Name: detection of homologous recombination intermediates via proximity ligation and quantitative pcr in saccharomyces cerevisiae
Uploaded: 2022-09-11T13:00:00.000+00:00
Duration: 7 min 55 s
Description: Watch this Scientific Journal Video about Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae at JoVE.com

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.

1. Pre-growth, DSB induction, and sample collection
NOTE: Supplementation of all media with 0.01% adenine is recommended for Ade- strains.
<ol>
	<li>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 &#176;C.</li>
	<li>Use a single colony to inoculate 5 mL of YPDA in a 15 mL glass culture tube. Grow cultures to saturation at 30 &#176;C with shaking or rotation for aeration.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Grow the culture for ~16 h at 30 &#176;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.</li>
	<li>For each time point, collect the appropriate volume of cells in a conical tube and place on ice. Typically, this is 1&#160;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&#160;cells (approximately 2.5 mL of culture at OD600 1.0) for the DLE assay.</li>
	<li>To ensure the accuracy of the OD600 values, prepare 1:5 dilutions for cultures with an OD600&#8805;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).</li>
	<li>DLC assay
	<ol>
		<li>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.</li>
		<li>Centrifuge the samples at 2,500 x g for 5 min at 4 &#176;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.</li>
		<li>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 &#8722;20&#176; 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.</li>
		<li>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 &#176;C, properly dispose of the supernatant, and store the pellet at &#8722;20&#176; C. Samples can be stored for up to 1 week before moving to the next step.</li>
	</ol>
	</li>
	<li>DLE assay
	<ol>
		<li>Centrifuge the samples at 2,500 x g for 5 min at 4 &#176;C. Wash the cell pellet in 2.5 mL of cold TE1 solution before repeating the spin and storing the pellets at &#8722;20 &#176;C. Samples can be stored for up to 1 week before moving to the next step.</li>
	</ol>
	</li>
	<li>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).</li>
</ol>
2. Cell spheroplasting, lysis, and restriction site restoration
<ol>
	<li>Thaw the samples on ice. Preheat a dry bath to 30 &#176;C.</li>
	<li>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.</li>
	<li>Add 3.5 &#181;L of zymolyase solution (2% glucose, 50 mM Tris-HCl pH 7.5, 5 mg/mL zymolyase 100T; 17.5 &#181;g/mL zymolyase final concentration). Mix gently by tapping or inversion. Incubate at 30 &#176;C for 15 min, and then place on ice. During the 15 min incubation, obtain liquid nitrogen or dry ice.</li>
	<li>Centrifuge for 3 min at 2,500 x g at 4 &#176;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 &#176;C.</li>
	<li>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 &#181;g/mL BSA pH ~8.0 at RT) and centrifuge for 3 min at 16,000 x g at 4 &#176;C. Place the samples on ice. Repeat the wash 1x.</li>
	<li>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 &#176;C.</li>
	<li>Resuspend one tube from each sample in 180 &#181;L of 1.4x restriction enzyme buffer with hybridizing oligos (see Table 2) and one tube in 180 &#181;L 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 &#8722;20 &#176;C in small aliquots at the working dilution. The concentration of the hybridizing oligos may require optimization; see Discussion.</li>
	<li>Snap freeze the samples in liquid nitrogen or dry ice/ethanol and store at &#8722;80 &#176;C. Samples can be stored at this stage for several months.</li>
</ol>
3. Restriction enzyme digest and intramolecular ligation
<ol>
	<li>Thaw the samples on ice. Preheat one dry bath to 65 &#176;C and another to 37 &#176;C.</li>
	<li>Pipet 36 &#181;L of the sample into a new 1.5 mL microcentrifuge tube on ice. Promptly return the remaining sample to &#8722;80 &#176;C for storage.</li>
	<li>Add 4 &#181;L of 1% SDS (0.1% final concentration) and mix by gently tapping the side of the tube. Incubate at 65 &#176;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.</li>
	<li>Add 4.5 &#181;L 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 &#176;C for 1 h with gentle agitation every 20-30 min. During this time, preheat a dry bath to 55 &#176;C and preset a water bath to 16 &#176;C.</li>
	<li>Add 8.6 &#181;L of 10% SDS (1.5% final concentration) to each sample and mix by pipetting and tapping. Incubate at 55 &#176;C for 10 min. Add 80 &#181;L of 10% Triton X-100 (6% final concentration) to each sample and mix by pipetting.</li>
	<li>Add 660 &#181;L of 1x ligation buffer without ATP (50 mM Tris-HCl pH 8.0, 10 mM MgCl2, 10 mM DTT, 2.5 &#181;g/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 &#176;C for 1.5 h with inversion every 30 min. Place the samples on ice immediately following the incubation.</li>
</ol>
4. DNA purification
<ol>
	<li>Preheat one dry bath to 65 &#176;C and another to 37 &#176;C. Add 1 &#181;L of 10 mg/mL proteinase K (prepared in 1x TE pH 8.0) to each sample (12.5 &#181;g/mL final concentration). Incubate at 65 &#176;C for 30 min and place the samples on ice immediately following the incubation until they have cooled.</li>
	<li>Transfer the samples to 2 mL tubes. Working in a fume hood, add an equal volume (~800 &#181;L) 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.</li>
	<li>Carefully remove 600 &#181;L of the upper phase of each sample into a new 1.5 mL tube. Properly dispose of the lower phase and 2 mL tubes.</li>
	<li>Precipitate the DNA by adding a 1/10 volume of 3 M sodium acetate pH 5.2 (~60 &#181;L) to each sample, followed by 1 volume of isopropanol (~660 &#181;L). Invert the samples 5x-10x and incubate at RT for 30 min.</li>
	<li>Place the samples on ice for 2 min, and then centrifuge the samples at 16,500 x g for 15 min at 4 &#176;C in a microcentrifuge. Return the samples to ice, pour off the supernatant, and drain the tube on a paper towel.</li>
	<li>Wash the DNA pellet with 200 &#181;L of 70% ethanol. Centrifuge at 16,500 x g for 3 min at 4 &#176;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 &#176;C for 15-20 min.</li>
	<li>Resuspend the DNA pellets in 50 &#181;L of 1x TE by vortexing. Incubate at RT for 30 min, vortex, and then incubate at 37 &#176;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 &#8722;20 &#176;C for several months, but it is advisable to proceed immediately for the decrosslinking (DLC only) and qPCR steps.</li>
</ol>
5. Psoralen crosslink reversal (for DLC assay only)
<ol>
	<li>Pipet 9 &#181;L of purified DNA into a PCR tube on ice. Add 1 &#181;L of 1 M KOH (0.1 M final concentration). Incubate the samples at 90 &#176;C for 30 min in a thermocycler.</li>
	<li>Add 19.73 &#181;L 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 &#8722;20 &#176;C for several months, but it is advisable to proceed immediately to the qPCR step.</li>
</ol>
6. Quantitative PCR, controls, and analysis
<ol>
	<li>Using 2 &#181;L of purified DNA, with or without crosslinking, set up a 20 &#181;L qPCR reaction according to the manufacturer&#39;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.</li>
	<li>qPCR cycling conditions need to be optimized for each qPCR kit.
	<ol>
		<li>Use the following DLC qPCR conditions, depending on the qPCR kits used: initial denaturation (95 &#176;C for 3 min); 50 rounds of amplification (95 &#176;C for 15 s, 61 &#176;C for 25 s, 72 &#176;C for 15 s with a single acquisition); melting curve analysis (95 &#176;C for 5 s, 65 &#176;C for 1 min, 97 &#176;C with continuous acquisition); and cooling (37 &#176;C for 30 s).</li>
		<li>Use the following qPCR conditions for the DLE assay: initial denaturation (95 &#176;C for 5 min); 50 rounds of amplification (95 &#176;C for 15 s, 60 &#176;C for 30 s, 72 &#176;C for 15 s with a single acquisition); melting curve analysis (95 &#176;C for 5 s, 65 &#176;C for 1 min, 97 &#176;C with continuous acquisition); and cooling (37 &#176;C for 30 s). Note that optimization for different qPCR machines/kits may be required.</li>
	</ol>
	</li>
	<li>DLC assay
	<ol>
		<li>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.
		<ol>
			<li>Genomic DNA at ARG4: Use olWDH1760/olWDH1761 to amplify dsDNA located at ARG4. Use this reaction as a loading control&#160;and normalize all other reactions except the DLC signal reaction to this control.</li>
			<li>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.</li>
			<li>DSB induction: Use olWDH1766/olWDH1767 to amplify a region that spans the induced DSB.</li>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>DLC signal: Use olWDH1764/olWDH1765 to amplify the chimeric DNA molecule created by intramolecular ligation of the resected (invading) strand and the donor.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>DLE assay
	<ol>
		<li>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).
		<ol>
			<li>Genomic DNA at ARG4: See section 6.3.1.1.</li>
			<li>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.</li>
			<li>DSB induction: See section 6.3.1.3.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psoralens+as+photoactive+probes+of+nucleic+acid+structure+and+function:+Organic+chemistry,+photochemistry,+and+biochemistry.">Psoralens as photoactive probes of nucleic acid structure and function: Organic chemistry, photochemistry, and biochemistry.</a> Annual Review of Biochemistry. 54 (1), 1151-1193 (1985).</li><li>Yeung, A. T., Dinehart, W. J., Jones, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alkali+reversal+of+psoralen+cross-link+for+the+targeted+delivery+of+psoralen+monoadduct+lesion.">Alkali reversal of psoralen cross-link for the targeted delivery of psoralen monoadduct lesion.</a> Biochemistry. 27 (17), 6332-6338 (1988).</li><li>Shi, Y. B., Spielmann, H. P., Hearst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Base-catalyzed+reversal+of+a+psoralen-DNA+cross-link.">Base-catalyzed reversal of a psoralen-DNA cross-link.</a> Biochemistry. 27 (14), 5174-5178 (1988).</li><li>Prakash, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+Mph1+helicase+dissociates+Rad51-made+D-loops:+Implications+for+crossover+control+in+mitotic+recombination.">Yeast Mph1 helicase dissociates Rad51-made D-loops: Implications for crossover control in mitotic recombination.</a> Genes &amp; Development. 23 (1), 67-79 (2009).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Srs2+promotes+synthesis-dependent+strand+annealing+by+disrupting+DNA+polymerase+&#948;-extending+D-loops.">Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase &#948;-extending D-loops.</a> eLife. 6, 22195 (2017).</li><li>Fasching, C. L., Cejka, P., Kowalczykowski, S. C., Heyer, W. -. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top3-Rmi1+dissolve+Rad51-mediated+D+loops+by+a+topoisomerase-based+mechanism.">Top3-Rmi1 dissolve Rad51-mediated D loops by a topoisomerase-based mechanism.</a> Molecular Cell. 57 (4), 595-606 (2015).</li><li>Shah, S. S., Hartono, S. R., Ch&#233;din, F., Heyer, W. -. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisulfite+treatment+and+single-molecule+real-time+sequencing+reveal+D-loop+length,+position,+and+distribution.">Bisulfite treatment and single-molecule real-time sequencing reveal D-loop length, position, and distribution.</a> eLife. 9, 59111 (2020).</li></ol>

The authors have nothing to disclose.

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 factor Rad54, and is, thus, more likely to represent a transient association between the Rad51-ssDNA filament and dsDNA rather than a D-loop intermediate10,11. In contrast, the DLC signal depends on DSB formation, Rad51, Rad52, Rad54, and shared sequence homology between the DSB and the donor site assayed8. Moreover, increased DLC signals are observed in the absence of the Mph1 and Srs2 helicases, and the Sgs1-Top3-Rmi1 helicase-topoisomerase complex, consistent with previous reports that these three factors can disassemble Rad51/Rad54-made nascent D-loops in vitro8,25,26,27. The DLE assay similarly represents an improvement over previous methods to follow recombination-associated DNA synthesis, as it can distinguish between D-loop extension and BIR product formation19.
As discussed above, the qPCR controls, including those for the genomic DNA, DSB induction, psoralen cross-linking, intramolecular ligation, and oligonucleotide hybridization, are critical to the success and reproducibility of these assays. Raw genomic DNA qPCR values should be approximately equivalent across samples. Low Cp values for the ARG4 genomic DNA control indicate excess DNA, and the number of cells collected should be adjusted. High Cp values for this control indicate insufficient DNA recovery or contamination with reagents that interfere with qPCR. Following spheroplasting, cell lysis can be observed using a standard light microscope and equal volumes of sample and sterile water. If insufficient lysis is observed upon the addition of water, the zymolyase solution must be remade, or the incubation at 30 &#176;C should be prolonged. Sample&#160;can also be lost or contaminants introduced during DNA purification by P/C/IA extraction. For the efficient recovery of DNA, one should ensure that the pH of the P/C/IA has been adjusted to ~8.0 and that the bottom phase is not disturbed while removing the upper phase. Lastly, inefficient resuspension of the DNA pellet in 1x TE can result in low Cp values. A longer incubation at 37 &#176;C and vortexing will improve the resuspension of the DNA pellet.
In addition to the genomic DNA genomic DNA control, the DSB induction and restriction enzyme cleavage control reactions should also be similar across samples. HO endonuclease or restriction enzyme cutting at the site of the DSB or restriction enzyme recognition site prevents amplification across this region; therefore, typical normalized qPCR values for these controls are near zero, and a high qPCR value indicates insufficient cleavage. If a high signal at the site of the DSB is observed, the galactose solution should be remade. For mutants with a known cell cycle defect, DSB induction should be quantified by plating equal amounts of culture grown according to the protocol (see section 1) on YPDA and YPA media supplemented with galactose. Colonies that grow on media containing galactose represent yeast in which end-joining created an uncleavable HOcs. If there are significantly more end-joining events in a mutant of interest relative to the wild type, a correction must be applied to compensate for this difference in DSB induction, which will affect the DLC/DLE signal.
Three primer pairs (olWDH1764/olWDH1768, olWDH2010/olWDH2012, and olWDH2009/olWDH2011) assess restriction enzyme site restoration by the hybridizing oligos and cutting by the EcoRI-HF and HindIII-HF restriction enzymes. Moreover, the intramolecular ligation controls also depend on adequate restriction enzyme digestion. Thus, a sample with low intramolecular ligation efficiency and a high signal for one of these three primer pairs has insufficient restriction enzyme cutting. Additional restriction enzyme should be provided in subsequent preparations, and the efficacy of the restriction enzyme should be assessed on genomic DNA. The olWDH1769/olWDH1763 primer pair represents an additional control for the DLC assay, which measures EcoRI cleavage at DAP2, where intramolecular ligation efficiency is also measured. A sample with an adequate intramolecular ligation signal but a high signal for one of these three primer pairs has inadequate restriction enzyme site restoration by the hybridizing oligos. To address this problem, duplicate samples should be collected and the concentration of the affected hybridizing oligo(s) should be varied. Typical qPCR values obtained for these reactions with and without hybridizing oligos can be found in Figure 3 and Figure 4 and in Piazza et al.8 and Piazza et al.9. 
For both the DLC and the DLE assay, an intramolecular ligation efficiency of 0.15-0.35&#160;as normalized to the genomic DNA control is considered normal. As the detection of nascent and extended D-loops and the BIR product is dependent on efficient ligation, samples with low ligation signals must be discarded. The 10x ligation buffer lacking ATP should be stored at 4 &#176;C for no more than 6 months. Collecting too many cells can lead to intermolecular ligation, which will result in low intramolecular ligation efficiency and DLC/DLE signal.
Though these controls for the DLC and DLE assays report on nearly all the sensitive steps, it is still possible to obtain non-physiological values for the DLC or DLE signal when these controls are within the appropriate range. A low DLC or DLE signal may result from errors in the cell spheroplasting step, which is extremely sensitive. One should process only a few samples in parallel and keep them at 4 &#176;C at all times. A high/low DLC/DLE signal can also result from collecting too many/few cells at each time point. This problem can be addressed by collecting multiple OD600s of cells at each time point for each sample.
There are several technical and conceptual limitations to the DLC and DLE assays in their present form. First, the psoralen-mediated inter-strand crosslink density is ~1 in 500 bp8. Therefore, an increased DLC signal can either indicate that there are more D-loops in the population, that the average length of the D-loops in the population has increased (assuming that D-loops can be smaller than 500 bp), or both. Furthermore, the likelihood that a D-loop will be captured by the DLC assay decreases with decreasing D-loop length. Given that very short D-loops may account for a significant fraction of the total D-loop population in certain mutant backgrounds, this limitation of the assay must be considered when interpreting results. Second, the DLC assay requires DNA crosslinking, whereas the DLE assay does not. Previously, for a given experiment, this meant that DLC and DLE samples had to be collected and analyzed separately. The method shown in Figure 5 achieves robust crosslink reversal, alleviating the need to collect multiple samples from the same culture. The introduction of a second EcoRI restriction enzyme site on the broken strand, downstream of the HindIII recognition site, will enable sequential DLC and DLE analysis.
In addition to these technical limitations, the DLC and DLE assay system currently does not permit the recovery of viable HR products because the right side of the inducible DSB lacks homology to the donor. To better understand the kinetics and mechanism of second end engagement and synthesis, the system could be modified such that repair using a proximal or distal region of homology shared between the second end of the break and the donor is feasible (Figure 6). Looking forward, it may prove insightful to combine the DLC and DLE assays with other technologies, such as ChIP-qPCR, high-throughput chromosome conformation capture (Hi-C), and in vivo D-loop mapping, to achieve a comprehensive analysis of the kinetics and regulation of the steps in the HR pathway, including break formation, end resection, Rad51 filament formation, nascent D-loop formation, D-loop extension, D-loop reversal, second end engagement, second end synthesis, and resolution28.
In summary, the DLC and DLE assays permit the quantification of nascent and extended D-loops, D-loop extension, and BIR product formation using the principle of proximity ligation. These assays represent major advancements in the field, as they are the first to permit the semi-quantitative measurement of D-loop formation and extension independent of cellular viability.

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&#39; 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&#160;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 &#948; 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&#39; 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&#39; end of the broken molecule to the newly extended 3&#39; 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.

<table><tbody><tr><td>&lt;strong&gt;1. Pre-growth, DSB induction, and sample collection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15 and 50 mL conical tubes</td><td></td><td></td><td></td></tr><tr><td>15 mL glass culture tubes</td><td></td><td></td><td></td></tr><tr><td>250 mL, 500 mL, or 1 L flasks</td><td></td><td></td><td></td></tr><tr><td>60 mm x 15 mm optically clear petri dishes with flat bottom</td><td></td><td></td><td>Suggested: Corning, Catalog Number 430166; or Genesee Scientific, Catalog Number 32-150G</td></tr><tr><td>Benchtop centrifuge with 15 and 50 mL conical tube adapters</td><td></td><td></td><td></td></tr><tr><td>Benchtop orbital shaker or tube rotator/revolver</td><td></td><td></td><td></td></tr><tr><td>Rotator</td><td></td><td></td><td></td></tr><tr><td>UV crosslinker or light box with 365 nm UV bulbs set atop an orbital shaker</td><td></td><td></td><td>Suggested: Spectrolinker XL-1500 UV Crosslinker (Spectronics Corporation) or Vilbert Lourmat BLX-365 BIO-LINK, Catalog Number 611110831</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>60% w/w sodium DL-lactate syrup</td><td>Sigma-Aldrich</td><td>L1375</td><td>For media preparation</td></tr><tr><td>Agar</td><td>Fisher</td><td>BP1423500</td><td>For media preparation</td></tr><tr><td>Bacto peptone</td><td>BD Difco</td><td>211840</td><td>For media preparation</td></tr><tr><td>Bacto yeast extract</td><td>BD Difco</td><td>212750</td><td>For media preparation</td></tr><tr><td>D-(+)-glucose</td><td>BD Difco</td><td>0155-17-4</td><td>For media preparation</td></tr><tr><td>Trioxsalen</td><td>Sigma-Aldrich</td><td>T6137</td><td>For psoralen cross-linking</td></tr><tr><td>&lt;strong&gt;2. Spheroplasting, lysis, and restriction enzyme site restoration&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>Dry bath</td><td></td><td></td><td></td></tr><tr><td>Liquid nitrogen or dry ice/ethanol</td><td></td><td></td><td></td></tr><tr><td>Refrigerated microcentrifuge or microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>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)</td><td></td><td></td><td></td></tr><tr><td>Zymolyase 100T</td><td>US Biological</td><td>Z1004</td><td>For spheroplasting</td></tr><tr><td>&lt;strong&gt;3. Restriction enzyme digest and intramolecular ligation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EcoRI-HF</td><td>New England Biolabs</td><td>R3101</td><td>Restriction enzyme digest for DLC assay</td></tr><tr><td>HindIII-HF</td><td>New England Biolabs</td><td>R3104</td><td>Restriction enzyme digest for DLE assay</td></tr><tr><td>T4 DNA ligase</td><td>New England Biolabs</td><td>M0202</td><td>Intramolecular ligation</td></tr><tr><td>&lt;strong&gt;4. DNA purification&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1.5 and 2 mL microcentrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Phenol/chloroform/isoamyl alcohol (P/C/IA) at 25:24:1</td><td>Sigma-Aldrich</td><td>P2069</td><td>DNA purification</td></tr><tr><td>&lt;strong&gt;5. Psoralen cross-link reversal&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Thermocycler/PCR machine</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;6. qPCR&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Lightcycler 480</td><td>Roche</td><td>5015278001</td><td>qPCR machine used by the authors</td></tr><tr><td>Lightcycler 96</td><td>Roche</td><td>5815916001</td><td>qPCR machine used by the authors</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>LightCycler 480 96-Well Plate, white</td><td>Roche</td><td>4729692001</td><td>96-well plates for qPCR</td></tr><tr><td>SsoAdvanced Universal SYBR Green Super Mix</td><td>BioRad</td><td>1725271</td><td>qPCR kit used by the authors</td></tr><tr><td>SYBR Green I Master Mix</td><td>Roche</td><td>4707516001</td><td>qPCR kit used by the authors</td></tr></tbody></table>

detection of homologous recombination intermediates via proximity ligation and quantitative pcr in saccharomyces cerevisiae

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.

Watch this Scientific Journal Video about Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae at JoVE.com

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

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.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

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 ...

detection-homologous-recombination-intermediates-via-proximity

Nanyang Technological University

Research

JoVE Journal

Genetics

1.7K Views.  University of California, Davis. De D-loop capture en extension assays zijn de eerste die onbevooroordeelde en directe kwantificering van de D-loop vorming en extensiestappen mogelijk maken tijdens homologe recombinatie in mitotisch groeiende gistcellen. Deze levensvatbaarheidsonafhankelijke techniek maakt het mogelijk om eiwitten in het D-loop-metabolisme te bestuderen die anders niet mogelijk zouden zijn om te ontwarren van latere rollen, alleen kijkend naar BIR-producten. In de toekomst voorzien we de overgang van deze te...

Video: Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Biochemistry

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors contribute to alterations in the normal aging process. These factors intersect in complex ways, as evidenced by the wealth of documented links identified and conserved in many organisms. Most of these studies focus on loss-of-function, null mutants that allow for rapid screening of many genes simultaneously. There is much less work that focuses on characterizing the role that overexpression of a gene in this process. In the present work, we present a straightforward methodology to identify and clone genes in the budding yeast, Saccharomyces cerevisiae, for study in suppression of the short-lived chronological lifespan phenotype seen in many genetic backgrounds. This protocol is designed to be accessible to researchers from a wide variety of backgrounds and at various academic stages. The SIR2 gene, which codes for a histone deacetylase, was selected for cloning in the pRS315 vector, as there have been conflicting reports on its effect on the chronological lifespan. SIR2 also plays a role in autophagy, which results when disrupted via the deletion of several genes, including the transcription factor ATG1. As a proof of principle, we clone the SIR2 gene to perform a suppressor screen on the shortened lifespan phenotype characteristic of the autophagy deficient atg1&#916; mutant and compare it to an otherwise isogenic, wild type genetic background.

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Here is a protocol to identify genetic interactions through an increased copy number suppressor screen in Saccharomyces cerevisiae. This method allows researchers to identify, clone, and test suppressors in short-lived yeast mutants. We test the effect of the copy number increase of SIR2 on lifespan in an autophagy null mutant.

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Biology

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

Cancer Research

Locus-Specific Chromatin Isolation from Yeast Cells

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone octamer. Chromatin is defined as the entity of NCPs and numerous other protein complexes, including transcription factors, chromatin remodeling and modifying enzymes. It is still unclear how these protein-DNA interactions are orchestrated at the level of specific genomic loci during different stages of the cell cycle. This is mainly due to the current technical limitations, which make it challenging to obtain precise measurements of such dynamic interactions. Here, we describe an improved method combining site-specific recombination with an efficient single-step affinity purification protocol to isolate a single-copy gene locus of interest in its native chromatin state. The method allows for the robust enrichment of the target locus over genomic chromatin, making this technique an effective strategy for identifying and quantifying protein interactions in an unbiased and systematic manner, for example by mass spectrometry. Further to such compositional analyses, native chromatin purified by this method likely reflects the in vivo situation regarding nucleosome positioning and histone modifications and is, therefore, amenable to further structural and biochemical analyses of chromatin derived from virtually any genomic locus in yeast.

Author Spotlight: Advancing Chromatin Research and Overcoming Limitations with a High-Enrichment Locus-Specific Chromatin Isolation Protocol 

This protocol presents a locus-specific chromatin isolation method based on site-specific recombination to purify a single-copy gene locus of interest in its native chromatin context from budding yeast, Saccharomyces cerevisiae.

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone ...

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms1-3. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs4. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases5, as well as cancer in humans6-9.
Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange10,11. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. 
In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-&Delta;3', contains a 3' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-&Delta;5', is located at the LEU2 locus on one copy of chromosome III, and contains a 5' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1 locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3 coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3 allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system12-14.

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

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