The work in this manuscript was supported by funds from Dept. of Radiation Oncology and Huntsman Cancer Institute and the National Institute of Health grant (R01-CA188520) to SB. I thank Danielle Johnson and Steven Bennett in my lab for demonstrating the protocol and explaining its benefits. I am grateful to Dr. Mahesh Chandrasekharan (Huntsman Cancer Institute) for critical comments on the manuscript.

1. Chromatin immunoprecipitation (ChIP) following BrdU Labeling
<ol>
	<li>Culture NIH3T3 cells in the presence of either DMSO or HDAC1,2-selective inhibitors as described previously6.</li>
	<li>Following treatment with DMSO or HDAC1,2 selective inhibitors, add BrdU to the cells in a sterile tissue culture hood. Incubate 2 x 106 NIH3T3 cells plated in 10 ml NIH3T3 media with a 20 &#181;M final concentration of bromodeoxyuridine (BrdU) for 60 min in a sterile 37 &#176;C tissue culture incubator.</li>
	<li>Crosslink intracellular proteins to DNA by adding formaldehyde directly to the culture media at a final concentration of 1%. Incubate 2 x 106 NIH3T3 cells with formaldehyde at RT&#160;for 10 min on a shaker.</li>
	<li>Quench the cross linking by the addition of glycine to a final concentration of 125 mM. Incubate at RT for 5 min on a shaker.</li>
	<li>Remove the media and collect cells in PBS in a 15 ml centrifuge tube. Spin cells at 956 x g for 5 min at 4 &#176;C. Wash the cell pellet twice with ice-cold PBS. Remove PBS from the tube. Store the cross-linked cell pellet without PBS (the supernatant) at -80 &#176;C until use.</li>
	<li>Prepare the lysate from the crosslinked cell pellet by adding 500 &#181;l FA140 buffer (50 mM of HEPES KOH pH 7.5, 140 mM of NaCl, 1 mM of EDTA (pH 8.0), 1% Triton-X-100 and 0.1% sodium deoxycholate) supplemented with protease inhibitor cocktail.</li>
	<li>Shear the chromatin by sonicating the samples five times at 35% power and with a 15 sec&#160;pulse, 0.9 sec on and 0.1 sec off setting. Keep the tubes on ice after every round of sonication to avoid heating of the samples.
	<ol>
		<li>Check the shearing on a DNA gel as the shearing efficiency will vary between different sonicators. To check sonication, reverse crosslink by adding NaCl to a concentration of 0.16 M and perform steps 1.17.1. - 1.22. Run eluted 10 &#181;l DNA on a 1.5% agarose gel to check whether the shearing is between 300 - 700 bp with an average intensity at 500 bp.</li>
	</ol>
	</li>
	<li>Following sonication, centrifuge samples for 10 min at 17,949 x g at 4 &#176;C and transfer the supernatant to a new tube.
	<ol>
		<li>Perform protein estimation using the commercial protein assay kit according to manufacturer&#39;s protocol. Use equal amount of lysate for ChIP of control and experimental samples. Generally, use 1 mg of total lysate per ChIP reaction.</li>
	</ol>
	</li>
	<li>Prepare 50% slurry of protein A-agarose using FA140 buffer containing protease inhibitor cocktail. Add 1/10 volume of the protease inhibitor cocktail. Calculate the volume of beads needed based on the number of samples in total.
	<ol>
		<li>Wash Protein A-agarose beads with FA140 buffer twice to remove the storage buffer and add an equal volume to FA140 buffer to the beads to make 50% slurry.</li>
		<li>To remove proteins that non-specifically bind to the beads, add 50 &#181;l of 50% slurry of Protein A-agarose and incubate the samples at 4 &#176;C with constant end-over-end rotation in a rotisserie mixer for 30 min.</li>
	</ol>
	</li>
	<li>Spin the samples at 956 x g at 4 &#176;C for a minute. Collect the supernatant and perform immunoprecipitation by adding either 8 &#956;l H4K16ac antibody or 5 &#956;l (5 &#956;g) of 1 mg/ml SMARCA5 antibody to the supernatant. Incubate at 4 &#176;C O/N&#160;with constant end-over-end rotation in a rotisserie mixer.</li>
	<li>Save 1/10th of the extract for &#39;input&#39; control and set it as aside at -20 &#176;C. The input needs to be reverse cross-linked along with the IP samples.</li>
	<li>Use equal amount of rabbit IgG (5&#160;&#956;l of 1 mg/ml) as the negative control immunoprecipitation sample.</li>
	<li>The following day, add 50 &#956;l of 50% protein A-agarose slurry to the extract and incubate for 1 hr at 4 &#176;C with rotation.</li>
	<li>Pellet agarose beads by centrifugation at 956 x g for 2 min at 4 &#176;C, carefully remove the supernatant and wash the beads with the following buffers sequentially. In buffer preparations, add protease inhibitors right before use.
	<ol>
		<li>Wash with Low Salt Immune Complex Wash Buffer (FA140; final concentration of 50 mM of HEPES KOH pH 7.5, 140 mM of NaCl, 1 mM of EDTA (pH 8.0), 1% Triton-X-100 and 0.1% Sodium deoxycholate) for 5 min at 4 &#176;C with end-over-end rotation.</li>
		<li>Wash with High Salt Immune Complex Wash Buffer (FA500; final concentration of 50 mM of HEPES KOH pH 7.5, 500 mM of NaCl, 1 mM of EDTA (pH 8.0), 1% Triton-X-100 and 0.1% Sodium deoxycholate) for 5 min at 4 &#176;C with end-over-end rotation.</li>
		<li>Wash with LiCl Immune Complex Wash Buffer (final concentration of 10 mM of Tris-Cl pH 8.0, 250 mM of LiCl, 0.5% NP40 and 0.5% Sodium deoxycholate) for 5 min at 4 &#176;C with end-over-end rotation.</li>
	</ol>
	</li>
	<li>Following washes as described above, add 200 &#181;l elution solution (final concentration of 1% SDS and 100 mM Sodium bicarbonate) and incubate at RT for 15 min with end-over-end rotation at RT.</li>
	<li>Spin the samples at 956 x g at RT and transfer the eluate to a new 1.5 ml tube. Repeat step 1.15 with additional 200 &#181;l elution solution.</li>
	<li>To 400 &#181;l of the eluate, add NaCl to a concentration of 0.16 M and mix well.
	<ol>
		<li>In addition, add 400 &#181;l elution solution to 10 &#181;l input that was set aside (step 1.11) and add NaCl to a concentration of 0.16 M to reverse crosslink input samples as well. Reverse crosslink the samples by incubating in 65 &#176;C water bath for 5 hr.</li>
	</ol>
	</li>
	<li>Add 1 ml&#160;of 100% EtOH to the reverse-crosslinked eluate. Mix well and incubate at -80 &#176;C for 2 - 3 hr or at -20 &#176;C O/N. Spin the samples at 17,949 x g for 15 min and discard ethanol.</li>
	<li>Add 800 &#181;l 70% ethanol to wash the pellet. Spin at 17,949 x g for 15 min at 4 &#176;C and discard ethanol. Spin briefly to remove any residual ethanol. Air-dry the samples at 37 &#176;C for 10 min to remove residual ethanol.</li>
	<li>Dissolve DNA in 90 &#181;l RNase and DNase free water and add 2 &#181;l 10 mg/ml RNase at a 0.2 mg/ml final concentration. Incubate at 37 &#176;C in water bath for 30 min.</li>
	<li>Add 10 &#181;l 10x&#160;Proteinase K Buffer (100 mM Tris-Cl pH 8.0, 50 mM EDTA pH 8.0, 5% SDS). Mix well and add 1 &#181;l Proteinase K. Incubate at 42 &#176;C for 1 hr.</li>
	<li>Purify input and ChIP DNA using commercial PCR purification kit according to manufacturer&#39;s protocol and elute DNA in 50 &#181;l water. Store DNA at -20 &#176;C until further use.</li>
</ol>
2. Slot Blot Analysis of ChIP DNA
<ol>
	<li>Measure the yield of input and ChIP DNA eluted in 50 &#181;l water as described in step 1.22 using a spectrophotometer at 280 nm according to manufacturer&#39;s protocol.</li>
	<li>Take equal volumes (50 &#181;l) of input or immunoprecipitated DNA that are within the linear range of detection.
	<ol>
		<li>For instance, if input DNA yield is 1.5 ng/&#181;l, then take 30 &#181;l (or 45 ng total DNA) and make volume to 50 &#181;l with water. For factor ChIP, take the entire 50 &#181;l eluate from Step 2.1 and proceed to step 2.3.</li>
	</ol>
	</li>
	<li>Denature 50 &#181;l input or immunoprecipitated DNA by adding 2.5 volumes of 0.4 N NaOH, briefly vortex and centrifuge for 956 x g for 10 sec, and incubate at RT for 30 min.</li>
	<li>Neutralize the denatured DNA by adding 175 &#181;l of 1 M Tris-HCl, pH 6.8, vortex, centrifuge for 956 x g for 10 sec and place the samples on ice.</li>
	<li>Prepare a serial dilution of input and immunoprecipitated DNA for the Slot Blot assay as described below.
	<ol>
		<li>Follow steps 2.3 - 2.4 to get a total sample volume of 350 &#181;l (i.e., 50 &#181;l Input/ChIP DNA, 125 &#181;l 0.4 N NaOH, 175 &#181;l 1 M Tris-HCl, pH 6.8). 
		Note: For the instance stated above, 45 ng of input DNA will end up being 0.129 ng/ &#181;l.</li>
		<li>For input DNA, label three tubes as 100, 50 and 25 and add 100 &#181;l, 50 &#181;l and 25 &#181;l of the denatured and neutralized DNA sample. Make the final volume to 100 &#181;l with nuclease-free water. For ChIP DNA, label three tubes as 200, 100 and 50 and add 200 &#181;l, 100 &#181;l and 50 &#181;l of the denatured and neutralized DNA sample. Make final volume to 200 &#181;l&#160;with nuclease free water.</li>
	</ol>
	</li>
	<li>Cut the membrane to the size of the Slot Blot apparatus. Pre-wet the membrane and blotting papers in 20x SSC before and prior to adding DNA. Assemble them into the Slot Blot apparatus according to the manufacturer&#39;s protocol.</li>
	<li>Pipet DNA from step 2.5.2 into appropriate slots. Apply vacuum slowly to pull the DNA onto the zeta-probe membrane. Stop vacuum after all samples have passed through the apparatus.
	<ol>
		<li>Disassemble the apparatus and immediately immobilize the DNA onto the membrane using a UV crosslinker. Have the DNA side up in the UV crosslinker and use the autocrosslink setting in the crosslinker. Follow manufacturer&#39;s protocol.</li>
	</ol>
	</li>
	<li>Detect BrdU on the slot blot by performing western blotting.
	<ol>
		<li>Block the membrane with 5% non-fat dry milk in PBS containing 0.1% Tween-20 (PBST) for 1 hr&#160;at RT to prevent the non-specific binding of antibodies to the membrane.</li>
		<li>Add primary anti-BrdU antibody (1:500 dilution) diluted in 1% non-fat dry milk in PBST and incubate the blot with the primary antibody for 3 hr at RT on a shaker. Wash the membrane with PBST three times after the primary antibody incubation.</li>
		<li>Add HRP-conjugated anti-mouse secondary antibody to the blot (1:5,000 dilution) and incubate at RT for 1 hr. Wash the membrane with PBST three times after the secondary antibody incubation. 
		NOTE: Use enough volume of the antibodies to completely cover the membrane during primary and secondary antibody incubations.</li>
	</ol>
	</li>
	<li>Prepare the ECL mix and add it to the blot according to the manufacturer&#39;s protocol. Incubate the membrane with ECL mix for 5 min at RT.
	<ol>
		<li>Place the membrane in an X-ray cassette, expose the membrane to the X-ray film and develop the blot using the manufacturer&#39;s protocol.</li>
		<li>Quantify the signal obtained for input and ChIPed DNA after scanning the blots using the Image J software according to manufacturer&#39;s protocol. For quantitation, use the signal that is within the linear range of detection in order to ensure accuracy of measurement.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Podhorecka, M., Skladanowski, A., Bozko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2AX+Phosphorylation:+Its+Role+in+DNA+Damage+Response+and+Cancer+Therapy.">H2AX Phosphorylation: Its Role in DNA Damage Response and Cancer Therapy.</a> J nucleic acids. , (2010).</li><li>Groth, A., Rocha, W., Verreault, A., Almouzni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+challenges+during+DNA+replication+and+repair.">Chromatin challenges during DNA replication and repair.</a> 128, 721-733 (2007).</li><li>Demeret, C., Vassetzky, Y., Mechali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+remodelling+and+DNA+replication:+from+nucleosomes+to+loop+domains.">Chromatin remodelling and DNA replication: from nucleosomes to loop domains.</a> Oncogene. 20, 3086-3093 (2001).</li><li>Bhaskara, S., 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=Deletion+of+histone+deacetylase+3+reveals+critical+roles+in+S+phase+progression+and+DNA+damage+control.">Deletion of histone deacetylase 3 reveals critical roles in S phase progression and DNA damage control.</a> Mol Cell. 30, 61-72 (2008).</li><li>Bhaskara, S., Hiebert, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+histone+deacetylase+3+in+maintenance+of+genome+stability.">Role for histone deacetylase 3 in maintenance of genome stability.</a> Cell Cycle. 10, 727-728 (2011).</li><li>Bhaskara, S., 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=Histone+deacetylases+1+and+2+maintain+S-phase+chromatin+and+DNA+replication+fork+progression.">Histone deacetylases 1 and 2 maintain S-phase chromatin and DNA replication fork progression.</a> Epigenetics Chromatin. 6, 27 (2013).</li><li>Bhaskara, S., 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=Hdac3+is+essential+for+the+maintenance+of+chromatin+structure+and+genome+stability.">Hdac3 is essential for the maintenance of chromatin structure and genome stability.</a> Cancer Cell. 18, 436-447 (2010).</li><li>Johnson, D. P., 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=HDAC1,2+inhibition+impairs+EZH2-+and+BBAP-mediated+DNA+repair+to+overcome+chemoresistance+in+EZH2+gain-of-function+mutant+diffuse+large+B-cell+lymphoma.">HDAC1,2 inhibition impairs EZH2- and BBAP-mediated DNA repair to overcome chemoresistance in EZH2 gain-of-function mutant diffuse large B-cell lymphoma.</a> Oncotarget. 6, 4863-4887 (2015).</li><li>Bhaskara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylases+1+and+2+regulate+DNA+replication+and+DNA+repair:+potential+targets+for+genome+stability-mechanism-based+therapeutics+for+a+subset+of+cancers.">Histone deacetylases 1 and 2 regulate DNA replication and DNA repair: potential targets for genome stability-mechanism-based therapeutics for a subset of cancers.</a> Cell Cycle. 14, 1779-1785 (2015).</li><li>Taddei, A., Roche, D., Sibarita, J. B., Turner, B. M., Almouzni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplication+and+maintenance+of+heterochromatin+domains.">Duplication and maintenance of heterochromatin domains.</a> J Cell Biol. 147, 1153-1166 (1999).</li><li>Alabert, C., Groth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+replication+and+epigenome+maintenance.">Chromatin replication and epigenome maintenance.</a> Nat Rev Mol Cell Biol. 13, 153-167 (2012).</li><li>Clapier, C. R., Cairns, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+ISWI+involves+inhibitory+modules+antagonized+by+nucleosomal+epitopes.">Regulation of ISWI involves inhibitory modules antagonized by nucleosomal epitopes.</a> Nature. 492, 280-284 (2012).</li><li>Sirbu, B. M., 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=Analysis+of+protein+dynamics+at+active,+stalled,+and+collapsed+replication+forks.">Analysis of protein dynamics at active, stalled, and collapsed replication forks.</a> Genes Dev. 25, 1320-1327 (2011).</li></ol>

I have no competing financial interests.

The protocol described in this manuscript is a relatively quick method to demonstrate the presence of proteins or their post-translationally modified forms on newly replicated or nascent DNA. Additionally, this technique permits one to measure the association-dissociation kinetics of a protein or its modified form with nascent DNA. This technique is complementary to the elegant iPOND technology13. In the iPOND technology, newly synthesized DNA is labeled with ethyl deoxyuridine (EdU). A biotin conjugate is the...

Defective DNA repair and/or DNA replication are a major cause of genome instability, which can trigger cell death. A single unrepaired double strand break is sufficient to cause cell death1. Chromatin organization is transiently altered during both replication and repair2,3, and failure to maintain epigenetic information during these processes will result in a threat to genome integrity. Loss of HDAC3 or HDAC1,2 function impedes S-phase progression,&#160;DNA replication and repair leading to genotoxic stress (DNA damage) and cell&#160;death4-9. It is therefore a practical strategy to use selective HDAC inhibitors to disrupt replication, repair and chromatin in cancer cells and cause DNA damage, which in turn can stop growth and induce cell death selectively in rapidly growing cancer cells.
During DNA replication, chromatin is rapidly disassembled and then reassembled following DNA duplication. Newly synthesized histone H4 is acetylated at K5 and K12 residues (H4K5K12ac) and following deposition onto chromatin, they are rapidly deacetylated by histone deacetylases (HDACs)10,11. Failure of cells to maintain nascent chromatin integrity by histone deacetylation can lead to fork collapse, which in turn can result in DNA damage and cell death. We recently showed that selective inhibition of HDAC1,2 increases histone acetylation (H4K5ac, H4K12ac and H4K16ac) and inhibits SMARCA5 chromatin remodeler activity on nascent chromatin, which correlates with reduced replication fork progression, increased fork collapse and increased replication stress-induced DNA damage6. Thus, HDAC inhibitor treatment can alter nascent chromatin structure to trigger DNA damage and death very quickly in cancer cells, as these cells cycle rapidly and pass many times through the S-phase. It is therefore important to understand how HDACs function to regulate histone acetylation and protein binding to maintain DNA replication in mammalian cells.
To quantitatively measure the amount of histone acetylation and SMARCA5 chromatin remodeler associated with nascent DNA during DNA replication, we devised a modified ChIP assay called the BrdU-ChIP-Slot-Western technique. Following chromatin immunoprecipitation (ChIP) of a desired protein or histone modification, the amount of nascent DNA (labeled using thymidine analog, BrdU) in the ChIP sample can be detected using western analysis of BrdU-labeled ChIP DNA transferred onto a membrane using a Slot Blot apparatus. Using this technique, we showed that H4K16ac (a mark involved in chromatin packaging) and the ISWI family member SMARCA5 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin) chromatin remodeler are associated with nascent DNA in S-phase cells6. We also found that the nascent H4K16ac mark is deacetylated by HDAC1,2 during DNA replication6. H4K16ac inhibits chromatin remodeler activity of the ISWI family member SMARCA512. Hence, using the BrdU-CHIP-Slot-Western technique, we could connect the functions for HDAC1,2 in regulating chromatin remodeling during DNA replication. Hence, the BrdU-ChIP-Slot-Western Blot technique is a powerful approach to quantitatively measure the association and dissociation dynamics of proteins or their post-translational modifications that are bound to nascent DNA.

<table><tbody><tr><td>Anti-BrdU (westerns)</td><td>BD Biosciences</td><td>B555627</td><td></td></tr><tr><td>Anti-SMARCA5</td><td>Abcam</td><td>ab3749</td><td></td></tr><tr><td>Anti-H4K16ac</td><td>Active Motif</td><td>39167</td><td></td></tr><tr><td>Zeta-Probe GT Membrane</td><td>Bio-Rad</td><td>162-0197</td><td></td></tr><tr><td>Formaldehyde</td><td>Fisher</td><td>BP531-500</td><td></td></tr><tr><td>Protein A agarose</td><td>Millipore</td><td>16-156</td><td></td></tr><tr><td>Rnase A</td><td>Qiagen</td><td>19101</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma</td><td>P4850</td><td></td></tr><tr><td>PCR Purification Kit</td><td>Qiagen</td><td>28106</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>G7403</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Roche</td><td>11836170001</td><td></td></tr><tr><td>BrdU</td><td>Sigma</td><td>B9285</td><td></td></tr><tr><td>Rabbit IgG&amp;nbsp;</td><td>Millipore</td><td>12-370</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S-3014</td><td></td></tr><tr><td>Triton-X-100</td><td>Sigma</td><td>93443</td><td></td></tr><tr><td>NP40</td><td>USB Corporation</td><td>19628</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma</td><td>D6750</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S-4019</td><td></td></tr><tr><td>Ethanol</td><td>Decon Laboratories</td><td>04-355-222</td><td></td></tr><tr><td>DEPC-treated water</td><td>Sigma</td><td>95284</td><td></td></tr><tr><td>Tris</td><td>Fisher</td><td>BP-152-5</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen</td><td>15575-020</td><td></td></tr><tr><td>SDS</td><td>Ambion</td><td>AM9820</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Mallinckrodt GenAR&amp;nbsp;</td><td>MAL7772-06</td><td></td></tr><tr><td>20x SSC</td><td>Life Technologies</td><td>15557-036</td><td></td></tr><tr><td>Non-fat dry milk</td><td>Lab Scientific Inc</td><td>M0841</td><td></td></tr><tr><td>ECL</td><td>Thermo Fisher</td><td>80196</td><td></td></tr><tr><td>X-ray film</td><td>Genesee Sci</td><td>30-101</td><td></td></tr><tr><td>Developer</td><td>Konica Minolta</td><td>SRX-101A</td><td></td></tr><tr><td>UV Crosslinker</td><td>Stratagene</td><td>XLE-1000</td><td></td></tr><tr><td>Sonicator</td><td>Branson Sonifier&amp;nbsp; Digital Ultrasonic Cell Disruptor</td><td>Model: 450</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>Model: 5810R</td><td></td></tr><tr><td>Water bath</td><td>Fisher Scientific Isotemp</td><td>Model: 2322</td><td></td></tr><tr><td>NanoDrop 1000 spectrophotometer</td><td>ThermoScientific</td><td>Model: 1000</td><td></td></tr><tr><td>Slot Blot apparatus&amp;nbsp;</td><td>Schleicher and Schuell Minifold II</td><td>44-27570</td><td></td></tr><tr><td>Tissue Culture Incubator</td><td>Thermo Scientific</td><td>Series II 3110 Water-Jacketed CO2 Incubators</td><td></td></tr></tbody></table>

examination of proteins bound to nascent dna in mammalian cells using  brdu-chip-slot-western technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

To determine the specificity of HDAC1,2-selective inhibitors, Hdac1,2FL/FL and Hdac3FL/FL fibrosarcoma cells were used. Adenovirus-containing Cre recombinase (Ad-Cre) was used to delete Hdac1,2 and Hdac3 in these cells. Following Ad-Cre infection of Hdac1,2FL/FL cells, a robust increase in H4K5ac was observed. Treatment of Hdac1,2&#160;knockout cells with 233 or 898 did not result in...

Watch this Scientific Journal Video about Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique at JoVE.com

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

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9.1K Views.  University of Utah School of Medicine. In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA. 

Video: Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Genetics

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed.
To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. 
This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying ...

Biochemistry

Measuring Ubiquitylated, SUMOylated, and ADP-Ribosylated DNA-Protein Crosslinks

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

Author Spotlight: Quantitative Detection of DNA Protein Crosslinks and Their Post-Translational Modifications  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects.

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs for cancer treatment. These targets are poly(ADP-ribose) polymerases (PARPs), which initiate various forms of DNA repair. Inhibiting these enzymes using PARP inhibitors (PARPi) achieves synthetic lethality by conferring a therapeutic vulnerability in homologous recombination (HR)-deficient cells due to mutations in breast cancer type 1 (BRCA1), BRCA2, or partner and localizer of BRCA2 (PALB2). 
Cells treated with PARPi accumulate DNA double-strand breaks (DSBs). These breaks are processed by the DNA end resection machinery, leading to the formation of single-stranded (ss) DNA and subsequent DNA repair. In a BRCA1-deficient context, reinvigorating DNA resection through mutations in DNA resection inhibitors, such as 53BP1 and DYNLL1, causes PARPi resistance. Therefore, being able to monitor DNA resection in cellulo is critical for a clearer understanding of the DNA repair pathways and the development of new strategies to overcome PARPi resistance. Immunofluorescence (IF)-based techniques allow for monitoring of global DNA resection after DNA damage. This strategy requires long-pulse genomic DNA labeling with 5-bromo-2&#8242;-deoxyuridine (BrdU). Following DNA damage and DNA end resection, the resulting single-stranded DNA is specifically detected by an anti-BrdU antibody under native conditions. Moreover, DNA resection can also be studied using cell cycle markers to differentiate between various phases of the cell cycle. Cells in the S/G2 phase allow the study of end resection within HR, whereas G1 cells can be used to study non-homologous end joining (NHEJ). A detailed protocol for this IF method coupled to cell cycle discrimination is described in this paper.

In the present protocol, we demonstrate how to visualize DNA double-strand end resection during S/G2 phase of the cell cycle using an immunofluorescence-based method.

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs ...

Detection of G1 Phase Single-Stranded DNA Foci via RPA2 Immunofluorescent Staining

Visualizing Single-Stranded DNA Foci in the G1 Phase of the Cell Cycle

DNA has dedicated cellular repair pathways capable of coping with lesions that could arise from both endogenous and/or exogenous sources. DNA repair necessitates collaboration between numerous proteins, responsible for covering a broad range of tasks from recognizing and signaling the presence of a DNA lesion to physically repairing it. During this process, tracks of single-stranded DNA (ssDNA) are often created, which are eventually filled by DNA polymerases. The nature of these ssDNA tracks (in terms of both length and number), along with the polymerase recruited to fill these gaps, are repair pathway-specific. The visualization of these ssDNA tracks can help us understand the complicated dynamics of DNA repair mechanisms.
This protocol provides a detailed method for the preparation of G1 synchronized cells to measure ssDNA foci formation upon genotoxic stress. Using an easy-to-utilize immunofluorescence approach, we visualize ssDNA by staining for RPA2, a component of the heterotrimeric replication protein A complex (RPA). RPA2 binds to and stabilizes ssDNA intermediates that arise upon genotoxic stress or replication to control DNA repair and DNA damage checkpoint activation. 5-Ethynyl-2'-deoxyuridine (EdU) staining is used to visualize DNA replication to exclude any S phase cells. This protocol provides an alternative approach to the conventional, non-denaturing 5-bromo-2'-deoxyuridine (BrdU)-based assays and is better suited for the detection of ssDNA foci outside the S phase.

Author Spotlight: Visualizing Single-Stranded DNA During DNA Repair for Therapeutic Insights 

The following protocol presents the detection of single-stranded DNA foci in the G1 phase of the cell cycle utilizing cell cycle synchronization followed by RPA2 immunofluorescent staining.

DNA has dedicated cellular repair pathways capable of coping with lesions that could arise from both endogenous and/or exogenous sources. DNA repair ...

Detection of Nuclear Blebbing and DNA Leakage in Mammalian Cells by Immunofluorescence

The nuclear lamina is a network of filaments underlying the nuclear membrane, composed of lamins and lamin-associated proteins. It plays critical roles in nuclear architecture, nuclear pore positioning, gene expression regulation, chromatin organization, DNA replication, and DNA repair. Mutations in genes involved in the expression or post-translational processing of lamin proteins result in genetic disorders known as laminopathies. Specifically, mutations in the LMNA or ZMPSTE24 genes can lead to the accumulation of incompletely processed forms of lamin A that retain farnesyl and methyl groups, which are absent in fully processed lamin A. These incompletely processed lamin A proteins localize to the inner nuclear membrane instead of the nuclear lamina, where mature lamin A resides. Mislocalized lamin proteins profoundly disrupt nuclear function and structure, often resulting in nuclear blebbing. In severe cases, nuclear rupture can occur, causing a loss of compartmentalization and leakage of genomic DNA into the cytosol. Abnormal nuclear structure and compartmentalization loss can be identified through indirect immunofluorescence (IF) on fixed cells. This study outlines such a method, employing specific antibodies against a lamin protein and double-stranded DNA (dsDNA) to simultaneously visualize the nuclear envelope and DNA. This approach enables a rapid assessment of nuclear structural integrity and the potential leakage of nuclear DNA into the cytosol.

Laminopathy disorders often lead to changes in the nuclear envelope, which may cause nuclear blebbing and leakage. This study presents an immunofluorescence method to visualize the nuclear lamina along with double-stranded DNA (dsDNA), providing a means to assess nuclear structure and integrity in mammalian cells.

The nuclear lamina is a network of filaments underlying the nuclear membrane, composed of lamins and lamin-associated proteins. It plays critical roles in ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique

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Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

Analysis of Cell Cycle Position in Mammalian Cells

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

In Vitro Analysis of E3 Ubiquitin Ligase Function

Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging

Visualizing Single-Stranded DNA Foci in the G1 Phase of the Cell Cycle

Detection of Nuclear Blebbing and DNA Leakage in Mammalian Cells by Immunofluorescence