α, RFC, PCNA and RPA. Other accessory proteins such as Mcm10 and the Mrc1-Tof1-Csm3 complex will stimulate replication but are not an absolute requirement for activity. The purification and storage conditions of these proteins is beyond the scope of this protocol and can vary by specific experimental requirements, however, full purification details of the proteins are described in (Georgescu et al., 2014 and 2015) and (Langston et al., 2014). Make note of the concentrations and salt concentration of all protein stocks. Aliquot desired fractions into small (~10-25 μl) aliquots, flash freeze in liquid nitrogen, and store at -80 °C.
    We prefer to use 25 μl as a standard experimental volume to minimize protein consumption while keeping the working volumes manageable. Volumes as low as 15 μl or as high as 40 μl will also work fine. For the protocols listed here, we often separate into three phases: 1) Helicase loading on the substrate. 2) Polymerase-clamp loading, i.e., polymerase, RFC clamp loader, PCNA clamp, and 2 or 3 dNTPs that allow ‘idling’ (repeated cycles of nucleotide incorporation and proofreading) of the polymerase on the template to prevent primer degradation, and 3) Replication initiation by adding the full complement of 4 dNTPs.
    The first two stages may be combined if necessary, but ideally CMG loads onto forked DNA (or RPA coats ssDNA in the case of the lagging strand model) during incubation for 5-10 min or longer.
    On the other hand, the second stage contains exonuclease proficient polymerases, which will degrade the substrate and/or its primer, so at least two dNTPs and a moderate incubation time with these enzymes is necessary.
    The third stage initiates ongoing replication, by adding all remaining dNTPs, and in the case of fork assays, also RPA. In the examples given here, the first substrate loading phase is prepared and split into tubes as necessary, the second enzymatic phase is subsequently added, and the third initiation phase is added as the reaction timer starts. This staging method helps minimize errors derived from pipetting of small volumes, and then the splitting the mixed phases into relatively large volumes.
    Because enzymes are added with respect to the final volume, it may also facilitate use of low volume such that CMG loading onto DNA can be performed at relatively high concentration. Since each phase is prepared separately before mixing, it is important to independently add salts and buffers (i.e., TDBG, MK, and any additional salts required) to each phase before adding proteins in order to prevent precipitation, aggregation, etc. that may occur if adding proteins directly to low ionic strength, depending on the protein behavior/solubility requirements.
    Assuming there are differences in protein concentrations between experimental groups (e.g., during a protein titration), it is important to correct for incoming salt by adding the appropriate amount of salt to the tubes with lower protein concentrations–replication reactions are sensitive to salt conditions. Therefore, it is essential to ensure that salt has been correctly balanced.
    1. Salt calibration
      Make a standard curve of conductivity vs. salt concentration for each type of salt used in the experiment and/or protein storage, and correct experiments.
      1. Prepare a serial dilution of salt, e.g., 0.1 M to 1 M.
      2. Add 10 μl each concentration to 990 μl of ddH2O.
      3. Measure conductivity, in mS/cm using a conductivity meter and plot against salt concentration.
      4. Compare conductivity of protein and/or experimental sample to standard curve. Adjust reactions with the appropriate salt as necessary.
    2. Leading and/or lagging strand replication; example 3-point timecourse leading/lagging (3nbf) experiment.
      Note: The following protocol is identical for the 3kbf (forked linear template), apart from the addition of radionucleotides in the 3nbf experiment, as noted. Do not initiate replication until each phase and time point stop tube has been prepared.
      1. Substrate loading (see Table 1A).

        Table 1A. Substrate loading phase of leading/lagging strand replication


        Load 0.5-2.0 nM 3nbf substrate with 10-30 nM CMG helicase (phase 1; 11 μl each).
        Example: In the following order, add 32.0 μl ddH2O, 13.2 μl 5x TDBG (see Recipes) (1x final), 6.6 μl 10x MK (see Recipes) (1x final), 11.3 μl 3kbf/3nbf (1.5 nM final), and 3.0 μl 1 μM CMG (20 nM final) for a final volume of 2 groups of 3 x 11 μl = 66 μl. Incubate at 30 °C for 10 min in the water bath. Split into two groups of 33 μl.
        Note: When splitting phases into separate groups, it may help to add one reaction volume to account for volume loss, e.g., split a volume that is sufficient for 7 reactions into 2 groups of 3.
      2. Enzyme loading (see Table 1B).

        Table 1B. Enzyme loading phase of leading/lagging strand replication


        Add RFC/PCNA/Pol ε/α/δ.
        Example: In the following order, add 40.0 μl ddH2O, 14.0 μl 5x TDBG (1x final), 7.0 μl 10x MK (1x final), 2.5 μl dA/dC mix (see Recipes) (60 μM final), 1.3 μl 1 μM RFC (5 nM final), 1.3 μl 5 μM PCNA (25 nM final), 1.3 μl 2 μM Pol α (10 nM final), 1.3 μl 2 μM Pol ε (10 nM final), and 1.3 μl 2 μM Pol δ (10 nM final) for a final volume of 10 x 7 μl reactions = 70 μl. Add 21 μl (3 x 7 μl) to each reaction tube, mixing thoroughly with pipette. Incubate at 30 °C for 1 min.
        Note: The addition of dATP and dCTP enables polymerase idling on the primed substrate while allowing RFC to load PCNA with dATP.
      3. Replication initiation (see Table 1C).

        Table 1C. Replication initiation phase of leading/lagging strand replication


        Initiate replication by adding remaining dNTPs and RPA. Monitor with 32P-α-dCTP (leading strand) or 32P-α-dGTP (lagging strand) incorporation.
        Example: To each of two tubes, in the following order, add 14.0 μl ddH2O, 5.6 μl 5x TDBG (1x final), 2.8 μl 10x MK (1x final), 0.6 μl dT/dG mix (see Recipes) (60 μM final), 5 μl ATP (5 mM final), 2 μl of T/G/C mix (see Recipes) (200 μM final), and 3 μl 20 μM RPA (600 nM final). Spike the first tube (leading strand mix) with 1.0 μl 32P-α-dCTP (10 μCi/reaction). Add 1.0 μl 32P-α-dCTP to the second tube (lagging strand mix). Final volume of both initiation solutions is 28 μl, sufficient to collect time points for 4 reactions. Set a timer for 15 min. Add 21 μl of leading strand mix to the first tube; start the timer while mixing thoroughly with pipette. Add 21 μl of the lagging strand mix to the second tube. Remove 20 μl of reaction from each tube at 5, 10, and 15 min and quickly transfer to each of six tubes pre-filled with 10 μl of stop solution (see Recipes).
    3. Model of lagging strand replication using a primed ssDNA substrate
      This is a similar protocol to the above, except CMG is not included and the substrate is a primed ssDNA circle, typically phage phiX174 or M13 (or an M13 derivative). Thus, no unwinding is required (i.e., CMG is on the leading strand, not the lagging strand). The clamp, clamp loader and DNA polymerases are all stimulated by pre-coating the ssDNA with single strand binding protein, allowing one to coat the substrate with RPA beforehand, which otherwise prevents CMG loading. The primer is chosen such that a run of dC and dT is just downstream on the template, making dATP and dGTP the included nucleotides for polymerase idling.
      1. Substrate loading (see Table 2A).

        Table 2A. Substrate loading phase of lagging strand model


        Load 0.5-2.0 nM lagging strand model substrate (phiX174:Pr1B) with 600 nM RPA (phase 1; 11 μl each).
        Example: In the following order, add 16.7 μl ddH2O, 6.6 μl 5x TDBG (1x final), 3.3 μl 10x MK (1x final), 0.75 μl phiX174:Pr1b (1.5 nM final), 1.5 μl 3 mM dATP (60 μM final), 1.5 μl 3 mM dGTP (60 μM final), 0.4 μl 100 mM ATP (0.5 mM final), and 2.3 μl 20 μM RPA (600 nM final) for a final volume of 3 x 11 μl = 33 μl. Incubate at 30 °C for 10 min in the water bath.
      2. Enzyme loading (see Table 2B).

        Table 2B. Enzyme loading phase of lagging strand mode


        Load RFC/PCNA/α/δ.
        Example: In the following order, add 23.3 μl ddH2O, 7.0 μl 5x TDBG (1x final), 3.5 μl 10x MK (1x final), 0.6 μl 1 μM RFC (5 nM final), 0.6 μl 5 μM PCNA (25 nM final), 0.6 μl 2 μM Pol α (10 nM final), and 0.6 μl 2 μM Pol δ (10 nM final) for a final volume of 5 x 7 μl reactions = 35 μl. Add 21 μl (3 x 7 μl) to each reaction tube, mixing thoroughly with a pipette tip. Incubate at 30 °C for 1 min.
      3. Replication initiation (see Table 2C).

        Table 2C. Replication initiation phase of lagging strand model


        Initiate replication by adding the nucleotides that had been withheld during preincubation.
        Example: in the following order, add 10.5 μl ddH2O, 4.2 μl 5x TDBG (1x final), 2.1 μl 10x MK (1x final), 0.5 μl 3 mM dCTP (20 μM final), 1.5 μl 3 mM dTTP (60 μM final), 1.5 μl T/C/G mix (200 μM final), 2 μl of T/G/C mix (200 μM final). Spike with 0.75 μl 32P-α-dCTP (10 μCi/reaction). Set a timer for 3 min. Add 21 μl of initiation mix; start timer while mixing thoroughly with pipette. Remove 20 μl of reaction at 1, 2, and 3 min and quickly transfer to each of three tubes pre-filled with 10 μl of stop solution.
    4. Replication product visualization
      For 3nbf experiments, leading strand products should look similar to those in Figure 1b of Georgescu et al. (2015); i.e., products whose length increases over time and goes to full length (3.2 kb) by ~10 min or less. Using an end-labeled primer to monitor primer extension on the 3kbf substrate, results should look similar to those in Figure 3b of Schauer et al. (2017); i.e., products whose length increases over time and goes to full length (2.8 kb). Replisome-directed lagging strand replication should appear similar to those in Figure 6 of Schauer et al. (2017); i.e., ~200 bp products. For all CMG-directed experiments, subtraction of either CMG or ATP from the experiment is a good negative control and should yield no extended product. Using the lagging strand model substrate, the experiments should look similar to those in Figure 1b in Schauer et al. (2017); i.e., robust full length (5.4 kb) product in under 3 min.
      1. Prepare ladder
        Follow manufacturer’s protocols to 5’-end-label the 1 kb DNA ladder with T4 kinase and 32P-γ-ATP. Typically, we label 20 μl of the ladder in 50 μl total with 5 μl T4 PNK and 4 μl 32P-γ-ATP. Purify away free 32P-γ-ATP with a G-25 column that has been equilibrated with TE, according to the manufacturer’s protocol.
      2. Prepare alkaline agarose gel
        Add 2.6 or 1.6 g LE agarose to 200 ml distilled H2O (1.3% for 3nbf/3kbf substrates, 0.8% for lagging strand model substrates). Microwave until boiling. Cool to at least 60 °C and add 0.5 ml 0.5 M EDTA and 0.6 ml 10 N NaOH before pouring the gel. Let cool to room temperature (RT) over ~2 h. Submerge in alkaline running buffer (see Recipes) in horizontal gel box.
      3. Load samples and DNA standard in gel. Run for 16 h at 35 V.
      4. Remove free radionucleotides
        Remove gel from gel box and expose to phosphorimaging screen for 15 min. Scan on phosphorimager and print gel at full size. Cut the saturated portion off of the printout, overlay it on the gel, and cut the bottom part of the gel off that contains free radionucleotides (usually the bottom third or fourth) in order to reduce exposure of yourself and your lab equipment. The identification of this region is particularly important when monitoring lagging strand synthesis as free radionucleotides do not run too far below Okazaki fragments. Free radionucleotides can also be run off the gel, but it contaminates the equipment, creates further radioactive waste, and they tend to diffuse in the buffer and can leach back into the entire gel and contribute to background signal.
      5. Compress the gel
        Wrap plastic wrap tightly around a flat surface like the bottom of a plexiglass radiation shield. Place the gel slab on the saran wrap. Place C-fold paper towels flat; one adjacent to each gel edge. Place one sheet of DEAE-cellulose paper over gel. Place one or two sheets of Whatman 3MM chromatography transfer paper over this. Stack 2 horizontal rows of ~25 C-fold paper towels over this. Cover paper towels with something flat and rigid (e.g., a flat plexiglass shield) to distribute weight. Gently rest a heavy weight (e.g., a lead brick) on the center of this assembly and press thin (few mm thick) for 6-12 h. We find this procedure superior to using a gel dryer, and it doubles as a step to allow DNA to transfer and immobilize on the DEAE-cellulose paper.
      6. Remove all paper except DEAE-cellulose, wrap in plastic wrap, and expose gel face to phosphorimaging screen for 2-36 h, depending on isotope strength.
      7. Scan phosphorimaging screen on a phosphorimager. 

Data analysis

Analysis of primer extension experiments is straightforward and well-documented elsewhere, and most operations can be handled by the Gels package in ImageJ (see https://imagej.nih.gov/ij/docs/menus/analyze.html#gels). Parameters of interest may be velocity, primer uptake, product length, etc., and will depend on the experiment. In addition to reading the documentation of the Gels package, we strongly encourage the reader to read through the ImageJ User Guide (https://imagej.nih.gov/ij/docs/guide/) for general help with image analysis before analyzing gel data.
When using radionucleotide incorporation, it is crucial to correct the bias of 32P incorporation using the DNA ladder as a reference point, as longer products will have much more incorporated nucleotide signal on a molar basis of short relative to long DNA molecules (Kurth et al., 2013 and Georgescu et al., 2014). The consequence of not performing this step is thus an overestimation of longer replication products and an underestimation of shorter ones. Use the following routine to quantify the gels, as in as in Figure 1c of Georgescu et al. (2015).

  1. Extract the lane intensity profiles (i.e., average pixel intensity vs. vertical distance) from the gel scans using the ImageJ.
    1. Rotate the gel horizontally so that lanes run left to right. Image>Transform>Rotate 90° right.
    2. Select the rectangle icon on the ImageJ toolbar and make a rectangle around the first lane.
    3. Plot the integrated intensity profile (average intensity in a.u. vs. distance in cm). Analyze>Plot Profile (or Ctrl+K).
    4. A new window will appear. Save profile as a text file by clicking save.
    5. Move rectangle down to next lane with the down arrow and repeat step 1c. Keeping an identical rectangle size and horizontal position assures the gel lanes will line up correctly.
    6. Import the intensity profiles as tab delimited data into spreadsheet software.
  2. Convert the distance in the intensity profiles to DNA lengths.
    1. Fit the lane with the molecular weight (MW) standard with a probability distribution containing the same number of Gaussian peaks as the number of bands that appear in the DNA MW standard.
    2. Plot the length of the dsDNA standards (bp) vs. the mean vertical distance (cm) obtained from the Gaussian fits, and fit the data with a logarithmic function, making note of the equation of the fitted line.
    3. In a spreadsheet, convert vertical distance (cm) to DNA (bp) using the equation obtained in Data analysis step 1c.
  3. Correct the lane intensity profiles for molecular weight. Normalize the intensity of the products on a pixel-by-pixel basis by dividing each intensity value by its DNA length (as determined in step 2) and multiplying by the full template length (in bp). The resulting profiles can be curve fit with any software capable of nonlinear curve fitting, and analyzed as necessary on a case-by-case basis.

Notes

  1. In preparing the 3nbf substrate, it is critical to remove all potential sources of end-labeling, including nicks in the template and flaps due to inefficient fork ligation. We therefore recommend extremely gentle handling of the template including minimal vortexing, gentle pipetting, etc.
  2. Extended ligations during the fork ligation step are useful. End labeling presents as a full-length band that appears at early timepoints of replication assays; this should be absent or only marginally detectable if the substrate has been correctly prepared. As a control, perform reactions lacking CMG and/or ATP to assess the degree of end-labeling; without CMG unwinding activity there should be no appearance of full-length product at any time, since Pol ε cannot strand displace.
  3. Nucleases pose a major problem for replication assays as they degrade the substrates and also contribute to end labeling, which can be indistinguishable from full length primer extension if the signal intensity is too high. It is therefore critical to check all protein preps and reagents for nucleases. Microbial contamination is another major culprit for bringing in nucleases, so ensure that all reagents are autoclaved, filter sterilized, aliquotted for single use, and frozen. There are commercial kits available to test DNAse and RNAse activity, however, a timecourse of incubation of your reagent of interest with a 32P-labeled oligo (such as the primer created for 3kbf) will also be informative. Nuclease activity will appear as a time-dependent disappearance of 32P signal on a nondenaturing polyacrylamide gel.

Recipes

Note: Store all solutions at -20 °C unless otherwise specified. Buffers used in replication assays should not be stored longer than ~6 weeks.

  1. 6x gel loading dye
    60% glycerol
    0.1% bromophenol blue
    0.1% xylene cylanol
    Store at RT
  2. TE buffer, pH 8.0
    10 mM Tris-HCl, pH 8.0
    1 mM EDTA (Ethylenediaminetetraacetic acid, disodium salt)
  3. 10x TBE
    1 M Tris base
    1 M boric acid
    20 mM EDTA
  4. DNA elution buffer
    100 mM NaCl
    10 mM Tris-HCl, pH 8.0
    1 mM EDTA
  5. 20x SSC
    3 M NaCl
    0.3 M sodium citrate
  6. 1-Butanol saturated water
    50 ml 1-butanol
    5 ml ddH2O
    Shake vigorously and store at 4 °C
  7. 5x TDBG
    125 mM Tris-acetate, pH 7.5
    25% glycerol
    200 μg/ml BSA
    10 mM TCEP (Tris(2-carboxyethyl)phosphine) pH 7.5
    15 mM DTT
    0.5 mM EDTA (Ethylenediaminetetraacetic acid)
  8. 10x MK
    500 mM potassium glutamate
    100 mM magnesium acetate
  9. dA/dC mix
    10 mM dATP
    10 mM dCTP
    10 mM Tris-HCl, pH 7.5
    1 mM EDTA
  10. dT/dG mix
    10 mM dTTP
    10 mM dGTP
    10 mM Tris-HCl, pH 7.5
    1 mM EDTA
  11. T/G/C mix
    10 mM TTP
    10 mM GTP
    10 mM CTP
    10 mM Tris-HCl, pH 7.5
    1 mM EDTA
  12. Stop solution
    1% SDS
    40 mM EDTA
    10% glycerol
    0.02% bromophenol blue
    0.02% xylene cylanol
    Store at RT
  13. Alkaline running buffer
    1.125 mM EDTA
    30 mM NaOH

Acknowledgments

This work was supported by grants from the National Institutes of Health (T32 CA009673 to G.S; and R01 GM115809 and the Howard Hughes Medical Institute to M.O.D.). The protocols herein were adapted from those originally used in Georgescu et al., 2014, Georgescu et al., 2015, and Schauer et al., 2017.

References

  1. Georgescu, R. E., Langston, L., Yao, N. Y., Yurieva, O., Zhang, D., Finkelstein, J., Agarwal, T. and O’Donnell, M. E. (2014). Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork. Nat Struct Mol Biol 21(8): 664-670.
  2. Georgescu, R. E., Schauer, G. D., Yao, N. Y., Langston, L. D., Yurieva, O., Zhang, D., Finkelstein, J. and O’Donnell, M. E. (2015). Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation. Elife 4: e04988.
  3. Kurth, I., Georgescu, R. E. and O'Donnell, M. E. (2013). A solution to release twisted DNA during chromosome replication by coupled DNA polymerases. Nature 496(7443): 119-122.
  4. Langston, L. D. and O’Donnell, M. (2008). DNA polymerase δ is highly processive with proliferating cell nuclear antigen and undergoes collision release upon completing DNA. J Biol Chem 283(43): 29522-29531.
  5. Langston, L. D., Zhang, D., Yurieva, O., Georgescu, R. E., Finkelstein, J., Yao, N. Y., Indiani, C. and O’Donnell, M. E. (2014). CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication. Proc Natl Acad Sci U S A 111(43): 15390-15395.
  6. Schauer, G. D. and O'Donnell, M. E. (2017). Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork. Proc Natl Acad Sci U S A 114(4): 675-680.
  7. Stodola, J. L. and Burgers, P. M. (2016). Resolving individual steps of Okazaki-fragment maturation at a millisecond timescale. Nat Struct Mol Biol 23(5): 402-408.

简介

真核生物复制品是重复DNA的多蛋白复合物。 复制品被雕刻成连续的前导链合成与不连续的滞后链合成,主要通过DNA聚合酶ε和δ以及解旋酶,聚合酶α-引发酶,DNA滑动夹,夹带载体和许多其它蛋白质进行。 我们以前已经建立了聚合酶ε和δ靶向其“正确”链的机制,以及在与“不正确”链相关联时驱赶聚合酶的质量控制机制。 在这里,我们提供了使用纯蛋白质在体外差异测定前导和滞后链复制的实用指南。
Using pure proteins from Saccharomyces cerevisiae, our lab was the first to reconstitute a functional eukaryotic DNA replisome, a ~2 MDa complex that includes the 11-subunit CMG helicase (complex of Cdc45, Mcm2-7, GINS heterotetramer), the 4-subunit DNA polymerase (Pol) ε, the 4-subunit Pol α-primase, the PCNA (Proliferating Cell Nuclear Antigen) clamp homotrimer ring shaped processivity factor that encircles duplex DNA, the 5-subunit clamp loader RFC (Replication Factor C) that uses ATP to open and close the PCNA sliding clamp ring onto primed sites for polymerase processivity, and the RPA (Replication Protein A) heterotrimeric single-strand DNA binding protein that removes secondary structure obstacles to DNA polymerase progression. In our initial studies we discovered that Pol ε is targeted to CMG on the leading strand after priming by Pol α-primase, while Pol δ is targeted to PCNA clamps on the lagging strand primed sites (Georgescu et al., 2014; Langston et al., 2014). We next reconstituted a functional coupled leading/lagging strand replisome which included the 4-subunit Pol α-primase and 3-subunit Pol δ, in which we demonstrated that Pol ε is inactive on the lagging strand and Pol ε is inactive on the leading strand (Georgescu et al., 2015). Interestingly, the Pol α-primase, which lacks proofreading activity, was active with CMG on both strands, but when either Pol ε or Pol δ are present, which both contain a proofreading 3’-5’ exonuclease for high fidelity synthesis, they take over from the low fidelity Pol α-primase on either strand. However, Pol ε and Pol δ only performed optimal synthesis when on their respective correct strands (Georgescu et al., 2015). In a subsequent study we characterized the unprecedented quality control mechanisms that exclude these polymerases from incorrect strands, a job that bacterial replisomes do not need to do because they utilize identical polymerases for both strands (Schauer et al., 2017). We found that on the lagging strand, Pol ε is excluded from primed sites by competition with the RFC clamp loader for the primer terminus, while CMG binds and protects Pol ε from RFC inhibition on the leading strand. In contrast Pol δ is preferentially targeted to PCNA on lagging strand primed sites through a tight binding affinity to PCNA clamps that is over 20-fold greater than the PCNA affinity to Pol ε and is unaffected by competition by the RFC clamp loader (Schauer et al., 2017). Interestingly, no stabilizing interaction with CMG exists for Pol δ (Schauer et al., 2017). Furthermore, Pol δ is less stable on a completed DNA than when idling at a primer terminus or extending a primer. Specifically, Pol δ is known to be stable for over a half hour with PCNA, consistent with its high processivity, but upon completing replication of a section of DNA, and bumping into a completed dsDNA region, it dissociates rapidly (i.e., < 1 min) from PCNA-DNA in a process referred to as collision release (Langston and O’Donnell, 2008; Langston et al., 2014).This inherent instability of Pol δ-PCNA upon completing replication may serve as a quality control to destabilize Pol δ-PCNA on the leading strand because Pol δ-PCNA is much faster than CMG unwinding and will be in a constant state of having completed DNA and collision with CMG (Schauer et al., 2017). Destabilization of Pol δ-PCNA when there is no more DNA to be extended should not be taken to mean that Pol δ instantly ejects from PCNA. For example, Pol δ-PCNA remains on DNA for a few seconds to fill-in short gaps upon RNA removal at 5’ ends of Okazaki fragments (Stodola and Burgers, 2016).
In interrogating these various activities, we observed that CMG does not load onto small (100-200 bp) rolling circle replication substrates, which are often used to study replisome behavior in bacterial systems. Thus, we turned to linear DNA fork assays as an alternative to address biochemical mechanisms in eukaryotic replication. These assays enable one to easily separate leading from lagging strand replication activity by synthesis of a long linear DNA that has no dC in one strand, and thus no dG in the other strand. By doing so, one can specifically monitor leading or lagging strand synthesis depending on the radioactive deoxyribonucleoside triphosphate (dNTP) used in the assay.

关键字:真核DNA复制, 复制体测定, CMG解旋酶, DNA聚合酶, RFC钳装载器, PCNA滑动钳, 先导链, 后随链

材料和试剂

  1. 剃刀刀片
  2. 1.57mm OD聚乙烯管(例如,粘土亚当斯公司,Intramedic ,BD,目录号:427431)
  3. Sephadex微量离心柱(Illustra Microspin G-25)(GE Healthcare,目录号:27-5325-01)
  4. 塑料包装(例如,,Fisherbrand Clear Plastic Wrap,Fisher Scientific,目录号:22-305654)
  5. C折纸巾(例如,Scott纸巾,KCWW,Kimberly-Clark,目录号:01510)
  6. 带正电荷的尼龙DNA印迹膜(Hybond-N +,30.0×50.0cm)(GE Healthcare,目录号:RPN3050B)
  7. 色谱转印纸(Whatman 3MM,46.0 x 57.0 cm)(GE Healthcare,目录号:3030-917)
  8. 注射针头(例如,,B-D 18 G 1 1/2精准度针/针)(BD,目录号:305196)
  9. phiX174病毒粒子DNA,1mg / ml(New England Biolabs,目录号:N3023L)
  10. Phi29 DNA聚合酶(New England Biolabs,目录号:M0269S)
  11. 100mM dNTP(脱氧核苷酸三磷酸)组(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0181)
  12. 1μMCMG(Cdc45 Mcm2-7纯金)解旋酶(见Georgescu等人。[2014])以获得细节)
  13. pUC19,1mg / ml(New England Biolabs,目录号:N3041L)
  14. Bsa 使用CutSmart缓冲液的I-HF(New England Biolabs,目录号:R3535L)
  15. 'blockLd'oligo *
  16. 'blockLg'oligo *
  17. 'Pr1B'oligo *
  18. '160Ld'oligo *
  19. '91Lg'oligo *
  20. 'Fork primer'oligo *
  21. 核苷酸偏置模板(由Biomatik,Wilmington DE合成)*

*注意:请参阅 补充文件1

  1. T4连接酶,包括10x连接酶缓冲液(New England Biolabs,目录号:M0202M)
  2. 100mM ATP(GE Healthcare,目录号:27-2056-01)
  3. 0.5M EDTA,二钠盐(Sigma-Aldrich,目录号:E5134)
  4. 5M NaCl(Sigma-Aldrich,目录号:S9888)
  5. Sepharose 4B尺寸排阻色谱树脂(GE Healthcare,目录号:17012001)
  6. 1 kb MW标记(New England Biolabs,目录号:N3232L)
  7. 溴化乙锭(EthBr,10mg / ml)(Thermo Fisher Scientific,Invitrogen TM,目录号:15585011)
  8. T4激酶和10x T4激酶缓冲液(New England Biolabs,目录号:M0201L)
  9. P-γ-ATP,3,000Ci / mmol,3.3μM(PerkinElmer,目录号:BLU002A)
  10. XI型低熔点琼脂糖(Sigma-Aldrich,目录号:A3038)
    注意:本产品已停产。
  11. CI(新英格兰Biolabs,目录号:R0647L)
  12. β-琼脂糖酶I(New England Biolabs,目录号:M0392L)
  13. 3 M乙酸钠(CH 3 COONa),pH 5.2(Sigma-Aldrich,目录号:S2889)
  14. 异丙醇(Sigma-Aldrich,目录号:190764)
  15. 糖原,分子生物学级(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0561)
  16. 乙醇(Sigma-Aldrich,目录号:E7023)
  17. 1μMRFC(复制因子C;参见Georgescu等人。 [2014],用于净化细节)
  18. 5μMPCNA(增殖细胞核抗原;见Georgescu等人。[2014]),用于纯化细节)
  19. 2μMPolε(参见Georgescu等人。[2014],用于纯化细节)
  20. 2μMPolδ(参见Georgescu et al。[2014],用于纯化细节)
  21. 2μMPolα(参见Georgescu et al。[2014]),用于纯化细节)
  22. 20μMRPA(复制蛋白A;见Georgescu等人)[2014],用于纯化细节)
  23. P-α-dCTP,3,000Ci / mmol,3.3μM(PerkinElmer,目录号:BLU013H)
  24. P-α-dGTP,3,000Ci / mmol,3.3μM(PerkinElmer,目录号:BLU514H)
  25. LE琼脂糖(BioExpress,GeneMate,目录号:E-3120-500)
  26. 10N氢氧化钠(NaOH)(Fisher Scientific,目录号:SS255)
  27. 甘油
  28. 二甲苯甲醇
  29. Tris-HCl,pH 8.0
  30. Tris碱(RPI,目录号:T60040-500.0)
  31. 硼酸(RPI,目录号:B32050-5000.0)
  32. 柠檬酸钠
  33. 1丁醇
  34. Tris-醋酸盐,pH7.5
  35. 牛血清白蛋白(BSA)(New England Biolabs,目录号:B9000S)
  36. 三(2-羧乙基)膦(TCEP)pH7.5
  37. 100mM二硫苏糖醇(DTT)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0861)
  38. 谷氨酸钾
  39. 醋酸镁
  40. 1%SDS
  41. 6x凝胶加载染料(见配方)
  42. TE缓冲液,pH 8.0(参见食谱)
  43. 10x TBE(Tris /硼酸盐/ EDTA;参见食谱)
  44. DNA洗脱缓冲液(参见食谱)
  45. 20x SSC(见配方)
  46. 1-丁醇饱和水(见食谱)
  47. 5x TDBG(见配方)
  48. 10x MK(见配方)
  49. dA / dC混合(见配方)
  50. dT / dG混合(见配方)
  51. T / G / C混合(见配方)
  52. 停止解决方案(请参阅食谱)
  53. 碱性运行缓冲液(见配方)

设备

  1. 加热块(例如,,VWR,目录号:12621-084)
  2. 馏分收集器(例如,,Gilson,型号:F203B)
  3. 可变模式凝胶成像仪(例如,,GE台风)
  4. UV-vis分光光度计(例如,Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDropTM 2000)
  5. 真空干燥器(例如,Thermo Fisher Scientific,Thermo Scientific,Sup。TM,目录号:5309-0250)
  6. 紫外灯箱
  7. UV阻挡面罩(例如,Sigma-Aldrich,目录号:F8142)
    注意:本产品已停产。
  8. 微量离心机
  9. 电导率计(例如,,辐射计医疗,型号:CDM 80)
  10. 带微量离心机托盘的温度控制水浴(例如,LabX型号:Lauda E100和Brinkman 30x x 1.5ml)
    制造商:LAUDA-Brinkmann,型号:E100。
  11. 磷光成像屏幕(GE Healthcare)
  12. 荧光成像仪(例如,,GE台风)
  13. 重的重量
    注意:我们使用我们发现的巨型铅块;一个〜50磅的哑铃会奏效。
  14. 使用ImageJ和电子表格软件(例如,,Apache Open Office)的计算机安装了
  15. 1×30cm玻璃柱(例如,玻璃Econo-Columns)(Bio-Rad Laboratories,目录号:7371032)
  16. 100 ml琼脂糖凝胶电泳仪
  17. 泡沫塑料盒足够大,适合100 ml琼脂糖凝胶电泳仪
  18. 电泳电源(例如,,Pharmacia Biotech,目录号:EPS 3500 XL)
  19. 分析结果
  20. 保护性有机玻璃样品屏蔽
  21. 20×14cm水平琼脂糖凝胶电泳装置(C.B.Science,目录号:SGU-014T-02)

软件

  1. ImageJ( https://imagej.nih。 GOV / IJ /文档/菜单/ analyze.html#凝胶

程序

  1. 线性分叉DNA模板构建
    我们主要使用两种类型的线性叉状DNA底物来测定引导/滞后链复制:基于线性pUC的天然序列底物(以下称为3kbf,用于3kb叉)和3.2kb核苷酸偏向的前导/滞后链底物,其合成生成,使得一条链不具有dC,另一条链不具有dG(称为3nbf,对于3kb核苷酸偏置的叉)。 3nbf底物可用于领先和滞后的链测定,以独立于另一条来监测每条链(参见图1)。我们有时候更喜欢使用5' - sup 32标记的引物来定量引导链复制,因为它是使用引物延伸的直接报告基因,与使用 32 P-dNTPs因为它缺乏潜在的终端标签工件。尽管两种底物都可以适应引物,但我们通常只使用3kbf底物用于此目的,因为制备起来要简单得多(例如,不需要从低浓度纯化3kb合成核苷酸偏向部分熔融凝胶)。尽管这些底物之间的模板长度和组成不同,但是我们使用与这些底物连接的相同的小型合成叉结构。
    叉子由一个160杆的前导链组成,一个91杆的后备链。前导链含有用于CMG负载的3'(dT)亚基末端,37个核苷酸引物退火位点,其后是缺少dC和dA残基的4个核苷酸区域以及缺少dC和dT残基的10个核苷酸区域(参见寡核苷酸组合物的配方)。引物上游的4个核苷酸区域允许通过仅包括dATP和dCTP(即,以防止Polε的3'-5'核酸外切酶活性降解)来引导链上的Polε"空转"引物)。 10个核苷酸区域阻止潜在的错配(即,即使是dTTP被扣留)之后的"失控"Polε。按照这些顺序,我们在设计成适应CMG(〜10nm)的覆盖区的叉结上包括(dT)<40>间隔物。滞后链含有(dT)60'末端和与前导链的5'末端互补的35bp。前导链叉模板寡核苷酸通常含有四个3'硫代磷酸酯键,以通过用硫原子替代寡核苷酸主链中的非桥连磷酸酯氧原子来保护DNA聚合酶中固有的外切核酸酶活性(可作为DNA合成公司的修饰)作为IDT)。核苷酸偏置底物的示意图见图1

    图1.核苷酸偏置底物的示意图。 显示"3nbf"底物,包括文中描述的寡核苷酸,其相关特征突出显示。偏置被设计为使得在前导链合成期间将仅引入 32 P-α-dCTP,而在滞后链合成期间仅包含P-α-dGTP 。虚线表示文中讨论的限制酶切位点。注意,使用相同合成叉构建但使用具有天然(无偏)序列的2,686bp模板的"3kbf"底物未显示。

    3kbf底物构建(通常用于使用 32引物的引物链反应)
    1. 在TE缓冲区中重新悬浮"160Ld"至20μM(请参阅食谱)
    2. 在TE缓冲区中将"91Lg"重悬浮至500μM
    3. 在1x CutSmart缓冲液中,将37 pmol pUC19与4μlBsa I-HF(20U /μl)一起消化,共37℃,4小时。
    4. 在75°C下热灭活I-HF 30秒。
    5. 在TE中加入17.5μl(350pmol)160Ld和4.2μl(2100pmol)91Lg,并加入238.3μlTE至400μl。
      注意:为了防止线性化pUC19的串联化,需要10:1过量的叉模板,并且将6L超过91Lg至160Ld,以确保所有连接的160Ld退火到91Lg。
    6. 退火通过加热至95°C 5秒钟,使热板在台面上冷却至RT模板
    7. 通过加入15μlT4连接酶,36μl10x CutSmart缓冲液,4μl100 mM ATP(最终为1 mM)将引导叉引导到线性pUC19上,加入TE至400μl体积。在16℃下孵育≥16小时。
    8. 加入10μl0.5 M EDTA,螯合Mg 2 +
    9. 加热使T4连接酶在65°C下灭活20秒
    10. 加入10μl5M NaCl,使[NaCl]至> 100 mM(必需防止非特异性结合Sepharose 4B色谱柱)
    11. 倒入〜20ml Sepharose 4B柱,并平衡>冷的DNA洗脱缓冲液的2个柱体积(CV)(参见食谱)。
    12. 将注射器针头调整到柱的末端。将聚乙烯管道连接到注射器末端,并将其连接到馏分收集器,使装置达到所有管道所需的最小长度。
    13. 向20ml Sepharose 4B柱中加入420μl反应液,确保在加载反应体积之前,缓冲液已完全渗入柱床。
    14. 样品完全穿透柱床后,用冷DNA洗脱缓冲液加入并洗脱
    15. 收集〜227μl的分数(10滴/ fxn为我们使用的油管)。在这些条件下,模板应该在馏分20上洗脱。
    16. 在1%TBE琼脂糖凝胶上运行7μl每个级分,同时在100 V下加入1 kb MW标记(带有凝胶加载缓冲液),直至溴酚蓝标记达到2/3。
    17. 用1×TBE中的2-5μlEthBr染色并在凝胶成像仪上观察。
      注意:
      1. 线性化质粒从结扎叉DNA的凝胶转移非常小,所以不要期待看到差异。然而,连接的叉DNA将比过量的游离合成分叉DNA更早地洗脱,其可以在适当染色/脱色的凝胶上显现。
      2. 我们用放射性标记的叉进行量化叉结,高效(> 95%)高效,因此3kb模板在上述条件下连接到DNA叉。
      3. 尽管如此,我们鼓励用户使用32P标记的叉DNA实际监测其连接效率,以确保连接,并使用phi29 DNA聚合酶延伸32P标记的引物(即参见下文)以进一步确保结扎效率。
    18. 保存含有〜2.7 kb谱带的级分(这将是所有级分中唯一可见的谱带),并使用UV / Vis分光光度计通过A 260测量DNA浓度。
    19. 按照制造商的方案,用T4激酶和 32 P-γ-ATP对引物进行5'-末端标记。我们通常使用25μl反应,终浓度为400 nM。使用已经用TE平衡的G-25柱,根据制造商的方案,将游离的 32 P-γ-ATP净化。
    20. 为了用 32 P引物引入3kbf底物,加入所需量的3kbf底物与等摩尔量的 P引物。最小化总体积,加入20x SSC(见配方)至最终1x浓度,加热至95°C,并在台式机上冷却至RT。

    3nbf基板构造
    1. 在TE缓冲区中将"160Ld"重新悬浮至20μM。
    2. 在TE缓冲区中将"91Lg"重悬浮至500μM
    3. 含有3.2 kb核苷酸偏置模板的Digest〜400μg质粒(请参阅补充文件1 ) 7 h。
    4. 在上一步骤完成之前,在0.8%TBE琼脂糖凝胶上以100V运行0.5μl,直到溴酚蓝达到2/3,以确保完全切割。如果切割不完整,请添加更多的Bsa I-HF并消化更多;重复直到切割完成。
    5. 加入12μlBts CI(20 U /μl)加上足够的10x CutSmart缓冲液,并补充添加的体积。在50°C孵育6 h。
    6. 在消化完成之前,如步骤4(程序A,3nbf底物构建)在凝胶上运行0.5μl,以确保完整的消化。
    7. 加入21.5μl0.5 M EDTA
    8. 将样品置于真空干燥器中6-8小时,减少体积约1/3
    9. 在1.2%TBE低熔点琼脂糖凝胶上运行整个样品(共15个梳齿牙齿中的8个)。加入2μl10 mg / ml EthBr至100ml凝胶。预冷TBE,每100 ml运行缓冲液中加入2μl10 mg / ml EthBr。运行在40-50 V,仪器坐在装有冰水的聚苯乙烯泡沫塑料箱(冷室不足),直到溴酚蓝达到凝胶的三分之一。穿上紫外线防护面罩,在UV灯箱上显现,并用剃须刀切出最大的(3,260bp)带。称量凝胶切片进行下一步骤。
    10. 在65°C的熔体凝胶切片。
    11. 称量熔融切片,加入1/9 V Tris-HCl,pH 6.8和1/224 V 0.5 M EDTA,粗糙度近似为凝胶密度为1 g / ml。
    12. 冷却至42°C。
    13. 每克凝胶加入〜5μlβ-琼脂糖酶I。在42℃孵育5-7小时。
    14. 重复步骤13(程序A,3nbf底物构造)。
    15. 异丙醇沉淀DNA片段 加入体积为3M的乙酸钠(pH5.2)的1/10,然后加入等体积的异丙醇。在-20°C孵育1小时。在微量离心机中以≥15,000x g旋转30分钟。
      注意:如果太多的琼脂没有被足够小的低聚糖消化,你会看到很多白色沉淀。您将需要完成沉淀,重新悬浮在水溶液中,并返回到步骤13(程序A,3nbf底物结构),以进行进一步的β-琼脂糖酶I消化。
    16. 弃去上清并在TE pH 8.0中重悬沉淀。
    17. 通过用等体积的1-丁醇饱和水提取除去EthBr(参见食谱)。轻轻涡旋混合。以1,500 x g旋转,节省底部解决方案。继续提取,直到没有可见的粉红色。提取3次以上。
    18. 乙醇沉淀物
      加入1μl糖原(20mg / ml),1/10体积的3M磷酸钠pH 5.2,然后加入等体积的冷乙醇。在-20°C孵育1小时。旋转微量离心机≥15,000x g 30分钟。
    19. 将DNA重悬于150μlTE pH 8.0
    20. 在0.8%TBE琼脂糖凝胶上运行2μl,4μl和8μl1:10和1:100稀释液以估计浓度。
    21. 使用35 pmol的这种材料用于后续步骤。
    22. 在TE中加入350 pmol 160Ld和2100pmol 91Lg,并加入TE至300μl。
      注意:需要10:1过量的叉到模板以防止串联,并且6:1超过91Lg到160Ld是确保所有连接的160Ld退火到91Lg。
    23. 退火通过加热至95°C 5秒钟,使热板在台面上冷却至RT模板
    24. 通过加入15μlT4连接酶,36μl10x CutSmart缓冲液,4μl100 mM ATP(最终为1 mM)将引导叉引导到线性pUC19上,加入TE至400μl体积。在16°C孵育至少16小时。
    25. 加入10μl0.5 M EDTA,螯合Mg 2 +
    26. 加热使T4连接酶在65°C下灭活20秒
    27. 加入10μl5M NaCl以使[NaCl]至> 100mM(为防止非特异性结合Sepharose 4B柱所必需)。
    28. 将注射器针头调整到柱的末端。将聚乙烯管道连接到注射器末端,并将其连接到馏分收集器,使装置达到所有管道所需的最小长度。
    29. 倒入〜20ml Sepharose 4B柱,并平衡>冷的DNA洗脱缓冲液的2个柱体积(CV)
    30. 向20ml Sepharose 4B色谱柱中加入420μl反应液,确保缓冲液在添加前已完全渗入柱床。
    31. 样品完全渗透入柱床后,用冷DNA洗脱缓冲液洗脱
    32. 收集〜227μl分数(10滴/分)
    33. 在1%TBE琼脂糖凝胶上运行7μl每个级分,并加入1 kb MW标记(含凝胶加载缓冲液),直至溴酚蓝标记达到约2/3。
    34. 在1×TBE中用2-5μl溴化乙锭染色,并在凝胶成像仪上显色。
      注意:线性化质粒从叉子束缚的凝胶移动非常小,所以不要指望看到差异。这个凝胶上的自由叉也很难看出。我们用放射性标记的叉子量化了叉结,并且极其(> 95%)的效率,因此模板应该有叉连接。
    35. 保存含有〜3.2 kb谱带(这应该是唯一可见光谱带)的分数,并通过分光光度计上的A2 260测量浓度。
    36. 用阻断寡核苷酸钝化分叉模板的非叉末端。在TE pH 8.0中将"blockLd"和"blockLg"重新悬浮至10μM。以2:1寡核苷酸:模板摩尔比添加每个寡核苷酸。加热至85°C 5秒钟,冷却至台式温度。
      注意:此步骤是为了防止在 32 P-α-dNTP并入期间BtsCI切割位点的最终标记(即,Pol可以使用未连接的5'突出端作为模板),然而,可以更实际地订购在该区域由钝切位点去除的核苷酸偏向序列。 />
    37. 通过加入10μlT4连接酶来阻断寡核苷酸。加入1/10体积的10x T4连接酶缓冲液并用1mM ATP补充。在16°C孵育至少16小时。
    38. 加热使T4连接酶在65℃灭活20秒。

    启动phiX174(模型滞后链)构造
    除了CMG导向的前导/滞后链合成之外,我们还发现使用引发剂的RPA涂覆的环状5.4kb ssDNA(phiX174病毒体环状ssDNA)作为模型滞后链,以便分离潜在的滞后链活性没有完整的复制上下文。
    1. 在TE pH 8.0中将Pr1B重悬至5μM(参见食谱)
    2. 向10 pmol phiX174 DNA(1.2:1引物:模板摩尔过量)中加入12 pmol Pr1B。加入缓冲液TE pH 8.0至57μl,加入3μl20x SSC。加热至95°C 5秒,冷却至台式温度。
  2. 复制分析
    我们假设读者已经净化了重组真核生物复制品所需的全部蛋白质,包括最小的CMG解旋酶Polsε,δ和α,RFC,PCNA和RPA。其他辅助蛋白如Mcm10和Mrc1-Tof1-Csm3复合物将刺激复制,但不是活性的绝对要求。这些蛋白质的纯化和储存条件超出了本协议的范围,可以根据具体实验要求而变化,然而,蛋白质的全部纯化细节在(Georgescu et al。,2014和2015)中有描述)和(Langston等人,,2014)。记录所有蛋白质库存的浓度和盐浓度。将所需级分分成小(〜10-25μl)等分试样,在液氮中闪蒸冷冻,并储存在-80℃。
    我们更喜欢使用25μl作为标准实验体积,以最大限度地减少蛋白质消耗,同时保持工作量的可控性。低至15μl或高达40μl的体积也可以正常工作。对于这里列出的协议,我们经常分为三个阶段:1)基质上的反应物加载。 2)聚合酶钳位载体,即聚合酶,RFC夹紧载体,PCNA钳位和2或3个dNTP,其允许聚合酶在模板上"空转"(核苷酸掺入和校对的重复循环)以防止引物降解,以及3)通过添加4个dNTP的全部补体来启动复制。
    如果需要,可以组合前两个阶段,但是理想地,在孵育5-10分钟或更长时间期间,CMG负载到叉状DNA(或在滞后链模型的情况下的RPA涂层ssDNA)。
    另一方面,第二阶段含有外切核酸酶专业聚合酶,其将降解底物和/或其引物,因此至少需要两种dNTP和与这些酶的适度孵育时间。
    第三阶段通过添加所有剩余的dNTP启动正在进行的复制,而在叉分析的情况下,也是RPA。在本文给出的实施例中,制备第一底物负载相并根据需要分成管,随后加入第二酶相,并且随着反应定时器开始,添加第三起始阶段。这种分期方法有助于最小化从小量移液中产生的错误,然后将混合相分解成相对较大的体积。
    由于相对于最终体积添加酶,因此也可以促进低体积的使用,使得可以以相对高的浓度进行对DNA的负载。由于每个相在混合前分别制备,因此在加入蛋白质之前,为了防止沉淀,在各相中独立地加入盐和缓冲剂(,即,TDBG,MK和任何其它所需的盐)是重要的,聚集,等等。根据蛋白质行为/溶解度要求,可能会直接将蛋白直接加入到低离子强度中。
    假设在蛋白质滴定过程中实验组之间的蛋白质浓度有差异(例如,),重要的是通过向具有较低蛋白质浓度的复制的管中加入适量的盐来校正进入的盐反应对盐条件敏感。因此,必须确保盐已正确平衡。
    1. 盐校准
      制作实验和/或蛋白质储存中使用的每种类型的盐的电导率对盐浓度的标准曲线,并进行正确的实验。
      1. 准备连续稀释的盐,例如,0.1 M至1 M。
      2. 加入10μl各浓度至990μlddH 2 O。
      3. 测量电导率,以mS / cm为单位,使用电导率计和阴离子浓度
      4. 将蛋白质和/或实验样品的电导率与标准曲线进行比较。根据需要调整与适当的盐的反应
    2. 引导和/或滞后链复制;例如3点时间段领先/滞后(3nbf)实验。
      注意:除了在3nbf实验中添加放射性核苷酸之外,以下协议对于3kbf(分叉线性模板)是相同的,如前所述。在每个阶段和时间点停止管已准备好之前,不要启动复制。
      1. 基板加载(见表1A)。

        表1A。引导/滞后链复制的底物加载阶段


        用10-30nM CMG解旋酶(阶段1;每个11μl)加载0.5-2.0nM 3nbf底物。
        例如: 按照以下顺序,添加32.0μlddH 2,将13.2μl5x TDBG(参见食谱)(1x最终),6.6μl 10×MK(参见食谱)(1x最终),11.3μl3kbf / 3nbf(最终1.5nM)和3.0μl1μMCMG(终浓度为20nM),终浓度为3×11μl=66μl的最终体积。在水浴中在30℃下孵育10分钟。分成两组,每组33μl。
        注意:当将阶段分成不同的组时,可能有助于添加一个反应体积来解释体积损失,例如,将一个足够的7个反应的体积分成2组3。 >
      2. 酶加载(见表1B)
        表1B。引导/滞后链复制的酶装载阶段


        添加RFC / PCNA / Polε/α/δ。
        例如: 按照以下顺序,加入40.0μlddH <2> O,14.0μl5x TDBG(1x final),7.0μl10x MK最终),2.5μldA / dC混合物(参见食谱)(最终60μM),1.3μl1μMRFC(最终5nM),1.3μl5μMPCNA(终浓度25nM),1.3μl2μMPolα(10nM最终),1.3μl2μMPolε(10nM最终)和1.3μl2μMPolδ(10nM终),终体积为10×7μl反应=70μl。向每个反应管中加入21μl(3 x 7μl),用移液管彻底混合。在30°C孵育1分钟。
        注意:添加dATP和dCTP可以在引发底物上聚合酶空转,同时允许RFC使用dATP加载PCNA。
      3. 复制启动(见表1C)
        表1C。引导/滞后链复制的复制开始阶段


        通过添加剩余的dNTP和RPA来启动复制。使用P-α-dCTP(前导链)或 32 P-α-dGTP(滞后链)并入进行监测。
        示例: 按照以下顺序向两个管中的每一个添加14.0μlddH 2 O,5.6μl5x TDBG(1x最终)将2.8μl10x MK(1x最终),0.6μldT / dG混合物(参见食谱)(最终60μM),5μlATP(最终5mM),2μlT / G / C混合物(参见食谱)(200μM最终)和3μl20μMRPA(终浓度为600nM)。将第一管(前导链混合物)与1.0μl(32C)P-α-dCTP(10μCi/反应)分开。向第二管(滞后链混合物)中加入1.0μl 32 P-α-dCTP。两种起始溶液的最终体积为28μl,足以收集4个反应的时间点。设置一个定时器15分钟。向第一根管中加入21μl前导链混合物;启动定时器,同时用移液器彻底混合。将21μl滞后链混合物加入到第二管中。在5,10和15分钟内从每个管中取出20μl的反应物,并快速转移到预填充10μl终止液的6个试管中(见食谱)。
    3. 使用引发的ssDNA底物的滞后链复制模型
      这是与上述类似的方案,不包括CMG,底物是引发的ssDNA圆,通常是噬菌体phiX174或M13(或M13衍生物)。因此,不需要退绕(即,即,CMG在主链上,而不是滞后链)。钳夹,钳装载体和DNA聚合酶均通过用单链结合蛋白预包被ssDNA来刺激,允许用RPA预先涂覆底物,否则可防止CMG加载。选择引物使得dC和dT的运行恰好在模板的下游,使得dATP和dGTP是用于聚合酶空转的所包含的核苷酸。
      1. 底物加载(见表2A)。

        表2A。滞后链模型的底物加载阶段


        用600nM RPA(阶段1;每个11μl)负载0.5-2.0nM滞后链模型底物(phiX174:Pr1B)。
        例如: 按照以下顺序,添加16.7μlddH O,6.6μl5x TDBG(1x Final),3.3μl10x MK最终),0.75μlphiX174:Pr1b(最终1.5nM),1.5μl3mM dATP(最终60μM),1.5μl3mM dGTP(最终60μM),0.4μl100mM ATP(最终0.5mM)和2.3μl 20μMRPA(终浓度为600nM),终体积为3×11μl=33μl。在水浴中在30℃下孵育10分钟。
      2. 酶装载(见表2B)
        表2B。滞后链模式的酶装载阶段


        加载RFC / PCNA /α/δ。
        例如: 按照以下顺序,添加23.3μlddH 2,O,7.0μl5x TDBG(1x Final),3.5μl10x MK最终),0.6μl1μMRFC(最终5nM),0.6μl5μMPCNA(终浓度25nM),0.6μl2μMPolα(10nM终)和0.6μl2μMPolδ(10nM终),用于最终体积为5×7μl反应=35μl。向每个反应管中加入21μl(3×7μl),用移液管尖端充分混合。在30℃孵育1分钟。
      3. 复制启动(见表2C)
        表2C。滞后链模型的复制开始阶段


        通过添加在预孵育期间被保留的核苷酸来启动复制。
        示例: 按照以下顺序添加10.5μlddH 2 O,4.2μl5x TDBG(1x final),2.1μl10x MK最终),0.5μl3mM dCTP(最终20μM),1.5μl3mM dTTP(最终60μM),1.5μlT / C / G混合物(200μM终浓度),2μlT / G / C混合物μM最终)。尖峰与0.75μl P-α-dCTP(10μCi/反应)。设置一个计时器3分钟。加入21μl起始混合物;启动定时器,同时用移液器彻底混合。在1分钟,2分钟和3分钟内清除20微升的反应物,并快速转移到预先装入10μl终止液的三个试管中。
    4. 复制产品可视化
      对于3nbf实验,前导链产品应类似于Georgescu等人的图1b(2015);即,其长度随着时间的推移而增加到全长(3.2kb)〜10分钟以下的产品。使用末端标记的引物来监测3kbf底物上的引物延伸,结果应该类似于Schauer等人(2017)的图3b中的引物延伸;即,,长度随着时间的推移而增加(2.8 kb)的产品。替代指导的滞后链复制应该类似于Schauer等人(2017)图6中的那些。即,约200bp的产品。对于所有CMG导向实验,实验中的CMG或ATP的减法是良好的阴性对照,不应产生延长的产物。使用滞后链模型底物,实验应与Schauer等人的图1b类似。 (2017); ,在3分钟内稳健的全长(5.4kb)产品。
      1. 准备梯子
        按照制造商的方案,用T4激酶和 32 P-γ-ATP对1kb DNA梯度进行5'末端标记。通常,我们用50μl总共5μlT4 PNK和4μl P-γ-ATP标记20μl梯子。使用已经用TE平衡的G-25柱,根据制造商的方案,将游离的 32 P-γ-ATP净化。
      2. 准备碱性琼脂糖凝胶
        将2.6或1.6g LE琼脂糖加入200ml蒸馏的H 2 O(3nbf / 3kbf底物为1.3%,滞后链模型底物为0.8%)。微波直到沸腾冷却至至少60℃,加入0.5ml 0.5M EDTA和0.6ml 10N NaOH,然后倒入凝胶。在约2小时内冷却至室温(RT)。在水平凝胶盒中浸入碱性运行缓冲液(参见食谱)。
      3. 在凝胶中加载样品和DNA标准品。在35V下运行16小时
      4. 去除免费的放射性核素
        从凝胶盒中取出凝胶,暴露于荧光成像屏幕15分钟。在磷光体上扫描并以全尺寸打印凝胶。将打印输出部分的饱和部分切掉,将其覆盖在凝胶上,并将包含游离核素(通常为底部第三或第四)的凝胶底部切掉,以减少自身和实验室设备的暴露。当监测滞后链合成时,该区域的鉴定特别重要,因为游离放射性核苷酸不超过冈崎片段运行得太远。自由放射性核素也可以从凝胶中流出,但会污染设备,产生进一步的放射性废物,并且它们倾向于在缓冲液中扩散,并可以回到整个凝胶中并有助于背景信号。
      5. 压缩凝胶
        将塑料包裹在一个平坦的表面上,如有机玻璃辐射屏蔽的底部。将凝胶板放在纱布上。将C折纸巾平放;一个与每个凝胶边缘相邻。将一张DEAE纤维素纸放在凝胶上。在这上面放置一张或两张Whatman 3MM色谱转印纸。堆叠2个水平行〜25 C折纸巾。盖上平坦而刚性的纸巾(例如平面有机玻璃罩),以分配重量。在该组件的中心轻轻地放置重物(例如,,铅砖),并将薄(几毫米厚)压榨6-12小时。我们发现这个方法优于使用凝胶干燥器,它可以作为使DNA转移和固定在DEAE-纤维素纸上的一个步骤。
      6. 除去除DEAE纤维素外的所有纸张,包裹在塑料包装中,并根据同位素强度将凝胶面暴露于磷化筛网2-36小时。
      7. 在磷光显像仪上扫描荧光成像屏幕。

数据分析

引物扩展实验的分析在其他地方是简单直观的,大多数操作可以通过ImageJ中的Gels包处理(参见 https://imagej.nih.gov/ij/docs/menus/analyze.html#gels )。感兴趣的参数可以是速度,引物摄取,产品长度,等等,并且将取决于实验。除了阅读Gels软件包的文档外,我们强烈建议读者阅读ImageJ用户指南( https://imagej.nih.gov/ij/docs/guide/ ),以便在分析凝胶数据之前进行图像分析的一般帮助。
当使用放射性核苷酸掺入时,使用DNA梯度作为参考点来校正 32 P掺入的偏倚至关重要,因为较长的产物将具有更多的掺入核苷酸信号,摩尔数相对于长DNA分子(Kurth等人,2013和Georgescu等人,2014)。因此,不执行此步骤的后果是对较长复制产品的过高估计,以及对较短复制产品的低估。使用以下例程量化凝胶,如Georgescu等人(1c)图1c所示。

  1. 从使用ImageJ的凝胶扫描中提取车道强度分布(,即平均像素强度与垂直距离)。
    1. 水平旋转凝胶,使车道从左到右。图像&gt;变换&gt;向右旋转90°。
    2. 选择ImageJ工具栏上的矩形图标,并在第一个通道周围制作一个矩形。
    3. 绘制积分强度分布(平均强度a.u.对距离(厘米))。分析&gt;绘图配置文件(或Ctrl + K)。
    4. 将出现一个新窗口。单击保存将配置文件另存为文本文件。
    5. 使用向下箭头将矩形向下移动到下一个车道,并重复步骤1c。保持相同的矩形尺寸和水平位置确保凝胶泳道正确排列。
    6. 将强度配置文件作为制表符分隔数据导入电子表格软件。
  2. 将强度分布中的距离转换为DNA长度。
    1. 以分子量(MW)标准拟合车道,其概率分布包含与出现在DNA MW标准中的条带数相同的高斯峰数。
    2. 绘制dsDNA标准(bp)的长度与从高斯拟合得到的平均垂直距离(cm),并用对数函数拟合数据,注意拟合线的方程式。
    3. 在电子表格中,使用数据分析步骤1c中获得的公式将垂直距离(cm)转换为DNA(bp)。
  3. 校正分子量的泳道强度分布。通过将每个强度值除以其DNA长度(如步骤2中确定)并乘以完整模板长度(以bp计),逐个像素地归一化产品的强度。所得到的曲线可以与任何能够进行非线性曲线拟合的软件曲线拟合,并根据需要逐个分析。

笔记

  1. 在准备3nbf底物时,至关重要的是消除所有潜在的终端标记来源,包括由于低效的叉连接引起的模板和襟翼的缺口。因此,我们建议极其温和地处理模板,包括最小的涡旋,温和的移液,等。
  2. 在叉式连接步骤期间的扩展连接是有用的。末端标记呈现出在复制分析的早期时间点出现的全长带;如果衬底已被正确准备,则应该不存在或只能稍微检测。作为对照,进行缺乏CMG和/或ATP的反应来评估终端标记的程度;没有CMG展开活动,任何时候都不应该出现全长的产品,因为Polε不能变换。
  3. 当复制测定降解底物并且也有助于终止标记时,核酸酶构成重要的问题,如果信号强度太高,其可能与全长引物延伸无法区分。因此,检查核酸酶的所有蛋白质制剂和试剂是至关重要的。微生物污染是引入核酸酶的另一个主要原因,因此确保所有试剂均经过高压灭菌,过滤灭菌,等分以供单次使用,并冷冻。有可用于测试DNAse和RNAse活性的商业试剂盒,然而,您感兴趣的试剂与 32 P标记的寡核苷酸(例如为3kbf创建的引物)的孵育时间也将提供信息。在非变性聚丙烯酰胺凝胶上,核酸酶活性将显示为 32 P信号的时间依赖性消失。

食谱


注意:除非另有说明,将所有溶液储存在-20°C。复制测定中使用的缓冲液不应超过〜6周。

  1. 6x凝胶负载染料
    60%甘油
    0.1%溴酚蓝
    0.1%二甲苯甲醇
    存储在RT
  2. TE缓冲液,pH 8.0
    10mM Tris-HCl,pH8.0
    1mM EDTA(乙二胺四乙酸,二钠盐)
  3. 10倍TBE
    1 M Tris碱
    1 M硼酸
    20 mM EDTA
  4. DNA洗脱缓冲液
    100 mM NaCl
    10mM Tris-HCl,pH8.0
    1 mM EDTA
  5. 20x SSC
    3 M NaCl
    0.3 M柠檬酸钠
  6. 1-丁醇饱和水
    50ml 1-丁醇
    5毫升ddH 2 O
    大力摇动并在4°C储存
  7. 5x TDBG
    125mM Tris-乙酸盐,pH7.5
    25%甘油
    200μg/ ml BSA
    10mM TCEP(Tris(2-羧乙基)膦)pH7.5
    15 mM DTT
    0.5mM EDTA(乙二胺四乙酸)
  8. 10x MK
    500 mM谷氨酸钾
    100 mM醋酸镁
  9. dA / dC mix
    10 mM dATP
    10 mM dCTP
    10mM Tris-HCl,pH7.5
    1 mM EDTA
  10. dT / dG mix
    10 mM dTTP
    10 mM dGTP
    10mM Tris-HCl,pH7.5
    1 mM EDTA
  11. T / G / C mix
    10 mM TTP
    10 mM GTP
    10 mM CTP
    10mM Tris-HCl,pH7.5
    1 mM EDTA
  12. 停止解决方案
    1%SDS
    40 mM EDTA
    10%甘油
    0.02%溴酚蓝
    0.02%二甲苯甲醇
    存储在RT
  13. 碱性运行缓冲器
    1.125 mM EDTA
    30 mM NaOH

致谢

这项工作得到了国家卫生研究院(T32 CA009673至G.S; R01 GM115809和霍华德休斯医学研究所对M.O.D.)的资助。本文中的方案适用于Georgescu等人,2014年,Georgescu等人,2015年和Schauer等人最初使用的方案。 ,2017年。

参考

  1. Georgescu,RE,Langston,L.,Yao,NY,Yurieva,O.,Zhang,D.,Finkelstein,J.,Agarwal,T.and O'Donnell,ME(2014)。&lt; a class = -insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/24997598"target ="_ blank">在真核复制叉处的不对称聚合酶装配的机制 Nat结构Mol Biol 21(8):664-670。
  2. Georgescu,RE,Schauer,GD,Yao,NY,Langston,LD,Yurieva,O.,Zhang,D.,Finkelstein,J.and O'Donnell,ME(2015)。&nbsp; 重建真核复制体显示了定义前导/滞后链操作的抑制机制。 Elife 4:e04988。
  3. Kurth,I.,Georgescu,RE和O'Donnell,ME(2013)。&nbsp; 通过偶联DNA聚合酶在染色体复制过程中释放扭转DNA的解决方案。自然 496(7443):119-122。
  4. Langston,LD和O'Donnell,M。(2008)。
  5. Langston,LD,Zhang,D.,Yurieva,O.,Georgescu,RE,Finkelstein,J.,Yao,NY,Indiani,C.and O'Donnell,ME(2014)。&lt; a class =插入文件"href ="http://www.ncbi.nlm.nih.gov/pubmed/25313033"target ="_ blank"> CMG解旋酶和DNA聚合酶ε形成用于真核引物链DNA复制的功能性15-亚基全酶。
    Proc Natl Acad Sci USA 111(43):15390-15395。
  6. Schauer,GD和O'Donnell,ME(2017)。&nbsp; 质量控制机制排除来自真核复制叉的不正确的聚合酶。 Proc Natl Acad Sci USA 114(4):675-680。
  7. Stodola,JL和Burgers,PM(2016)。解决在几毫秒的时间内,冈崎片段成熟的各个步骤。 Nat Struct Mol Biol 23(5):402-408。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Schauer, G., Finkelstein, J. and O’Donnell, M. (2017). In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication. Bio-protocol 7(18): e2548. DOI: 10.21769/BioProtoc.2548.
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