The D-loop capture and extension assays are the first to allow unbiased and direct quantitation of the D-loop formation and extension steps during homologous recombination in mitotically growing yeast cells. This viability independent technique allows the studying of proteins in D-loop metabolism that would not otherwise be possible to disentangle from later roles, just looking at BIR products. In the future, we envision transitioning these assays to human cells to better understand the role of key proteins, including BRCA2 and the bloom and Warner kilo cases in homologous recombination.
Begin by centrifuging the samples for five minutes. Resuspend the pellet in 2.5 milliliters of 1x psoralen solution and transfer it to a 60 by 15 millimeter Petri dish. For no crosslinking control, resuspend the pellet in 2.5 milliliters of TE1 solution.
Next, set the UV light source the top in orbital shaker, set at 50 RPM. To cross-link the samples using a UV cross-linker with 365 nanometer long wave bulbs. Position the Petri dishes one to two centimeters below the UV light source for 10 minutes with gentle shaking on a pre chilled plastic or plexiglass plate.
Then transfer the sample into a new 15 milliliter tube. Rinse the Petri dish with 2.5 milliliters of TE1 solution and add this to the tube. Centrifuge the samples for five minutes.
Discard the supernatant and store the pellet at minus 20 degrees Celsius. Place the samples on ice for thawing. Simultaneously, preheat a dry bath to 30 degrees Celsius.
Resuspend the samples in one milliliter of spheroplasting buffer and transfer them to 1.5 milliliter micro centrifuge tubes. Then add 3.5 microliters of zymolyase solution and mix gently by tapping. Incubate at 30 degrees Celsius for 15 minutes.
And then place the tubes on ice. Centrifuge for three minutes and place the samples on ice. Wash the samples thrice in one milliliter spheroplasting buffer and centrifuge the samples for three minutes.
Resuspend the samples in one milliliter of cold restriction enzyme buffer. Centrifuge for three minutes and place the samples on ice. Repeat the wash once.
Resuspend the samples in one milliliter of cold 1x restriction enzyme buffer. Split the sample equally into two. Centrifuge the samples for three minutes at 16, 000 G at four degree Celsius.
Resuspend one tube from each sample in 180 microliters of 1.4x restriction enzyme buffer with hybridizing oligos and another tube in 180 microliters of 1.4x restriction enzyme buffer without hybridizing oligos. Snap freeze the samples in liquid nitrogen. Thaw the samples on ice.
Preheat one dry bath to 65 and another to 37 degrees Celsius. Aliquot 36 microliters of the sample into a new micro centrifuge tube and four microliters of 1%SDS and mixed by gently tapping the side of the tube. Incubate at 65 degrees Celsius for 15 minutes with gentle tapping every five minutes.
Place samples on ice immediately following the incubation. Add 4.5 microliters of 10%Triton X-100 and mixed by pipetting. Add 20 to 50 units of high fidelity EcoR1 or HindIII restriction enzyme to each sample.
And incubate at 37 degrees Celsius for one hour with gentle agitation every 20 to 30 minutes. During this time, preheated dry bath to 55 degrees Celsius and a water bath to 16 degrees Celsius. Add 8.6 microliters of 10%SDS to each sample and mix by pipetting and tapping.
Incubate at 55 degree Celsius for 10 minutes. Add 80 microliters of 10%Triton X-100 to each sample and mix by pipetting. Add 660 microliters of 1x ligation buffer without ATP, One millimolar ATP at pH 8.0 and eight units of T4 DNA ligase to each sample, and mix gently by inversion.
Incubate 16 degrees Celsius for 1.5 hours with an inversion every 30 minutes. Place the samples on ice immediately following the incubation. Following DNA purification as described in the protocol, use two microliters of purified DNA to set up a 20 microliter qPCR reaction according to the manufacturer's instructions.
For each sample, set up five control reactions and one quantification reaction and run them in duplicate. DLC assay analysis two hours after double stranded break or DSB induction showed that psoralen cross-linking is critical and efficiency depends on the time between sample collection and qPCR. ARG4 Cp values values were similar between the cross-linked samples but lower for the without cross-linking sample since DNA without cross-links amplifies more efficiently.
For all samples, the intermolecular ligation qPCR control was within the range. Robust DSB induction was evidenced by a low qPCR signal for the control that amplifies across the HO endonuclease recognition site. Similar results were observed for the EcoR1 qPCR control.
DLC signal with hybridizing oligo was calculated by normalizing to the intermolecular ligation control. DLE assay analysis performed at six hours post DSB induction showed that ARG4 CP values were similar between the with and without hybridizing oligos sample. The intermolecular ligation qPCR control revealed an acceptable signal for the with and without hybridizing oligos sample but a lower signal for the failed sample.
In all three samples, there was robust DSB induction. Since HindIII cleavage depends on restriction enzyme site restoration by the hybridizing oligo. There was a significant difference in amplification across the HindIII cleavage site on the resected strand between the width and without oligo samples.
A smaller difference in amplification across the site on the extended strand was observed because here HindIII cleavage also depends on extension. The samples and buffer need to be kept cold at all times. Samples must be flash frozen following resuspension in 1.4x cold restriction enzyme buffer with or without hybridizing oligos.
The effects downstream of D-loop formation and extension on product formation may be studied using Southern blotting or genetic endpoint assays. Effects on homology search may be investigated using high CP.Chromatin-immunoprecipitation can provide information on recruitment of a protein of interest.
