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Summary

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

Abstract

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Introduction

Homologous recombination (HR) is a high-fidelity mechanism of repair of DNA double-stranded breaks (DSBs), inter-strand cross-links, and ssDNA gaps, as well as a pathway for DNA damage tolerance. HR differs from error-prone pathways for DNA damage repair/tolerance, such as non-homologous end-joining (NHEJ) and translesion synthesis, in that it utilizes an intact, homologous duplex DNA as a donor to template the repair event. Moreover, many of the key intermediates in the HR pathway are reversible, allowing for exquisite regulation of the individual pathway steps. During the S, G2, and M phases of the cell cycle, HR competes with NHEJ for the repair of the two-ended DSBs1. In addition, HR is essential to DNA replication for the repair of replication-associated DNA damage, including ssDNA gaps and one-sided DSBs, and as a mechanism of DNA lesion bypass2.

A critical intermediate in the HR pathway is the displacement loop, or D-loop (Figure 1). Following end resection, the central recombinase in the reaction, Rad51, loads onto the newly resected ssDNA of the broken molecule, forming a helical filament2. Rad51 then carries out a homology search to identify a suitable homologous donor, typically the sister chromatid in somatic cells. The D-loop is formed when the Rad51-ssDNA filament invades a homologous duplex DNA, which leads to the Watson-Crick base pairing of the broken strand with the complementary strand of the donor, displacing the opposite donor strand. Extension of the 3' end of the broken strand by a DNA polymerase replaces the bases that were lost during the DNA damage event and promotes resolution of the extended D-loop intermediate into a dsDNA product through the synthesis-dependent strand annealing (SDSA), the double-Holliday junction (dHJ), or the break-induced replication (BIR) HR sub-pathways.

Assays that physically monitor the intermediates in the HR pathway permit the analysis of the genetic requirements for each step (i.e., pathway analysis). DSB formation, end resection, dHJs, BIR replication bubbles, and HR products are readily observed by Southern blotting3,4,5,6,7. Yet, Southern blotting fails to report on nascent and extended D-loops, and, thus, an alternative method to reliably measure these joint molecules is required4,8,9. One widely used strategy to analyze nascent D-loop formation is chromatin-immunoprecipitation (ChIP) of Rad51 coupled with quantitative PCR (qPCR)10,11. However, Rad51 association with dsDNA as measured by ChIP-qPCR is independent of sequence homology and the Rad51 accessory factor Rad5410,11. In contrast, an appreciable signal using the method of D-loop analysis presented here, called the D-loop capture (DLC) assay, depends on DSB formation, sequence homology, Rad51, and the Rad51 accessory proteins Rad52 and Rad548. The finding that Saccharomyces cerevisiae Rad51-promoted D-loop formation depends on Rad54 in vivo is in agreement with numerous in vitro reconstitution experiments indicating that Rad54 is required for homology search and D-loop formation by budding yeast Rad518,12,13,14,15.

Current approaches to measuring D-loop extension, primarily through semi-quantitative PCR, are similarly problematic. A typical PCR-based assay to detect D-loop extension amplifies a unique sequence, resulting from recombination between a break site and an ectopic donor and the subsequent recombination-associated DNA synthesis, via a primer upstream of the region of homology on the broken strand and another primer downstream of the region of homology on the donor strand. Using this method, the detection of recombination-associated DNA synthesis requires the non-essential Pol δ processivity factor Pol3216. This finding conflicts with the observation that POL32 deletion has only a mild effect on gene conversion in vivo17. Moreover, these PCR-based assays fail to temporally resolve D-loop extension and BIR product formation, suggesting that the signal results from dsDNA products rather than ssDNA intermediates17,18,19. The D-loop extension (DLE) assay was recently developed to address these discrepancies. The DLE assay quantifies recombination-associated DNA synthesis at a site ~400 base pairs (bp) downstream of the initial 3' invading end9. By this method, D-loop extension is independent of Pol32 and is detectable within 4 h post-DSB induction, whereas BIR products are first observed at 6 h. Indeed, a recent publication from the Haber and Malkova laboratories noted that using this method of preparation of genomic DNA singularly results in ssDNA preservation9,20.

Here, the DLC and DLE assays are described in detail. These assays rely on proximity ligation to detect nascent and extended D-loops in S. cerevisiae (Figure 2)8,9. BIR products can be quantified using this same assay system. For both assays, DSB formation at an HO endonuclease cut site located at the URA3 locus on chromosome (Chr.) V is induced by the expression of the HO endonuclease under the control of a galactose-inducible promoter. Rad51-mediated DNA strand invasion leads to nascent D-loop formation at the site of an ectopic donor located at the LYS2 locus on Chr. II. As the right side of the DSB lacks homology to the donor, repair via SDSA and dHJ formation is not feasible. Initial repair of the DSB by BIR is possible, but the formation of viable products is inhibited by the presence of the centromere21. This deliberate design prevents productive DSB repair, thereby avoiding the resumption of growth by cells with repaired DBSs, which could otherwise overtake the culture during the time course analysis.

In the DLC assay, psoralen crosslinking of the two strands of the heteroduplex DNA within the D-loop preserves the recombination intermediate. Following restriction enzyme site restoration on the broken (resected) strand and digestion, the crosslinking allows for ligation of the unique sequences upstream of the homologous broken and donor DNAs. Using qPCR, the level of chimeric DNA molecule present in each sample is quantified. In the DLE assay, crosslinking is not required, and restriction enzyme site restoration and digestion followed by intramolecular ligation instead link the 5' end of the broken molecule to the newly extended 3' end. Again, qPCR is used to quantify the relative amounts of this chimeric product in each sample. In the absence of restriction enzyme site restoration, the DLE assay reports on the relative levels of the BIR (dsDNA) product that is formed following D-loop extension.

Representative results for each assay using a wild type strain are shown, and readers are referred to Piazza et al.8 and Piazza et al.9 for the use of these assays for the analysis of recombination mutants8,9. The intent of this contribution is to enable other laboratories to adopt the DLC and DLE assays, and support for them is available upon request.

Protocol

1. Pre-growth, DSB induction, and sample collection

NOTE: Supplementation of all media with 0.01% adenine is recommended for Ade- strains.

  1. Streak out the appropriate haploid strains (see Table 1) on yeast peptone dextrose adenine (YPDA) (1% yeast extract, 2% peptone, 2% glucose, 2% agar, 0.001% adenine) and grow for 2 days at 30 °C.
  2. Use a single colony to inoculate 5 mL of YPDA in a 15 mL glass culture tube. Grow cultures to saturation at 30 °C with shaking or rotation for aeration.
  3. DLC assay: Prepare the 5x psoralen stock solution (0.5 mg/mL trioxsalen in 200-proof ethanol) in a fume hood by dissolving psoralen in a 50 mL conical tube wrapped in aluminum foil overnight at room temperature with continuous shaking or inversion. Seal screw top with a transparent film to prevent evaporation. Do not prepare more than 7 mL of 5x psoralen stock solution per 50 mL conical tube to ensure proper dissolution of the psoralen.
  4. The next day, use 5 mL of the YPDA grown overnight culture to inoculate 50-100 mL of YEP-lactate (1% yeast extract, 2% peptone, 2% w/w lactate, 0.001% adenine) in an appropriately sized flask (budding yeast grows optimally in a flask that is at least 5x the volume of the culture) to an OD600 of ~0.03.
  5. Grow the culture for ~16 h at 30 °C with shaking at 220 rpm. After ~16 h, measure the OD600 of the culture and it should be ~0.5-0.8. Do not use under- or overgrown cultures.
  6. For each time point, collect the appropriate volume of cells in a conical tube and place on ice. Typically, this is 1 x 108 cells (approximately 7.5 mL of culture at OD600 1.0 for a haploid wild type strain) for the DLC assay and 5 x 107 cells (approximately 2.5 mL of culture at OD600 1.0) for the DLE assay.
  7. To ensure the accuracy of the OD600 values, prepare 1:5 dilutions for cultures with an OD600≥0.2 to keep the OD reading at 0.2 or below. For wild type strains, optimal time points for DLC analysis are between 2 h and 6 h, and optimal time points for DLE analysis are between 4 h and 8 h (see Figure 3 and Figure 4).
  8. DLC assay
    1. Before each time point, prepare enough 1x psoralen solution (0.1 mg/mL trioxsalen, 50 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0, 20% ethanol) in a fume hood for all the samples in a 50 mL conical tube wrapped in foil. Leave at RT.
    2. Centrifuge the samples at 2,500 x g for 5 min at 4 °C. Resuspend the pellet in 2.5 mL of 1x psoralen solution in a fume hood and transfer to a 60 mm x 15 mm Petri dish. Alternatively, resuspend the pellet in 2.5 mL of TE1 solution (50 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0) for a no-crosslinking control.
    3. Crosslink the samples. For a UV crosslinker fit with long-wave (365 nm) bulbs, position the Petri dishes 1-2 cm below the UV light source with the lid removed atop a plastic or plexiglass plate that has been pre-chilled at −20° C. For a UV light box, place the Petri dishes directly atop the UV light source. Expose the samples for 10 min with gentle shaking.
      NOTE: It is recommended to set the UV light source atop an orbital shaker set at ~50 rpm.
    4. In a fume hood, transfer the sample into a new 15 mL tube. Rinse the Petri dish with 2.5 mL of TE1 solution and add this to the tube. Centrifuge the samples at 2,500 x g for 5 min at 4 °C, properly dispose of the supernatant, and store the pellet at −20° C. Samples can be stored for up to 1 week before moving to the next step.
  9. DLE assay
    1. Centrifuge the samples at 2,500 x g for 5 min at 4 °C. Wash the cell pellet in 2.5 mL of cold TE1 solution before repeating the spin and storing the pellets at −20 °C. Samples can be stored for up to 1 week before moving to the next step.
  10. For sample collection at 0 h, collect the samples prior to the addition of 20% galactose. For subsequent timepoints, induce DSB formation by adding 20% galactose to the cultures to a final concentration of 2%. Collect the remaining samples as described above, pellet, and freeze relative to the time post-DSB induction (i.e., the 4 h sample is collected 4 h after the addition of 20% galactose).

2. Cell spheroplasting, lysis, and restriction site restoration

  1. Thaw the samples on ice. Preheat a dry bath to 30 °C.
  2. Resuspend the samples in 1 mL of spheroplasting buffer (0.4 M sorbitol, 0.4 M KCl, 40 mM sodium phosphate buffer pH 7.2, 0.5 mM MgCl2) and transfer to a 1.5 mL microcentrifuge tube.
  3. Add 3.5 µL of zymolyase solution (2% glucose, 50 mM Tris-HCl pH 7.5, 5 mg/mL zymolyase 100T; 17.5 µg/mL zymolyase final concentration). Mix gently by tapping or inversion. Incubate at 30 °C for 15 min, and then place on ice. During the 15 min incubation, obtain liquid nitrogen or dry ice.
  4. Centrifuge for 3 min at 2,500 x g at 4 °C and place the samples on ice. Wash the samples 3x in 1 mL of spheroplasting buffer. Centrifuge the samples for 3 min at 2,500 x g at 4 °C.
  5. Resuspend the samples in 1 mL of cold 1x restriction enzyme buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 µg/mL BSA pH ~8.0 at RT) and centrifuge for 3 min at 16,000 x g at 4 °C. Place the samples on ice. Repeat the wash 1x.
  6. Resuspend the samples in 1 mL of cold 1x restriction enzyme buffer. Split the sample (0.5 mL each) into two 1.5 mL microcentrifuge tubes. Centrifuge the samples for 3 min at 16,000 x g at 4 °C.
  7. Resuspend one tube from each sample in 180 µL of 1.4x restriction enzyme buffer with hybridizing oligos (see Table 2) and one tube in 180 µL of 1.4x restriction enzyme buffer without hybridizing oligos. Each hybridizing oligo is resuspended in 1x TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) and used at a final concentration of 7 nM. The 1x TE replaces the hybridizing oligos in the 1.4x restriction enzyme buffer without hybridizing oligos.
    NOTE: The hybridizing oligos must be stored at −20 °C in small aliquots at the working dilution. The concentration of the hybridizing oligos may require optimization; see Discussion.
  8. Snap freeze the samples in liquid nitrogen or dry ice/ethanol and store at −80 °C. Samples can be stored at this stage for several months.

3. Restriction enzyme digest and intramolecular ligation

  1. Thaw the samples on ice. Preheat one dry bath to 65 °C and another to 37 °C.
  2. Pipet 36 µL of the sample into a new 1.5 mL microcentrifuge tube on ice. Promptly return the remaining sample to −80 °C for storage.
  3. Add 4 µL of 1% SDS (0.1% final concentration) and mix by gently tapping the side of the tube. Incubate at 65 °C for 15 min with gentle tapping every 5 min. Place samples on ice immediately following the incubation.
    NOTE: This SDS treatment promotes the denaturation of DNA-associated proteins, solubilization of the nuclear envelope, and chromatin accessibility in advance of the restriction enzyme digest and intramolecular ligation steps.
  4. Add 4.5 µL of 10% Triton X-100 (1% final concentration) and mix by pipetting. Add 20-50 U of restriction enzyme (EcoRI-HF or HindIII-HF) to each sample and incubate at 37 °C for 1 h with gentle agitation every 20-30 min. During this time, preheat a dry bath to 55 °C and preset a water bath to 16 °C.
  5. Add 8.6 µL of 10% SDS (1.5% final concentration) to each sample and mix by pipetting and tapping. Incubate at 55 °C for 10 min. Add 80 µL of 10% Triton X-100 (6% final concentration) to each sample and mix by pipetting.
  6. Add 660 µL of 1x ligation buffer without ATP (50 mM Tris-HCl pH 8.0, 10 mM MgCl2, 10 mM DTT, 2.5 µg/mL BSA) + 1 mM ATP pH 8.0 + T4 DNA ligase (8 U/sample) to each sample and mix by gentle inversion. Incubate at 16 °C for 1.5 h with inversion every 30 min. Place the samples on ice immediately following the incubation.

4. DNA purification

  1. Preheat one dry bath to 65 °C and another to 37 °C. Add 1 µL of 10 mg/mL proteinase K (prepared in 1x TE pH 8.0) to each sample (12.5 µg/mL final concentration). Incubate at 65 °C for 30 min and place the samples on ice immediately following the incubation until they have cooled.
  2. Transfer the samples to 2 mL tubes. Working in a fume hood, add an equal volume (~800 µL) of phenol/chloroform/isoamyl alcohol (P/C/IA; pH 8.0) to each sample. Vortex the samples for ~30 s and centrifuge the samples for 5-10 min at 16,000 x g in a microcentrifuge.
  3. Carefully remove 600 µL of the upper phase of each sample into a new 1.5 mL tube. Properly dispose of the lower phase and 2 mL tubes.
  4. Precipitate the DNA by adding a 1/10 volume of 3 M sodium acetate pH 5.2 (~60 µL) to each sample, followed by 1 volume of isopropanol (~660 µL). Invert the samples 5x-10x and incubate at RT for 30 min.
  5. Place the samples on ice for 2 min, and then centrifuge the samples at 16,500 x g for 15 min at 4 °C in a microcentrifuge. Return the samples to ice, pour off the supernatant, and drain the tube on a paper towel.
  6. Wash the DNA pellet with 200 µL of 70% ethanol. Centrifuge at 16,500 x g for 3 min at 4 °C, place the samples back on ice, pour off the supernatant, and remove the residual alcohol with a pipet. Dry the samples with the caps of the tubes open at 37 °C for 15-20 min.
  7. Resuspend the DNA pellets in 50 µL of 1x TE by vortexing. Incubate at RT for 30 min, vortex, and then incubate at 37 °C in a dry bath for 30 min. Vortex the samples again, and then place them on ice. Samples can be stored at this stage at −20 °C for several months, but it is advisable to proceed immediately for the decrosslinking (DLC only) and qPCR steps.

5. Psoralen crosslink reversal (for DLC assay only)

  1. Pipet 9 µL of purified DNA into a PCR tube on ice. Add 1 µL of 1 M KOH (0.1 M final concentration). Incubate the samples at 90 °C for 30 min in a thermocycler.
  2. Add 19.73 µL of sodium acetate solution (0.1 M sodium acetate, 9.6 mM Tris-HCl pH 8.0, 1.0 mM EDTA pH 8.0). Samples can be stored at this stage at −20 °C for several months, but it is advisable to proceed immediately to the qPCR step.

6. Quantitative PCR, controls, and analysis

  1. Using 2 µL of purified DNA, with or without crosslinking, set up a 20 µL qPCR reaction according to the manufacturer's instructions. Set up each reaction in duplicate. For both the DLC and DLE assays, there are five control reactions and one DLC/DLE quantification reaction, or a total of six reactions per sample, run in duplicate. Supplementary Table S1 and Supplementary Table S2 provide a template for setting up these reactions and analysis, and the sequences of the qPCR primers are listed in Table 3.
  2. qPCR cycling conditions need to be optimized for each qPCR kit.
    1. Use the following DLC qPCR conditions, depending on the qPCR kits used: initial denaturation (95 °C for 3 min); 50 rounds of amplification (95 °C for 15 s, 61 °C for 25 s, 72 °C for 15 s with a single acquisition); melting curve analysis (95 °C for 5 s, 65 °C for 1 min, 97 °C with continuous acquisition); and cooling (37 °C for 30 s).
    2. Use the following qPCR conditions for the DLE assay: initial denaturation (95 °C for 5 min); 50 rounds of amplification (95 °C for 15 s, 60 °C for 30 s, 72 °C for 15 s with a single acquisition); melting curve analysis (95 °C for 5 s, 65 °C for 1 min, 97 °C with continuous acquisition); and cooling (37 °C for 30 s). Note that optimization for different qPCR machines/kits may be required.
  3. DLC assay
    1. Controls: See the list of qPCR primers in Table 3. A map of the primer binding sites is shown in Figure S1. For supplementary sequence files for the relevant genomic features and amplicons, check the A plasmid Editor (ApE) files; Supplementary Sequence Files 1-5.
      1. Genomic DNA at ARG4: Use olWDH1760/olWDH1761 to amplify dsDNA located at ARG4. Use this reaction as a loading control and normalize all other reactions except the DLC signal reaction to this control.
      2. Intramolecular ligation efficiency at DAP2: Use the 1,904 bp fragment created by EcoRI digestion for intramolecular ligation in parallel with the DLC ligation. Amplification across this ligation junction reports on the intramolecular ligation efficiency and serves as a control to which the DLC signal is normalized.
      3. DSB induction: Use olWDH1766/olWDH1767 to amplify a region that spans the induced DSB.
      4. Psoralen crosslinking and resection: Use olWDH2019/olWDH2020 to amplify the unique PhiX region downstream of the EcoRI recognition site. Without crosslink reversal, use the ratio of the ssDNA (no crosslinking) over ARG4 (crosslinked dsDNA) to determine the crosslinking efficiency. With crosslink reversal, resection will lead to a progressive decrease from 1 to 0.5 of the signal relative to ARG4.
      5. EcoRI recognition site restoration and cutting: Use olWDH1768/olWDH1764 to amplify a region that spans the restored EcoRI recognition site upstream of the DSB on the resected strand. olWDH1769/olWDH1763 amplify a region that spans the EcoRI restriction enzyme site at DAP2. Perform EcoRI cleavage at this site to use as intramolecular ligation control.
    2. DLC signal: Use olWDH1764/olWDH1765 to amplify the chimeric DNA molecule created by intramolecular ligation of the resected (invading) strand and the donor.
    3. Analysis: Calculate the average and standard deviation of the Cp values for each of the duplicate reactions. Use the ARG4 genomic DNA qPCR Cp values as a reference to normalize all the other control qPCRs. Normalize the DLC signal to the intramolecular ligation control at DAP2. See Figure 3 for typical DLC signal values at 2 h.
  4. DLE assay
    1. Controls: See the list of qPCR primers in Table 3. A map of the primer binding sites is shown in Figure S1. For supplementary sequence files for the relevant genomic features and amplicons, check the A plasmid Editor (ApE) files (Supplementary Sequence Files 1-5).
      1. Genomic DNA at ARG4: See section 6.3.1.1.
      2. Intramolecular ligation efficiency at YLR050C: Use the HindIII digestion to create a 765 bp fragment that will undergo intramolecular ligation in parallel with the DLE ligation. Amplification across this ligation junction reports on the intramolecular ligation efficiency and serves as a control to which the DLE signal is normalized.
      3. DSB induction: See section 6.3.1.3.
      4. HindIII recognition site restoration and cutting: Use olWDH2010/olWDH2012 and olWDH2009/2011 to amplify a region that spans the HindIII restriction enzyme sites on the broken strand where it has been resected and extended, respectively.
    2. DLE signal: Use olWDH2009/olWDH2010 to amplify the chimeric DNA molecule created by intramolecular ligation of the resected end of the invading strand upstream of the DSB to the newly extended end downstream of the DSB.
    3. Analysis: Calculate the average and standard deviation of the Cp values for each of the duplicate reactions. Use the ARG4 genomic DNA qPCR Cp values as a reference to normalize all the other control qPCRs. Normalize the DLE signal to the intramolecular ligation control at YLR050C. Typical DLE signal values at 6 h are reported in Figure 4 and previous publications9.

Results

DLC assay
The DLC assay detects both nascent and extended D-loops formed by the invasion of a site-specific DSB into a single donor (Figure 2). Psoralen crosslinking physically links the broken strand and the donor via the heteroduplex DNA within the D-loop. Restriction enzyme site restoration with a long, hybridizing oligo on the resected strand of the break allows for restriction enzyme cleavage, followed by ligation of the broken strand to the proximal dono...

Discussion

The assays presented allow for the detection of nascent and extended D-loops (DLC assay), D-loop extension (DLE assay), and BIR product formation (DLE assay with no hybridizing oligonucleotides) using proximity ligation and qPCR. ChIP-qPCR of Rad51 to sites distant from the DSB has previously been used as a proxy for Rad51-mediated homology search and D-loop formation. However, this ChIP-qPCR signal is independent of the sequence homology between the break site and a potential donor, as well as the Rad51-associated facto...

Disclosures

The authors have nothing to disclose.

Acknowledgements

The work in the Heyer laboratory is supported by grants GM58015 and GM137751 to W.-D.H. Research in the Piazza laboratory is supported by the European Research Council (ERC-StG 3D-loop, grand agreement 851006). D.R. is supported by T32CA108459 and the A.P. Giannini Foundation. We thank Shih-Hsun Hung (Heyer Lab) for sharing his DLC/DLE assay results and for additionally validating the changes to the assays that are detailed in this protocol.

Materials

NameCompanyCatalog NumberComments
1. Pre-growth, DSB induction, and sample collection
Equipment
15 and 50 mL conical tubes
15 mL glass culture tubes
250 mL, 500 mL, or 1 L flasks
60 mm x 15 mm optically clear petri dishes with flat bottomSuggested: Corning, Catalog Number 430166; or Genesee Scientific, Catalog Number 32-150G
Benchtop centrifuge with 15 and 50 mL conical tube adapters
Benchtop orbital shaker or tube rotator/revolver
Rotator
UV crosslinker or light box with 365 nm UV bulbs set atop an orbital shakerSuggested: Spectrolinker XL-1500 UV Crosslinker (Spectronics Corporation) or Vilbert Lourmat BLX-365 BIO-LINK, Catalog Number 611110831
Materials
60% w/w sodium DL-lactate syrupSigma-AldrichL1375For media preparation
AgarFisherBP1423500For media preparation
Bacto peptoneBD Difco211840For media preparation
Bacto yeast extractBD Difco212750For media preparation
D-(+)-glucoseBD Difco0155-17-4For media preparation
TrioxsalenSigma-AldrichT6137For psoralen cross-linking
2. Spheroplasting, lysis, and restriction enzyme site restoration
Supplies
1.5 mL microcentrifuge tubes
Dry bath
Liquid nitrogen or dry ice/ethanol
Refrigerated microcentrifuge or microcentrifuge
Materials
10X restriction enzyme (CutSmart) buffer (500 mM potassium acetate, 200 mM Tris-acetate, 100 mM magnesium acetate, 1 mg/mL BSA, pH 7.8-8.0)
Zymolyase 100TUS BiologicalZ1004For spheroplasting
3. Restriction enzyme digest and intramolecular ligation
Supplies
Water bath
Materials
EcoRI-HFNew England BiolabsR3101Restriction enzyme digest for DLC assay
HindIII-HFNew England BiolabsR3104Restriction enzyme digest for DLE assay
T4 DNA ligaseNew England BiolabsM0202Intramolecular ligation
4. DNA purification
Supplies
1.5 and 2 mL microcentrifuge tubes
Materials
Phenol/chloroform/isoamyl alcohol (P/C/IA) at 25:24:1Sigma-AldrichP2069DNA purification
5. Psoralen cross-link reversal
Supplies
Thermocycler/PCR machine
6. qPCR
Supplies
Lightcycler 480Roche5015278001qPCR machine used by the authors
Lightcycler 96Roche5815916001qPCR machine used by the authors
Materials
LightCycler 480 96-Well Plate, whiteRoche472969200196-well plates for qPCR
SsoAdvanced Universal SYBR Green Super MixBioRad1725271qPCR kit used by the authors
SYBR Green I Master MixRoche4707516001qPCR kit used by the authors

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