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Oct 2019

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Site-specific DNA Mapping of Protein Binding Orientation Using Azidophenacyl Bromide (APB)
叠氮苯酰溴(APB)定位蛋白质结合方向的DNA图谱   

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Abstract

The orientation of a DNA-binding protein bound on DNA is determinative in directing the assembly of other associated proteins in the complex for enzymatic action. As an example, in a replisome, the orientation of the DNA helicase at the replication fork directs the assembly of the other associated replisome proteins. We have recently determined the orientation of Saccharalobus solfataricus (Sso) Minichromosome maintenance (MCM) helicase at a DNA fork utilizing a site-specific DNA cleavage and mapping assay. Here, we describe a detailed protocol for site-specific DNA footprinting using 4-azidophenacyl bromide (APB). This method provides a straightforward, biochemical method to reveal the DNA binding orientation of SsoMCM helicase and can be applied to other DNA binding proteins.

Keywords: MCM helicase (解旋酶MCM), DNA replication (DNA复制), Site-specific footprinting (Site-specific足迹分析), Orientation (定位), DNA translocation (DNA 转运), DNA mapping (DNA基因定位)

Background

DNA replication is the process in which the duplex genomic strands separate into two template strands, the leading and lagging strands. This function is executed by a ring-shaped hexameric helicase in all Domains of life. Like other ring-shaped hexameric helicases, MCM consists of two domains; an N-terminal domain (NTD) and a C-terminal domain (CTD). In theory, either of these domains could be oriented towards the replication fork during translocation and be consistent with the known 3′-5′ translocation directionality. The MCM helicase loads onto DNA origins as a double hexamer with NTDs facing each other. The orientation of the helicase during translocation determines whether the two hexamers dissociate away from each other or bypass one another during active unwinding. Our recent paper shows that the Saccharolobus solfataricus (SsoMCM) unwinds DNA with NTD leading the way (Perera and Trakselis, 2019).

In order to directly determine the translocation orientation, we utilized a combination of site-specific DNA footprinting, single turnover unwinding, and translocation assays. Here, we provide detailed protocols of site-specific DNA footprinting assays with 4-azidophenacyl bromide (APB) to analyze the translocation orientation of SsoMCM (Pendergrast et al., 1992, Kassabov and Bartholomew, 2004, Nodelman et al., 2017).

APB is a heterobifunctional photoactivatable crosslinking agent. Its bromide functional group reacts by S-alkylation with reduced thiols (i.e., cysteines) to form stable thioether products (Figure 1). After binding of the functionalized protein to DNA and then exposure to UV light, a reactive singlet nitrene forms that can crosslink to either protein or DNA (in close proximity) through multiple insertion or addition mechanisms (Figure 1). The resulting crosslinked protein-DNA complex can be cleaved at the crosslinked nucleotide(s) under induced alkali/heat treatment. The lengths of the resulting DNA fragments can be used to determine the orientation distribution of the SsoMCM helicase on DNA. This is a straightforward biochemical method that reveals unique positions of SsoMCM helicases on DNA. It has an important advantage over traditional footprinting in that it can determine protein binding orientation on DNA instead of just binding site size.

Useful applications of this method can be employed at instances where the 3D structure of the protein is known or can be predicted, but the 3D structure of the protein-DNA complex is unknown (Pendergrast et al., 1992). It is especially useful in determining the orientation of proteins that translocate along DNA or for those that bind to specific DNA sequences. Alternatively, the orientation of DNA-binding proteins can also be determined by other biochemical methods that employ localized hydroxyl radical Fenton footprinting reactions utilizing 1-(p-Bromoacetamidobenzyl) ethylenediamine N,N,N (Fe-BABE), similarly (Owens et al., 1998).


Figure 1. Conjugation of APB to free Cys on SsoMCM and UV induced crosslinking reaction mechanism to DNA

Materials and Reagents

  1. Cover slips (Fisher Scientific, catalog number: 12-546 )
  2. Glass Petri dish (Corning, catalog number: 3160100 )
  3. Kimwipe (Kimberly-Clark, catalog number: 06-666 )
  4. p-Azidophenacyl bromide (APB) (Sigma-Aldrich, catalog number: 57018-46-9 , storage: 4 °C or (Fisher Scientific, catalog number: 50-520-767 )
  5. Dimethylformamide (DMF) (EMD Millipore Corporation, catalog number: DX1730-6 )
  6. Tris Base (Fisher Scientific, catalog number: 77-86-1 )
  7. Glacial acetic acid (Mallinckrodt Baker, Inc., catalog number: UN 2789 )
  8. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-3 )
  9. Sodium hydroxide (NaOH) (Mallinckrodt Baker, Inc., catalog number: 7708-10)
  10. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144-212 )
  11. Magnesium chloride (MgCl2) (Spectrum Chemical, catalog number: M1035 )
  12. Glycerol (Fisher Scientific, catalog number: BP-229-4 )
  13. Sodium acetate (NaOAc) (Fisher Scientific, catalog number: S210-500 )
  14. Potassium acetate (KOAc) (EM Science Industries, catalog number: PX1330-1 )
  15. Magnesium acetate (MgOAc) (Sigma-Aldrich, catalog number: M-0631 )
  16. Ammonium acetate (NH4OAc) (Mallinckrodt Baker, Inc., catalog number: 0596-01 )
  17. Bovine serum albumin (BSA) (Fisher Scientific, catalog number: BP1600-100 )
  18. Dithiotheritol (DTT) (Fisher Scientific, catalog number: BP172-5 )
  19. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E5134 )
  20. Sodium dodecyl sulfate (SDS) (Fisher Scientific, catalog number: 151-21-3 )
  21. Salmon sperm DNA (Invitrogen, catalog number: 15632-011 , storage temperature: -20 °C)
  22. Ethanol (Fisher Scientific, catalog number: A962-4 )
  23. Poly-L-lysine (Sigma-Aldrich, catalog number: P4832 )
  24. Orange G dye (EMD Millipore Corporation, catalog number: 312-12 )
  25. SsoMCM protein
    SsoMCM protein was purified as previously described (McGeoch et al., 2005; Graham et al., 2011). Any DNA binding protein with a suitable single cysteine residue can be utilized with this approach. It will help if that cysteine residue is solvent accessible from structural data. If a single cysteine is not available, then site-directed mutagenesis can be used to add a cysteine at the appropriate position to test.
  26. Fluorescent (5′ or 3′) labelled (Cy3 or Cy5) DNA (purchased from IDT (Coralville, IA) or Sigma-Aldrich (St. Louis, MO)) (Synthesis scale–0.025 μmole, Purification: Desalt, Format: Dry).
    Note: PAGE purification of DNA can be performed depending on the purity of the DNA.
  27. DNA Sequences for the 3’-long arm fork substrates
    1. DNA 165-3 (5′ Cy3)
      5′ 3TCCCACCCAACCCGACCGGCATCTAGTCTGGTAGCGTGAGCGAACGGACC
    2. DNA 171
      5′ CTAACTGCGCCGGTCGGGTTGGGTGGGA
  28. 1x complex buffer (CB) (see Recipes)
  29. Annealing buffer (see Recipes)
  30. Post irradiation buffer (see Recipes)
  31. Glycerol loading buffer (see Recipes)
  32. 20% Native PAGE (see Recipes)

Equipment

  1. Pipettes
  2. -80 °C freezer
  3. Benchtop 3UV transilluminator (UVP, model: LMS-20E , catalog number: P/N 95-0220-01)
  4. Typhoon FLA 9000 imager (GE Healthsciences)
  5. PCR Instrument (BioRad laboratories (Hercules, CA)
  6. Benchtop microcentrifuge (Eppendorf, model: 5424R )
  7. Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels (Bio-Rad Laboratories)
  8. LightSafe micro centrifuge tubes (Sigma-Aldrich, catalog number: Z688312 )
  9. Dark room (Recommended)

Software

  1. Image Quant (v.5.0) (GE Healthsciences, (Pittsburgh, PA))

Procedure

  1. SsoMCM Protein labelling with APB
    1. In the dark, dissolve APB in 100% DMF at a concentration of 40 mM.
      Notes:
      1. Dark conditions can be provided by using LightSafe micro centrifuge tubes, covering Eppendorf tubes in foil or turning off the lights.
        1. Use LightSafe micro centrifuge tubes when preparing APB solutions.
        2. Use clear micro centrifuge tubes covered in foil in subsequent reactions for easy visualization of a pellet and/or to achieve complete discarding of a supernatant.
      2. When measuring APB, quantities as small as ~2 mg can be measured under dark conditions.
      3. Use freshly resuspended APB for each new experiment. Discard the unused APB.
    2. Dilute the APB to 4 mM in 20 mM Tris pH 7.5, 75 mM NaCl, 10% glycerol and 10% DMF.
    3. Add the diluted APB to a sample of the protein (~10 µM monomer) containing a single reduced cysteine (in 20 mM Tris [pH 7.5], 75 mM NaCl, 10% glycerol), to achieve a final concentration of 0.4 mM APB and 1% DMF.
      Note: Cysteines must be reduced before conjugation with APB, followed by gel filtration or dialysis to remove the reducing agent. Alternatively, tris(2-carboxyethyl)phosphine (TCEP) can be used without subsequent removal.
    4. Proceed labelling for 2-3 h at room temperature.

  2. Preparing silanized coversli
    1. Dilute poly-L-lysine solution with distilled water for 1:10.
    2. Place the coverslips (~15) in a Petri dish.
    3. Pour the solution (~20 ml) on the coverslips (make sure the coverslips sink in).
    4. Leave it in the solution for 15-30 min.
    5. Rinse the coverslips with water three times at Room Temperature (RT).
    6. Air dry the coverslips placing a kimwipe on top of the Petri dish (avoid touching the coverslips).
    7. Coverslips can be prepared ahead of time and can be stored at RT for several weeks.

  3. Preparing annealed fork DNA
    1. Prepare stock solutions (100 μM) of each complementary DNA strand in water (DNA 165-3 and DNA 171).
    2. Mix the DNA in equal 1:1 (5 μM) concentration in water or annealing buffer.
      Note: EDTA (chelates divalent metal ions) and salt in the annealing buffer facilitate duplex stability. But annealing DNA in water has also yielded in a complete duplex.
    3. Heat at 95 °C for 5 min and cool at a rate of 1 °C/min to room temperature in a PCR instrument.

  4. Preparing the crosslinked protein-DNA complex
    1. Incubate APB labelled SsoMCM with fluorescent (Cy3) fork DNA (150 nM) for 10-20 min in 1x CB buffer in 50 µl volumes (maintaining stochiometric ~1:1 MCM6:DNA ratio).
      Note: Fluorescently labelled DNA is used here, but 32P labelled DNA can also be utilized.
    2. Transfer the sample into silanized coverslips. Pipette 50 µl of the sample into the middle of the coverslip.
    3. UV irradiate for 15 s (302 nm UV-B).
    4. Transfer the sample into an Eppendorf tube.
    5. Add 150 µl of post irradiation buffer.
    6. Vortex at RT and place at 70 °C for 20 min.

  5. Separate the crosslinked protein-DNA complex
    1. Next, add 1 µl of 10 mg/ ml Salmon sperm DNA, 30 µl of 3.0 M NaOAc, 750 µl of ice cold 100% ethanol, vortex, and leave on ice for 1-2 h at -80 °C.
    2. Spin down the samples in microfuge at 4 °C, 16,000 x g for 30 min.
    3. Discard the supernatant and wash the pellet twice with ice cold 70% ethanol.
      Note: In most of the steps, the DNA pellets are invisible. Discard the supernatant completely and resuspend in the subsequent buffer.
    4. Spin down the samples in microfuge at 4 °C, 16,000 x g for 30 min.
    5. Remove ethanol and air dry the pellets by inverting on bench for 1 h.
    6. Resuspend the pellet in 100 µl: 20 mM NH4OAc, 2% SDS, 0.1 mM EDTA pH 8.0 by vortexing.
    7. Spin down the samples in microfuge at room temperature, 16,000 x g for 10 min.
    8. Transfer the supernatant into fresh tubes and place in heat block at 90 °C for 2 min.
    9. Then, add 1 µl of 2 M NaOH, vortex briefly, and incubate at 90 °C for 20 min.
    10. After incubation, pulse spin the samples, add 101 µl 20 mM Tris-HCl pH 8.0, 1 µl of 2 M HCl, 1 µl of 2 M MgCl2, 480 µl 100% ethanol, vortex, and place at -80 °C for 1-2 h.
    11. Pellet the samples in microfuge at 4 °C at 16,000 x g for 30 min, wash two times with ice cold 70% ethanol, and air dry on bench for 1 h.
    12. Resuspend the DNA pellet with 5 µl of 40% glycerol loading buffer containing Orange G dye for gel loading.
    13. Electrophorese the samples on a 20% TBE-PAGE (native PAGE) in 1x TBE (Tris base, Boric acid, EDTA) at constant 30 mA for 45 min, and visualize on a Typhoon FLA 9000 imager (GE Healthsciences).

Data analysis

  1. Quantifying the density of DNA bands on the gel (Figure 2A) is performed by ImageQuant software.
  2. Calculation of the footprinting percentages (Figure 2B) is performed by quantifying the relative density (minus background) for the labelled strand, divided at the midpoint on the ssDNA arm according to the following equation:




    Figure 2. SsoMCM orientation mapping onto 3′-(DNA171/165-3) long arm fork DNA substrate. A. APB orientation mapping of the 3′-encircled strand labelled at the 5′ duplex end with Cy3 on a 3′-long arm fork DNA substrate with a 20 base duplex. SsoMCM was labelled at C682 with APB specifically. B. Quantification of the relative amount of DNA cleavage for bases 20-35 or 36-50 from the 5′-end indicate the relative orientation for placing the N @duplex (orange) or C@duplex (blue), respectively closer to the duplex junction. DNA markers (M) indicate 18, 50 bases and fork DNA. Error bars represent standard error from 3-5 independent experiments. P-value is defined as ** < 0.01.

  3. A standard two-tailed equal variance Student’s t-test is used to determine significant differences of C@duplex versus N@duplex. P-values are reported for each experimental condition. Detailed explanation on data analysis can be found in the original research paper Figures 1C and 1F, 2B and 2D, 3C and 2E (Perera and Trakselis, 2019).

Notes

  1. Control experiments are performed with APB-MCM-DNA in the absence of UV light and/or with DNA alone in the absence of UV and APB. After activation by UV light, the crosslinking of APB to DNA bases is generally non-specific, yet we detected significant crosslinking and subsequent ssDNA cleavage even in the absence of direct UV light.
  2. Similarly, 1-(p-Bromoacetamidobenzyl) ethylenediamine N,N,N (Fe-BABE) (Dojindo, Rockville, Maryland) can also be used in orientation mapping studies (Figure 1B and 1E in Perera and Trakselis, 2019). FeBABE utilizes a localized hydroxyl radical Fenton footprinting reaction. Rapid reactions generating high yields, mild protein conjugation and cleavage reaction conditions, non-sequence specific DNA cleavage are some advantages of utilizing FeBABE (Ishihama, 2000).

Recipes

  1. 1x CB (complex buffer)
    20 mM TrisOAc
    25 mM KOAc
    10 mM MgOAc
    0.1 mg/ml BSA
    1 mM DTT
  2. Annealing buffer
    10 mM Tris pH 7.5
    50 mM NaCl
    1 mM EDTA
  3. Post irradiation buffer
    20 mM Tris-HCl pH 8.0
    0.1% SDS
    50 mM NaCl
  4. Glycerol loading buffer
    40% glycerol
    10 mM EDTA (pH 8.0)
    7.5% Orange G dye
  5. 20% Native PAGE
    1x TBE
    20% acrylamide
    0.05% APS
    4 μl TEMED

Acknowledgments

This work was supported by the National Science Foundation Division of Molecular and Cellular Biosciences [NSF1613534 to M.A.T.] and supported by Baylor University. We thank Gregory Bowman for introducing us to the feasibility of this technique for orientation mapping (Nodelman et al., 2017).

Competing interests

The authors declare that they have no conflicts of interest with the contents of this article.

References

  1. Graham, B. W., Schauer, G. D., Leuba, S. H. and Trakselis, M. A. (2011). Steric exclusion and wrapping of the excluded DNA strand occurs along discrete external binding paths during MCM helicase unwinding. Nucleic Acids Res 39: 6585-6595.
  2. Ishihama, A. (2000). Molecular anatomy of RNA polymerase using protein-conjugated metal probes with nuclease and protease activities. Chem Commun 13: 1091-1094. 
  3. Kassabov, S. R. and Bartholomew, B. (2004). Site-directed histone-DNA contact mapping for analysis of nucleosome dynamics. Methods Enzymol 375: 193-210.
  4. McGeoch, A. T., Trakselis, M. A., Laskey, R. A. and Bell, S. D. (2005). Organization of the archaeal MCM complex on DNA and implications for the helicase mechanism. Nat Struct Mol Biol 12:756-762.
  5. Nodelman, I. M., Bleichert, F., Patel, A., Ren, R., Horvath, K. C., Berger, J. M. and Bowman, G. D. (2017). Interdomain communication of the Chd1 chromatin remodeler across the DNA Gyres of the nucleosome. Mol Cell 65(3): 447-459 e446.
  6. Owens, J. T., Miyake, R., Murakami, K., Chmura, A. J., Fujita, N., Ishihama, A. and Meares, C. F. (1998). Mapping the σ70 subunit contact sites on Escherichia coli RNA polymerase with a σ70-conjugated chemical protease. PNAS 95(11): 6021-6026.
  7. Pendergrast, P. S., Chen, Y., Ebright, Y. W. and Ebright, R. H. (1992). Determination of the orientation of a DNA binding motif in a protein-DNA complex by photocrosslinking. Proc Natl Acad Sci U S A 89(21): 10287-10291.
  8. Perera, H. M. and Trakselis, M. A. (2019). Amidst multiple binding orientations on fork DNA, Saccharolobus MCM helicase proceeds N-first for unwinding. Elife 8: e46096.

简介

[摘要 ] 结合在DNA上的DNA结合蛋白的方向决定了复合物中其他相关蛋白的组装,以进行有意的作用。例如,在复制体中,复制叉处的DNA解旋酶的方向指导我们最近通过定点DNA切割和作图分析确定了DNA叉处的Saccharalobus solfataricus (Sso )微型染色体维持(MCM)解旋酶的方向。 使用4-叠氮苯甲酰溴(APB)进行位点特异性DNA足迹的详细协议。此方法提供了一种直接的生化方法来揭示Sso MCM解旋酶的DNA结合方向,并可应用于其他DNA结合蛋白。

[背景 ] DNA复制是其中基因组双链体链分离成两个模板链中,超前和滞后strands.This功能是通过在生命。如同其他环状六聚体的解旋酶结构域的所有的环状六聚体解旋酶的处理,从理论上讲,这些结构域中的任何一个都可以在易位期间朝向复制叉,并且与已知的3'-5 MCM包含两个结构域; N端结构域(NTD)和C端结构域(CTD)。' 易位directionality.T 他MCM解旋酶负载到DNA起源作为具有面向易位期间解旋酶的每个other.The取向管畸形双六聚体确定两个六聚体是否彼此或旁路彼此活性unwinding.Our最近的一篇论文中离解远表明Saccharolobus solfataricus (Sso MCM )通过NTD引导DNA解链(Perera和Trakselis ,2019)。

为了直接确定易位方向,我们结合了位点特异性DNA足迹,单周转码和易位测定的方法。在这里,我们提供了使用4-叠氮苯甲酰溴(APB)进行位点特异性DNA足迹测定的详细方案分析Sso MCM 的易位方向(Pendergrast 等,1992; Kassabov和Bartholomew,2004; Nodelman 等,2017)。

APB是异光活化交联剂。通过S-烷基化及其溴化物官能团反应具有降低的硫醇(即,半胱氨酸),以形成稳定的硫醚产物(图1 )。经过与DNA曝光官能蛋白结合,然后于UV光,生成的单联氮烯形式可以通过多种插入或添加机制与蛋白质或DNA交联(非常接近)(图1 )。可以在诱导的碱/条件下在交联的核苷酸处裂解所得的交联的蛋白质-DNA复合物。产生的DNA片段的长度可用于确定Sso MCM解旋酶在DNA上的方向分布,这是一种直接的生化方法,揭示了Sso MCM解旋酶在DNA 上的独特位置,与传统方法相比具有重要的优势。因为它可以确定蛋白质在DNA上的结合方向,而不仅仅是结合位点大小。

在已知或可以预测蛋白质3D结构但蛋白质-DNA复合物3D结构未知的情况下(Pendergrast 等人,1992),可以使用此方法的有用方法。确定蛋白质的取向沿着DNA或那些易位,其结合于特定DNA序列,此外,DNA结合蛋白的取向也可以通过其它生化方法确定雇用局部羟基自由基芬顿足迹利用反应1-(对类似地,溴乙酰胺基苄基)乙二胺N,N,N(Fe-BABE)(Owens 等,1998)。



D:\ Reformatting \ 2020-4-7 \ 1903051--1418 Michael Trakselis 856305 \ Figs jpg \ Fig 1.jpg

1图。共轭APB免费半胱氨酸在SSO MCM 和UV引发的交联反应机制,DNA

关键字:解旋酶MCM, DNA复制, Site-specific足迹分析, 定位, DNA 转运, DNA基因定位

材料和试剂


 


盖玻片(Fisher Scientific,目录号:12-546)
玻璃培养皿(Corning,目录号:3160100)
Kimwipe(金伯利-克拉克(Kimberly-Clark),目录号:06-666)
对- 叠氮基苯甲酰溴(APB)(Sigma-Aldrich ,目录号:57018-46-9 ,s 残留量:4 °C或(Fisher Scientific,目录号:50-520-767)
二甲基甲酰胺(DMF)(EMD密理博公司,目录号:DX1730-6)
Tris基地 (Fisher Scientific,目录号:77-86-1)
冰醋酸(Mallinckrodt Baker,Inc.,目录号:UN 2789)
氯化钠(NaCl)(Fisher Scientific,目录号:S271-3)
氢氧化钠(NaOH)(Mallinckrodt Baker,Inc.,目录号:7708-10)
盐酸(Fisher Scientific,目录号:A144-212)
氯化镁(MgCl 2 )(Spectrum Chemical,目录号:M1035)
甘油(Fisher Scientific,目录号BP-229-4)
乙酸钠(NaOAc )(Fisher Scientific,目录号:S210-500)
乙酸钾(KOAc )(EM Science Industries,目录号:PX1330-1)
醋酸镁(MgOAc )(Sigma-Aldrich,目录号:M-0631)
醋酸铵(NH 4 OAc)(Mallinckrodt Baker,Inc.,目录号:0596-01)
牛血清白蛋白(BSA)(Fisher Scientific,目录号:BP1600-100)
二硫代糖醇(DTT)(Fisher Scientific,目录号:BP172-5)
乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E5134)
十二烷基硫酸钠(SDS)(Fisher Scientific,目录号:151-2-3)
鲑鱼精子DNA(Invitrogen,货号:15632-011,储存温度:-20 °C )
乙醇(Fisher Scientific,目录号:A962-4)
聚-L-赖氨酸(Sigma-Aldrich ,目录号:P4832)
橙色G染料(EMD Millipore Corporation ,目录号312-12)
Sso MCM蛋白
Sso MCM蛋白按先前的描述进行纯化(McGeoch 等,2005; Graham 等,2011)。任何具有合适的半胱氨酸残基的DNA结合蛋白均可用于该方法,如果该半胱氨酸残基为溶剂,将有帮助如果没有单个半胱氨酸,则可以使用定点诱变在合适的位置添加一个半胱氨酸进行测试。


荧光(5 ' 或3 ' )标记的(Cy3或Cy5)DNA(购自IDT(爱荷华州科勒维尔)或Sigma-Aldrich(密苏里州圣路易斯))(综合量表– 0.025 微摩尔,纯化:脱盐,形式:干燥)。
注意:可以根据DNA的纯度进行PAGE纯化。


3'长臂叉底物的DNA序列
一个。DNA 165-3(5 ' 的Cy3)       


5 ' 3 TCCCACCCAACCCGACCGGCATCTAGTCTGGTAGCGTGAGCGAACGGACC


湾DNA 171      


5 ' CTAACTGCGCCGGTCGGGTTGGGTGGGA


1X复杂缓冲液(CB)(见ř ecipes)
退火缓冲液(见ř ecipes)
辐照缓冲液柱(请参阅R Ecipes)
甘油加载缓冲液(请参见食谱)
20%本机PAGE (请参阅食谱)
 


设备


 


P ipettes
-80°C冷冻室
台式3UV透照仪(UVP,型号:L MS-20E,目录号:P / N 95-0220-01 )。
台风FLA 9000成像仪(GE Healthsciences )
PCR仪器(BioRad 实验室(加利福尼亚州赫尔克里士)
台式微量离心机(Eppendorf ,型号:5424R)
用于微型预制凝胶的PROTEAN Tetra-Mini垂直电泳池(Bio - Rad L Aboratories)
LightSafe 微量离心管(Sigma-Aldrich ,目录号:Z688312)
暗室(推荐)
 


软件


 


Image Quant(v.5.0)(GE Healthsciences ,(宾夕法尼亚州匹兹堡)。)
 


程序


 


用APB标记Sso MCM 蛋白
在黑暗中,将APB溶解在100%DMF中,浓度为40 mM。
笔记:


a。可以通过使用LightSafe 微型离心管,用箔覆盖Eppendorf管或关闭灯来提供黑暗条件。       


制备APB溶液时,请使用LightSafe 微量离心管。
在随后的反应中,请使用覆盖在箔纸中的透明微型离心管,以轻松观察沉淀和/或完全丢弃上清液。
b。在测量APB时,可以在黑暗条件下测量约2 mg的量。      


C. 为每个新实验使用新近重悬的APB,并丢弃未使用的APB。       


在20 mM Tris pH 7.5、75 mM NaCl,10%甘油和10%DMF中将APB稀释至4 mM。
将稀释的APB加到含有单个还原半胱氨酸的蛋白质(〜10μM单体)样品中(在20 mM M Tris [pH 7.5],75 mM NaCl,10 %甘油中),最终浓度为0.4 mM APB和1%DMF。
注意:在与APB结合之前必须还原半胱氨酸,然后进行凝胶过滤或渗析以去除还原剂。可以轻松使用三(2- 羧乙基)膦(TCEP),而无需随后去除。


在室温下贴标签2-3小时。
 


准备硅烷化的盖玻片
用蒸馏水将聚L-赖氨酸溶液稀释为1:10。
联合广场Verslips(〜15)在一个P ETRI菜。
将溶液(约20 ml)倒在盖玻片上(确保盖玻片下沉)。
将其在溶液中放置15-30分钟。
在室温(RT)下用水冲洗盖玻片三次。
晾干盖玻片,将Kimwipe 放在P Etri盘顶部(避免触摸盖玻片)。
盖玻片可以提前准备,可以在室温下保存数周。
 


准备退火的叉子DNA
储备溶液准备(100 Myu M )水中的每个互补DNA链(DNA 165-3 和DNA 171)。
混合DNA在水中或退火缓冲液中的浓度等于1:1(5 Myu M )。
注意:退火缓冲液中的EDTA(螯合二价金属离子)和盐有助于双链体的稳定性,但是水中DNA的退火也可以形成完整的双链体。


在PCR仪中以95 °C 加热5分钟,然后以1 °C / min 的速度冷却至室温。
 


制备交联的蛋白质-DNA复合物
将APB标记的Sso MCM与荧光(Cy3)叉子DNA(150 nM )在1x CB缓冲液中以50 µl体积孵育10-20分钟(保持化学计量〜1:1 MCM 6 :DNA 比率)。
注意:此处使用了荧光标记的DNA,但也可以使用32 P标记的DNA。


将样品转移到硅烷化的盖玻片中,用移液管吸取50 µl样品到盖玻片的中间。
紫外线照射15 s(302 nm UV-B)。
将样品转移到Eppendorf管中。
加入150 µl辐照后缓冲液。
在室温下涡旋并在70°C下放置20分钟。
 


分离交联的蛋白质-DNA复合物
接下来,加入1 µl 10 mg / ml鲑鱼精子DNA,30 µl 3.0 M NaOAc ,750 µl冰冷的100%乙醇,涡旋振荡,然后在-80°C的冰上放置1-2 h。
在微量离心机中于4°C,16,000 x g 旋转样品30分钟。
弃去上清液,用冰冷的70 %乙醇洗涤沉淀两次。
注意:在大多数步骤中,DNA沉淀是不可见的,将其完全丢弃并重悬在随后的缓冲液中。


在微量离心机中于4°C,16,000 xg离心30分钟以旋转样品。
除去乙醇,在工作台上翻转1小时,风干沉淀。
通过涡旋将沉淀重悬于100 µl:20 mM NH 4 OAc,2%SDS,0.1 mM EDTA pH 8.0中。
降速的样品中微量离心在室温下,16000 ×g下FO -R 10分钟。
将印迹转移到新的试管中,并置于90°C的加热块中2分钟。
然后,加入1 µl 2 M NaOH,短暂涡旋,并在90°C下孵育20分钟。
温育后,将样品脉冲旋转,加入101 µl 20 mM Tris-HCl pH 8.0、1 µl 2 M HCl,1 µl 2 M MgCl 2 、、 480 µl 100%乙醇,涡旋,并于-80°C放置1-2小时
将样品在微量离心机中于4°C以16,000 xg沉淀30分钟,用冰冷的70 %乙醇洗涤两次,然后在工作台上风干1 h。
用5 µl 40 %含橙色G染料的甘油上样缓冲液重悬DNA沉淀,以进行凝胶上样。
以恒定的30 mA 在1 x TBE(Tris碱,硼酸,EDTA)中的20%TBE- PAGE(天然PAGE)上电泳样品45分钟,然后在Typhoon FLA 9000成像仪(GE Healthsciences )上观察。
 


数据分析


 


通过ImageQuant 软件对凝胶上DNA条带的密度进行定量(图2A)。
足迹百分比的计算(图2B )是通过量化标记链的相对密度(减去背景)进行的,根据以下公式在ssDNA臂的中点进行划分:
 






 


D:\ Reformatting \ 2020-4-7 \ 1903051--1418 Michael Trakselis 856305 \ Figs jpg \ Figure2 .jpg


2图。SSO MCM取向映射到3 “ - (DNA171 / 165-3)长臂叉DNA底物A. APB取向映射的3 ” -Encircled链标记在5 “ 双工结束用Cy3上的3 ” -长臂叉DNA底物具有20个碱基的双链体.Sso MCM在C682处特别用APB标记.B。从5' 端对20-35或36-50碱基的DNA切割相对量的定量表明相对方向分别将N双链(橙色)或C双链(蓝色)放置在更靠近双链结的位置.DNA标记(M)表示18、50个碱基和叉子DNA 。误差线代表3-5个独立实验的标准误差。值是-p定义为* <0.05,** <0.01 *** < 0.001。Ns。不重要。


 


使用标准两尾相等方差学生的T检验确定C Atto双工与N Atto双工的显着差异。每种实验条件均报告P 值。数据分析的详细说明可以在原始研究论文图中找到1C和1F,2B和2D,3C和2E (Perera和Trakselis,2019)。
 


笔记


 


对照实验是在没有紫外线的情况下用APB-MCM-DNA进行的,和/或在没有紫外线和APB 的情况下用单独的DNA进行的。在被紫外线激活后,APB与DNA碱基的连接通常是非特异性的,然而,即使在没有直接紫外线的情况下,我们也检测到了明显的交联和随后的ssDNA裂解。
类似地,1-(对- Bromoacetamidobenzyl )乙二胺-N,N ,N(FeBABE)(同仁化学中心,Rockville,马里兰)也可以使用的取向作图研究(图1B和1E中佩雷拉和Trakselis ,2019 )。FeBABE 利用利用FeBABE (Ishihama,2000)的优点是快速反应产生高产率,温和的蛋白质结合和裂解反应条件,非序列特异性DNA裂解是快速的反应(Ishihama,2000)。
 


菜谱


 


1个CB(复杂缓冲区)
20毫米TrisOAc


25毫米KOAc


10毫米氧化镁


0.1 mg / ml BSA


1毫米DTT


退火缓冲
10 mM Tris pH 7.5


50毫米氯化钠


1毫米EDTA


照射后缓冲液
20毫米Tris-HCl pH 8.0


0.1 %SDS


50毫米氯化钠


甘油加载缓冲液
40%甘油


10 mM EDTA(pH 8.0)


7.5 %橙色G染料


20%原生PAGE
1 x TBE


20%丙烯酰胺


0.05 %的APS


4 Myu L TEMED


 


致谢


 


这项工作得到了美国国家科学基金会分子与细胞生物科学部门的支持[MAT的NSF1613534]并得到了贝勒大学的支持,我们感谢Gregory Bowman向我们介绍了该技术用于定向作图的可行性(Nodelman et al。,2017)。 。


 


利益争夺


 


作者声明,他们与本文的内容没有利益冲突。


 


参考文献


 


Graham,BW,Schauer,GD ,Leuba ,SH和Trakselis ,M.A ..(2011)。在MCM解旋酶解链核酸过程中,沿着离散的外部结合途径发生的立体排斥和包裹的DNA链分离沿着核酸Res 39:6585-6595 。
Ishihama ,A。(2000)。使用具有核酸酶和蛋白酶活性的蛋白质缀合的金属探针对RNA聚合酶的分子解剖学, Chem Commun 13:1091-1094。              
Kassabov ,SR和缪,B.(2004)。对于核小体动力学的分析位点定向组蛋白DNA接触映射。方法酶学375:193-210。
McGeoch ,AT,Trakselis ,MA ,Laskey ,RA和Bell ,S. d 。(2005)组织的古菌MCM复杂的DNA和意义的解旋机制。纳特结构分子生物学12:756-762。
Nodelman ,IM,Bleichert ,F。,帕特尔,A.,仁,R.,Horvath的,KC,伯杰,JM和鲍曼,GD(2017)横跨核小体DNA环流。在染色质CHD1的改造商通信.Interdomain分子。单元格65(3):447-459 e446。
欧文斯,JT,三宅,R. ,村上,K. ,Chmura,AJ ,藤田,N. ,Ishihama ,A 。和Meares的,CF(1998).Mapping 所述σ 70 亚基接触部位上的大肠杆菌RNA聚合酶与σ 70 偶联的化学蛋白酶。PNAS 95(11):6021-6026。
Pendergrast ,PS,Chen,Y.,Ebright ,YW和Ebright ,RH(1992)。通过光交联法测定蛋白质-DNA复合物中DNA结合基序的方向。Proc Natl Acad Sci USA 89(21):10287- 10291。
Perera ,HM和Trakselis,MA(2019)。在叉子DNA上的多个结合方向中,Saccharolobus MCM解旋酶以N-先进行解链.Elife 8:e46096。
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Copyright Perera and Trakselis. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Perera, H. M. and Trakselis, M. A. (2020). Site-specific DNA Mapping of Protein Binding Orientation Using Azidophenacyl Bromide (APB). Bio-protocol 10(12): e3649. DOI: 10.21769/BioProtoc.3649.
  2. Perera, H. M. and Trakselis, M. A. (2019). Amidst multiple binding orientations on fork DNA, Saccharolobus MCM helicase proceeds N-first for unwinding. Elife 8: e46096.
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