The overall goal of this procedure is to rapidly examine proteins bound to nascent DNA with accuracy using BrdU labeling and chromatin precipitation. This method can answer key questions in the replication, repair in chromatin field, such as, what proteins are bound to nascent DNA during DNA replication and what repair proteins are bound to nascent DNA during replication stress. The main advantage of this technique is that it is a relatively quick method to detect proteins bound to nascent DNA in a highly quantitative manner.
To begin, set up cultures of two million NIH 3T3 cells in 10 milliliters of media with either DMSO or inhibitors. Also, add BrdU to a final concentration of 20 micromolar. Once plated, incubate the cells for 60 minutes.
After an hour, crosslink the intracellular proteins to the DNA by adding formaldehyde directly to the culture medium to a concentration of 1%by volume. Then, let the cells incubate at room temperature for 10 minutes with agitation. After 10 minutes, quench the reaction by adding glycine to the media to 125 millimolar and continue the incubation for five more minutes.
Next, collect the cells in PBS and wash them twice with PBS using centrifugation. The pellet can be frozen at 80 degrees Celsius per storage if desired. Now, prepare the lysate from the crosslinked cells, add 500 microliters of FA140 buffer supplemented with the protease inhibitor cocktail to the pellet and shear the chromatin using five 15-second pulses of sonication.
Keep the cells chilled between the pulses. Then, run 10 microliters of sample out on a 1.5%gel to diagnose the efficiency of the shearing. Once sheared, centrifuge the samples for 10 minutes, and transfer the supernatant to a new tube.
Then, estimate the protein concentration in the supernatant using a kit. Next, prepare a 50%slurry of Protein A Agarose beads with FA140 buffer containing protease inhibitor. Add 50 microliters of the slurry to each sample and incubate the samples at 4 degrees Celsius with constant end-over-end rotation for 30 minutes.
After half an hour, spin down the samples for a minute around 1000 G's in a refrigerated centrifuge. Then, collect the supernatant containing the labelled chromatin. Before proceeding, save 1/10th of the extract at 20 degrees Celsius to later serve as the input control.
To immunoprecipitate the chromatin, add either eight microliters of H4K16ac antibody or five micrograms of SMARCA5 antibody to the supernatants. Also, add five micrograms of rapid IgG to the extract that serves as the negative control sample. Incubate the chromatin at 4 degrees Celsius overnight on a rotisserie mixer.
The next day, add 50 microliters of the prepared 50%Protein A Agarose slurry to the immunoprecipitations and incubate the mixtures for an hour at 4 degrees Celsius with rotation. After an hour, pellet the Agarose beads and carefully discard the supernatant. Now, wash the beads using end-over-end rotations at 4 degrees Celsius in three sequential buffers.
First, use freshly prepared, low-salt immune complex wash buffer for five minutes. Second, use freshly prepared, high-salt immune complex wash buffer for five minutes. Third, use freshly prepared, Lithium chloride immune complex wash buffer for five minutes.
Following washes, add elution solution to the samples as described in the text. Spin the samples and transfer the eluate to a new 1.5 milliliter tube. Now, incubate the samples at 65 degrees Celsius for 5 hours to reverse the crosslinking.
Later, precipitate the DNA. Add one milliliter of 100%ethanol to the samples and mix well. Then, incubate them at 80 degrees Celsius for two to three hours or overnight at 20 degrees Celsius.
After the precipitation, remove the ethanol with a 15-minute ceintrifugation at 18000 G's. Then, following the text protocol, wash the pellet, treat the samples with RNase, then Proteinase K, purify DNA as described in the text, and finally, store at 20 degrees Celsius. To begin, measure the yield of input and ChIP DNA on a spectrophotometer.
Then, denature the samples. Add 2.5 volumes of 0.4 normal sodium hydroxide, briefly vortex and centrifuge and incubate the samples at room temperature for 30 minutes. Next, neutralize the denatured DNA by adding 175 microliters of one molar Tris hydochloride pH 6.8.
Vortex, centrifuge, and place the samples on ice. Now, prepare serial dilutions of the input and immunoprecipitated DNA and proceed with the slot-blot assay as described in the text protocol. After the DNA on the blot has been UV immobilized, detect the BrdU on the slot blot using western blotting.
First, block the membrane with 5%nonfat dry milk in PBST for an hour at room temperature to prevent nonspecific binding. Next, prepare a bath of 1%nonfat dry milk in PBST with added 1:500 diluted anti-BrdU antibody. Incubate the blot in this bath for three hours at room temperature on a shaker.
Afterwards, wash the membrane with PBST three times. Then, submerge the blot in a 1:5000 dilution of HRP conjugated anti-mouse secondary antibody in PBST. Incubate at room temperature for an hour with shaking.
Use three washes with PBST to remove the secondary and analyse the result. The BrdU H4K16ac ChIP slot western technique was used to determine the amount of H4K16ac associated with nascent DNA. A robust enrichment in H4K16ac associated with nascent DNA was observed when compared to the rapid IgG control.
Following inhibition of HDAC1/2 with compound 233, an increase in nascent-DNA-associated H4K16ac was observed. The same result was obtained using compound 898. Finally, the level of SMARCA5 chromatin remodeler on nascent DNA was determined using the BrdU SMARCA5 ChIP slot western technique.
Indeed, SMARCA5 associated with nascent DNA and compound 898 did not change the levels of SMARCA5. After watching this video, you should have a good understanding of the changes that occur in chromatin and chromatin-modifying enzymes during DNA replication in mammalian cells. After its development, this technique paved the way for researchers in the field of replication to explore chromatin changes on nascent DNA as well as dynamics of DNA repair proteins on nascent DNA.
In the future, this technique could also be used to study the dynamics of the DNA damage response and DNA repair proteins at the stalled fork in addition to studying the histone modifications and chromatin modifying enzymes at the fork.
