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. Author manuscript; available in PMC: 2020 Aug 18.
Published in final edited form as: Methods Mol Biol. 2018;1703:95–108. doi: 10.1007/978-1-4939-7459-7_7

The Use of Psoralen Photobinding to Study Transcription-Induced Supercoiling

Fedor Kouzine 1, Laura Baranello 2, David Levens 2
PMCID: PMC7433354  NIHMSID: NIHMS1617409  PMID: 29177736

Abstract

Proteins manipulating intracellular DNA necessarily impart torsional stress, which redistributes across the DNA. Overtwisting and undertwisting of the double helix result in the manifestation of positive and negative DNA supercoiling. A growing body of evidence indicates that DNA topology is an important player in the key regulatory steps of genome function, highlighting the need for biochemical techniques to detect dynamic changes in the DNA structure. Psoralen binding to DNA in vivo is proportional to the level of supercoiling, providing an excellent probe for the topological state of nuclear DNA. Here we describe a psoralen-based methodology to detect transcription-induced DNA supercoiling genome-wide. The DNA samples generated with this approach can be hybridized to microarray platforms or high-throughput sequenced to provide a topological snapshot of the whole genome.

Keywords: DNA topology, DNA supercoiling, Psoralen, Transcription, Topoisomerases, Chromatin, High-throughput genomics

1. Introduction

Rather than being a static helix, DNA possesses structural variability. DNA supercoiling plays a major role in the dynamic variation of the double helix. In supercoiled DNA, the torsional stress results in an excess or a deficiency in helical turns of the double helix. Notably, this torsional energy can be used during many critical steps of genes transcription [1, 2]. In eukaryotes, DNA supercoiling is believed to be generated dynamically and primarily by protein complexes, such as the RNA polymerase (RNAP) translocating along the double helix [3-5]. During transcription the active site of the RNAP tracks the helical path of DNA, which requires polymerase rotation relative to the DNA. The rotation of the polymerase may be hindered due to viscous drag or to tethering to nuclear structures (Fig. 1a), inducing overtwisting and undertwisting of the double helix in front of and behind the RNAP (positive and negative supercoiling, respectively). Increasing evidence suggests that biophysical mechanisms couple supercoiling to different DNA-dependent processes. Yet, we are still far from fully understanding the interplay between DNA topology and genome biology. The study of DNA topology has been hindered by experimental difficulties associated with detecting supercoiling and assessing its regulation in vivo, especially in eukaryotic cells [6].

Fig. 1.

Fig. 1

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Transcription-generated supercoiling and psoralen crosslinking as a probe for DNA supercoiling in vivo. (a) Transcription complex (shown as an oval) tracking along the DNA introduces negative and positive supercoiling. (b) Psoralen crosslinking density in vivo is high for negatively supercoiled DNA and low for positively supercoiled DNA. (c) Crosslinking density is a measure of DNA supercoiling in vivo

A powerful approach to detect DNA supercoiling relies on the properties of the molecule psoralen [7, 8]. Psoralen’s planar aromatic structure allows it to penetrate cellular membranes and to intercalate preferentially between the base pairs of negatively supercoiled DNA. Positively supercoiled DNA shows reduced psoralen binding (Fig. 1b). Upon exposure to UV light, the intercalated psoralen molecules crosslink complementary strands of the double helix via the formation of covalent bonds at each end of the molecules. The variation in the density of crosslinking can be used to detect and quantify DNA supercoiling in living cells (Fig. 1c). Though for several decades psoralen-based assays have been used to probe supercoiling averaged over entire genomes or in selective regions [9-11], only recently has this method been combined with genome-wide approaches, providing a global view of the functional dynamics of DNA supercoiling in vivo [12-16].

We have developed a methodology that is particularly suitable to detect and study transcription-induced dynamic supercoiling. Our method is based on the different electrophoretic mobility in agarose gel of crosslinked versus uncrosslinked DNA fragments after denaturation of sonicated genomic DNA from cells treated with psoralen and UV light. The crosslinked and uncrosslinked DNA fragments are separated, purified and can be tested for enrichment throughout the genome by microarray or direct sequencing (Fig. 2). Because psoralen intercalation is also affected by DNA sequence composition and DNA-protein interactions [8], corrections for sequence and chromatin structure must be made to detect and estimate supercoiling inside the cells. A crosslinking difference between intact cells and cells treated with a transcriptional inhibitor is intrinsically normalized for the effect of sequence composition and chromatin, providing a measure of transcription-generated supercoiling [13]. Genome-wide mapping of nucleosome occupancy is an important control experiment in our psoralen-based approach to insure that inhibition of transcription does not alter nucleosome deposition on the studied DNA regions, that otherwise might influence the level of crosslinking and confound the detection of DNA supercoiling [17]. Treatment of the cells with topoisomerases inhibitors (topoisomerases are enzymes which can relax DNA supercoiling) magnifies the results and helps to illuminate the mechanism of DNA topological homeostasis inside cells (Fig. 2). Details of the microarray hybridizations, high-throughput sequencing and transcriptome study as well as subsequent analysis have been reported elsewhere [13, 18, 19] and are not the focus of the protocol described here.

Fig. 2.

Fig. 2

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Flow chart of psoralen-based approach. (Middle) Grow two batches of cells in culture. Cells in the first batch are grown under normal condition. Cells in the second batch are treated with transcription inhibitor. Add psoralen and introduce DNA interstrands crosslinks by exposing the cells to UV light. Using gel electrophoresis, separate and purify crosslinked and non-crosslinked fragments of genomic DNA after glyoxal denaturation. The crosslinked fraction is enriched for DNA negatively supercoiled at the moment of UV-irradiation. Genomic mapping of crosslinked DNA is achieved by hybridization to oligonucleotide microarrays or by direct high-throughput sequencing. Construction of the genome-wide pattern of transcription-generated supercoiling as a difference in crosslinking density between normal cells and cells treated with transcription inhibitor. (Left) To confirm that the crosslinking difference is due to transcription-generated supercoiling, verify that chromatin structure does not change at genomic regions of interest. (Right) To reveal the dynamic character of supercoiling and to examine its regulation, treat cells with topoisomerases inhibitors. Dotted line marks experimental methods presented in this chapter

Our approach has already been adopted by other groups [20, 21]. It will allow researchers to study in detail the physical organization of chromosome and investigate the role of dynamic supercoiling in genome functioning.

2. Materials

2.1. Reagents

  1. Proteinase K (Solution, 20 mg/mL).

  2. Phenol–chloroform–isoamyl alcohol 25:24:1 v/v/v, Tris pH 8.0 saturated.

  3. Ethanol 100%.

  4. Sodium acetate 3.0 M, pH 5.2.

  5. 1 M CaCl2 solution.

  6. RNase, DNase-free, 500 μg/mL.

  7. 10% SDS.

  8. 0.5 MEDTA.

  9. 37% formaldehyde.

  10. Glycogen: 20 mg/mL stock solution.

  11. UltraPure Agarose (Invitrogen).

  12. NuSieve 3:1 Agarose (Cambrex).

  13. Dimethyl sulfoxide (DMSO), for molecular biology, >99.9%.

  14. Glyoxal aldehyde solution, for molecular biology, 40% in H2O.

  15. Amberlite Mixed Bed Exchanger Amberlite MB-150, Sigma.

  16. 5,6-dichloro-1-β-d-ribofuranosylbenzimide (DRB), 50 mM solution in DMSO.

  17. Camptothecin (CPT), 10 mM solution in DMSO.

  18. β-Lapachone (LAP), 40 mM solution in DMSO.

  19. 4,5′,8-trimethylpsoralen (psoralen), 0.9 mg/mL solution in ethanol.

  20. MNase.

  21. SYBR Green Nucleic Acid Gel Stain.

  22. Dialysis tube, cellulose tubular membrane, MWCO ~8000.

2.2. Buffers

  1. TE buffer: 10 mM Tris–HCl, 1 mM EDTA, pH 8.0.

  2. Denaturing solution: 0.5 M NaOH, 1.5 M NaCl.

  3. Neutralizing solution: 1.5 M NaCl, 500 mM Tris–HCl pH 8.0, 1 mM EDTA.

  4. TAE buffer: 40 mM Tris pH 7.6, 20 mM acetic acid, 1 mM EDTA.

  5. 0.1 M sodium phosphate buffer, pH 7.0.

  6. PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, without calcium and magnesium, pH 7.4.

  7. Lysis buffer: 10 mM Tris–HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40, 0.15 mM spermine, 0.5 mM spermidine.

  8. MNase digestion buffer: 10 mM Tris–HCl pH 7.4, 15 mM NaCl, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine.

2.3. Equipments

  1. Gel electrophoresis apparatus with a gel tray up to 20 cm.

  2. Ultraviolet lamp; model B-100 A, Ultra-Violet Products, Inc.

  3. Perkin Elmer MBA 2000 UV/VIS Spectrometer.

  4. Thermoblock.

  5. Sonicator, Ultrasonic processor XL, MISONIX Inc.

  6. Rocker, tube wheel rotator, vacuum dryer, water bath, aspirator, and thermomixer.

2.4. Kits

  1. QIAquick Gel Extraction Kit (Qiagen).

3. Methods

3.1. Preparing Genomic DNA from the Cells Treated with Psoralen and UV-Light

  1. Grow Raji cells in RPMI 1640 medium with 10% FBS and 2 mM of l-glutamine (see Note 1). Split cells into fresh medium at a density of 0.2 × 106 cell/mL in 50 mL (175 cm2 flask) and add DMSO (1.5% final concentration). Allow cells to grow for 4 days (see Note 2).

  2. Count cells and transfer the suspension to a centrifuge tube. Recover the cell pellet by centrifugation at 500 × g for 10 min at room temperature (RT). Remove the supernatant by aspiration and gently resuspend the cells in fresh medium without DMSO at a concentration of 1 × 106 cells/mL. Allow cells to grow for 6 h (see Note 3). For a typical experiment, we use 1 × 107 cells per condition.

  3. A typical experiment requires condition #I, cells not exposed to UV-light; #II, cells exposed to UV-light; #III, cells treated with an inhibitor of transcription (we chose DRB) and exposed to UV-light (see Note 4). Write these numbers on the bottom part of the 6 cm tissue culture dishes. If topoisomerase inhibitors will be used, prepare also sample #IV, cells treated with camptothecin and sample #V, cells treated with β-Lapachone. Other topoisomerase inhibitors can be used, according to the purpose of the experiment. For simplicity, in the protocol described below we will consider only treatments #I, #II, and #III.

  4. Transfer the cells to a centrifuge tube and recover them by centrifugation at 500 × g for 10 min at RT. Resuspend the cells to have 2 × 106 cell/mL and transfer 1 × 107 cells (5 mL) into 6 cm tissue culture dishes (see Note 5).

  5. Add 40 μM of DRB (final concentration) to dish #III. After 26 min of incubation at 37 °C, add 70 μL of psoralen solution to each dish (see Note 6), mix by rocking, and incubate for 4 min at 37 °C in shadow light environment (see Note 7).

  6. Place dishes #II and #III on an ice bed and expose them to ~3.6 kJ m−2 of 365-nm light (ultraviolet lamp; model B-100 A, Ultra-Violet Products, Inc. at a distance of 12 cm from the top of the light filter to the surface of the cell-containing media during 40 s) (see Note 8).

  7. Immediately lyse the cells in each dish by adding SDS, EDTA and Proteinase K, giving final concentrations of 0.5%, 100 mM, and 100 μg/mL, respectively.

  8. Transfer cellular lysates to the 50 mL Falcon tube, mix by shaking, and incubate the lysates for 5 h (or overnight) at 55 °C.

  9. Cool the solution to room temperature (RT) and add an equal volume of phenol-chloroform. Mix the two phases by shaking the tube for a few minutes, and separate the two phases by centrifugation at 3500 × g for 10 min at RT. Transfer the aqueous phase to a new 50 mL Falcon tube.

  10. Repeat step 3.1.9. Add to the aqueous phases 0.1 volume of 3 M sodium acetate and 2 volumes of ethanol stored at RT. Swirl the tubes to thoroughly mix solutions and precipitate the DNA by centrifugation at 3500 × g for 10 min. Remove as much of the ethanol solution as possible, using an aspirator.

  11. Add 5 mL of TE (pH 8.0) to each tube and 5 μg of RNase, DNase-free. Place the tubes on a tube wheel rotator and incubate the solution overnight at RT with gentle agitation until the DNA is completely dissolved. Repeat step 3.1.9 and the ethanol precipitation. Wash the DNA precipitates with 70% ethanol. Remove as much ethanol as possible and air-dry the pellet for 15 min.

  12. Dissolve the DNA pellet by vortexing it in 100 μL of TE (pH 8.0) for 1 hour and by heating it in a water bath to 55 °C. Save 5 μL of DNA to use in step 3.1.13 and fragment the remaining DNA by sonication. Sonication is performed on ice with an ultrasonic sonicator (Sonicator, Ultrasonic processor XL, MISONIX Inc. at 15% of power) by pulsing 20 times for 30 s, cooling in an ice-bath for 30 s between pulses.

  13. Quantify the DNA of both unsonicated and sonicated samples spectrophotometrically and run 0.3 μg of DNA on a 0.6% agarose gel. The average DNA length of sonicated DNA should be around 200–300 bp with the distribution of fragment size ranging from 100 bp up to 500 bp. Unsonicated DNA should not show any sign of degradation, evidenced by smearing of DNA below the high-molecular band.

3.2. Separation of Psoralen Crosslinked and Uncrosslinked DNA Fragments

  1. Run a preparatory 0.6% agarose gel electrophoresis in TAE buffer. Load DNA from each sample (#I, #II, #III) into the 1 cm wide gel wells, 5 μg of DNA per well. Run 20 cm gel for 2 h at 5 V/cm.

  2. Divide DNA distribution into two parts: part 1 is from 300 bp to 100 bp and part 2 is from 500 bp to 300 bp. Cut the gel into slices with the DNA falling into these ranges. Purify the DNA by QIAquick Gel Extraction Kit (see Note 9). Label purified DNA as #I-1, #I-2, #II-1, #II-2, #III-1, and #III-2. Add to the purified DNA 20 μg of glycogen, 2 volumes of ice-cold ethanol 100%, and 0.1 volume of 3.0 M sodium acetate. Incubate at −20 °C for 30 min and precipitate by centrifugation at 13,000 × g for 10 min at +4 °C. Wash DNA pellets with 70% ethanol. Air-dry the pellet and dissolve DNA in 18 μL of water.

  3. Incubate glyoxal aldehyde with “Amberlite Mixed Bed Exchanger Amberlite MB-150” (Sigma) at 2 to 1 volume ratio for 30 min at constant rotation (see Note 10). Prepare a master stock by mixing of 150 μL of 40% glyoxal aldehyde and 630 μL of DMSO right before use in next step.

  4. Add 9 μL of 100 mM sodium phosphate buffer (pH 7.0) to each tube with DNA. Mix samples by vortex. Boil the samples for 1 min. Immediately add 78 μL of master stock from step 3.2.3 to the denatured DNA (see Note 11). Incubate the samples for 1 h at 55 °C. Reduce the volume of the samples to approximately ¼ by vacuum dryer.

  5. Add the appropriate amount of loading buffer to the samples. Separate crosslinked and non-crosslinked DNA fragments by electrophoresis on a 3% agarose gel in 10 mM sodium phosphate buffer (pH 7.0) at 2 V/cm while recirculating the buffer for approximately 10 h (see Note 12). The length of the gel is 20 cm. To cast gel, use 1.2:1.8 mixture of UltraPure Agarose from Invitrogene and NuSieve 3:1 Agarose from Cambrex (see Note 13).

  6. Incubate the gel with denaturing solution at 65 °C for 3 h to reverse psoralen crosslinks. Then incubate the gel with neutralizing solution for 1 h with constant agitation. Equilibrate the gel in the TAE buffer overnight. Stain the DNA in the gel with SYBR Green in TAE buffer for 16–18 h while changing the buffer three times.

  7. Examine the stained gel by UV illumination. Locate the DNA “clouds” that belongs to the uncrosslinked (migrates faster) and crosslinked (migrates slower) DNA fragments. As a reference, chose the DNA “cloud” from the cells not treated with UV light (samples #I-1, #I-2). Use a sharp scalpel to cut out a slice of agarose containing the DNA of interest (Fig. 3). The ratio of non-crosslinked to crosslinked fragments should be ~3:1 to insure unsaturated conditions for crosslinking.

  8. Close one end of a dialysis tube with a clip. Fill the dialysis tube with TAE buffer and transfer the gel slice into the buffer-filled tube. Squeeze out most of the buffer and seal the tube with a second dialysis tube clip. Label the clip with the name of DNA sample. For example, #II-1-NC means uncrosslinked DNA from sample #II-1, #II-1-C means crosslinked DNA from sample #II-1. Immerse the tube in a horizontal electrophoresis tank and apply an electric current through the tube (5 V/cm) for 2 h. After finishing the elution, massage the tube to redistribute DNA into the buffer.

  9. Transfer the eluted DNA solution into a 15 mL Falcon tube and extract DNA twice with an equal volume of phenol–-chloroform. Aliquot the extracted DNA into the Eppendorf tubes (0.5 mLin each) and precipitate the DNA with 2 volumes of ice-cold ethanol 100% in the presence of 0.3 M sodium acetate and 20 μg of glycogen after incubation at −20 °C for 30 min. Dissolve DNA pellets from each Eppendorf tube in 30 μL of TE buffer and combine DNA corresponding to each sample. Repeat ethanol precipitation in the presence of 0.3 M sodium acetate.

  10. Wash DNA pellets with 70% ethanol, air-dry and dissolve DNA in 50 μL of TE buffer (pH 8.0). Combine purified DNA in the following order. C+ is crosslinked DNA from the untreated cells: #II-1-C and #II-2-C. NC+ is uncrosslinked DNA from the untreated cells: #II-1-NC and #II-2-NC. C- is crosslinked DNA from the cells treated with DRB: #III-1-C and #III-2-C. NC- is uncrosslinked DNA from the cells treated with DRB: #III-1-NC and #III-2-NC. Measure DNA concentrations by spectrometer (see Note 14).

  11. Repeat the experiment generating required number of biological replicates (see Note 15).

  12. The resulting DNA samples are ready for downstream steps, which can involve either hybridization on a microarray platform or high throughput sequencing of single-stranded DNA, accordingly the scheme presented in Fig. 4. In our laboratory, we performed DNA labeling, hybridization, detection, data extraction, and quality assessment using the commercial microarray service. Algorithm of computational data analysis can be found at Supplementary Information of Kouzine et al. [13].

Fig. 3.

Fig. 3

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Guide for preparative selection of crosslinked (-C) and uncrosslinked (-NC) DNA fragments on a hypothetical agarose gel. Uncrosslinked DNA from the cells not treated with UV light (samples # I-1 and I-2) serves as a reference marker to better resolve different DNA species in the experimental samples # II-1 and II-2. Use scalpel to excise the bands as shown by the black lines

Fig. 4.

Fig. 4

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Definition of DNA samples and equivalence of genomic data

3.3. Inhibition of Topoisomerases

  1. Starting from step 3.1.5 of the current protocol, having ready dishes #III, VI and V. Add 40 μM of DRB (final concentration) to dish # III. After 21 min of incubation at 37 °C, add 10 μM (final concentration) CPT to dish # IV. Add 10 μM (final concentration) LAP to dish #V (see Note 16). Mix by rocking for 30 s. After additional 5 min of incubation at 37 °C, add 70 μL of psoralen solution to each dish, mix by rocking, and incubate for 4 min at 37 °C in the shadow light environment.

  2. Follow steps 3.1.6-3.2.10 of the current protocol to obtain the following DNA samples: C-CPT is crosslinked DNA from the CPT treated cells; NC-CPT is uncrosslinked DNA from the CPT treated cells; C-L is crosslinked DNA from the LAP treated cells; NC-L is uncrosslinked DNA from the LAP treated cells.

  3. Continue to steps 3.2.11 and 3.2.12 of the current protocol (see Fig. 4).

3.4. Preparation of Nucleosomal DNA

  1. Use cells treated or not with DRB for 30 min. Cross-link 15–-20 million cells in each condition by adding formaldehyde (1% final concentration) directly to the medium and incubate for 5 min at RT (see Note 17). Wash the cells twice with ice-cold PBS and lyse the cells in 1 mL of ice-cold lysis buffer in Eppendorf tubes. After 5 min of incubation on ice, spin the tubes at 1000 × g for 5 min at 4 °C. Wash the pellets of nuclei twice with ice-cold MNase digestion buffer. Resuspend the nuclei in 1 mL of MNase digestion buffer and add CaCl2, giving 1 mM final concentration. Keep the nuclei on ice.

  2. Add 0, 0.01, 0.02, 0.04, 0.08, 0.15, 0.3, 0.6, 1.0, and 2.0 U of MNase to 10 Eppendorf tubes and aliquot 100 μL of nuclei solution into each tube. Incubate the reaction mixture at 37 °C for 10 min. Stop the reaction by adding SDS, EDTA, and Proteinase K, giving final concentrations of 0.5%, 100 mM, and 100 μg/mL, respectively. Incubate the mixture at 65 °C for overnight.

  3. Extract DNA twice with phenol–chloroform, add 20 μg of glycogen as a carrier and precipitate the DNA with 2 volumes of ethanol 100% in the presence of 0.3 M sodium acetate after incubation at −20 °C for 30 min. Wash DNA pellets with 70% ethanol, air-dry and dissolve DNA in 20 μL of TE buffer (pH 8.0).

  4. Add an appropriate amount of loading buffer to 10 μL of each sample and run 0.6% agarose gel electrophoresis in TAE buffer (20 cm gel for 2 h at 5 V/cm) to resolve the nucleosome ladder. Examine the gel stained with SYBR Green by UV illumination. Use a sharp scalpel to cut out a slice of agarose covering mononucleosome DNA only from lines containing 3–8 bands in the nucleosome ladder.

  5. Purify the DNA by QIAquick Gel Extraction Kit. Resulting DNA samples are ready for downstream analysis by using microarray platform or high throughput sequencing.

3.5. Transcription Activity Assays

To generate cellular transcriptome required to correlate the map of DNA supercoiling with transcriptional activity, in our experiments we used microarray-based method to quantify gene expression level from cDNA (complementary DNAs generated from the RNA) signal at the annotated genes [13]. However, a recently developed technique called RNA Sequencing (RNA-Seq) uses high-throughput sequencing to build transcriptome of genomes at a far higher resolution than in microarray-based methods [18]. Thus, we recommend using RNA-Seq assay to create a reference map for transcription-generated DNA supercoiling. An independent measure of transcriptional activity and pausing state of RNA polymerase II might be obtained by RNA polymerase ChIP-sequencing assay [19].

Footnotes

1.

The DMSO treatment for 96 h will synchronize Raji cells at early G1 cell cycle phase. While this step is not necessary, it might reduce variation in the crosslinking density due to other reasons than transcription (DNA replication, mitosis etc.).

2.

This protocol is designed for suspension cells. If adherent cells are used, grow them in 6 cm tissue culture dishes. The number of dishes should fulfill the requirement to have 10 million cells per condition: #I, #II and #III. Start the protocol for adherent cells from step 3.1.5, preferably working with no more than three dishes simultaneously.

3.

At this step, synchronized cells are in the middle of G1 phase of the cell cycle.

4.

DRB rapidly enters cells, specifically inhibits CDK9 phosphorylation of the C-terminal domain of RNA polymerase II, and blocks transcription within a minute after administration.

5.

The small size of the dish is chosen to ensure uniform irradiation of the cells. However, other commercially available light sources might accommodate larger dishes.

6.

Psoralen is light sensitive and its exposure to light should be minimized. It should be stored in the dark. When added to cells, we typically reduce lighting, avoid sunlight, etc. Psoralen is a powerful mutagen and carcinogen and should be handled with care.

7.

Following the UV irradiation, all subsequent manipulations of the cells, of the cellular lysates and of the DNA are performed under shadow light conditions, with the cells and DNA being subjected only to indirect lighting.

8.

If using other types of the fluorescent lamps or other type of cells, in a pilot experiment, expose dishes to various doses of 365-nm light, starting from 3.0 kJ m−2 with an incident light intensity of about 0.3 kJ m−2 up to 3.9 kJ m−2. Then go through all steps until step 3.2.7 and choose the conditions which satisfy 3:1 ratio between uncrosslinked and crosslinked DNA fragments to perform the main experiment.

9.

Be careful to not exceed the DNA binding capacity of the QIAquick membranes (10 μg).

10.

Glyoxal is readily air-oxidized. By mixing the glyoxal solution with the mixed bed, we remove oxidation products the same day as the DNA samples are prepared.

11.

Glyoxal reacts with DNA by introducing an additional ring onto guanosine residues, thus sterically hindering the formation of G-C base pairs and consequently the renaturation of DNA double helix.

12.

To better estimate the time of running, mix all leftover DNA samples in one tube and load the mixture in one well on the side of the gel. After few hours of electrophoresis, cut a slice of the gel containing the mixture of DNA loaded in this well, stain it with SYBR Green in TAE buffer, and calculate the exact time required to run the gel.

13.

It is important to use agarose with high melting temperature (NuSieve 3:1) to successfully perform step 3.2.19 with an addition of typical agarose (UltraPure Agarose) to insure durability of the gels in the subsequent steps.

14.

At this step DNA exist in the partially denatured state. Single-stranded DNA fragments might anneal with each other to form mesh-like structure complex. To measure accurately the DNA concentration, we boil DNA for 1 min and then transfer 1 μL of DNA solution to the 100 μL of ice-cold TE buffer. The concentration of resulting DNA solution is measured by Perkin Elmer MBA 2000 UV/VIS Spectrometer.

15.

Microarrays and high-throughput sequencing have been routinely used for the ChIP-Chip and ChIP-Seq experiments, where the enrichment of sequences is 10–100-fold higher than the background. The relative enrichment of psoralen crosslinking at negatively supercoiled DNA is only two folds in comparison to relaxed DNA. In addition, psoralen binding sites are not focal, rather continuously distributing across the genome. However, from our experience, even for high noise experiments such as microarrays, 3 replicates are sufficient to achieve a required level of accuracy. With high-throughput sequencing this number will come down further to two replicates, insuring the reproducibility of experiment.

16.

Different inhibitors act on different topoisomerases and affect different steps in the topoisomerase reaction cycle. Changes in crosslinking density will reflect their mode of action; confirming the results of the main experiment and revealing the mechanisms of topological homeostasis inside the cells (see also Fig. 4).

17.

The crosslinking with formaldehyde is required to avoid possible redistribution of nucleosomes upon nuclei preparation and MNase digestion.

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