Name: tools to study the role of architectural protein hmgb1 in the processing of helix distorting, site-specific dna interstrand crosslinks
Uploaded: 2016-11-10T13:00:00.000+00:00
Duration: 12 min 19 s
Description: Watch this Scientific Journal Video about Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks at JoVE.com

The authors would like to thank the members of Vasquez laboratory for helpful discussions. This work was supported by the National Institutes of Health/National Cancer Institute [CA097175, CA093279 to K.M.V.]; and the Cancer Prevention and Research Institute of Texas [RP101501]. Funding for open access charge: National Institutes of Health/National Cancer Institute [CA093279 to K.M.V.].

1. Preparation of TFO-directed ICLs on Plasmid Substrates
<ol>
	<li>Incubate equimolar amounts of plasmid pSupFG110,11 DNA (5 &#181;g plasmid DNA is a good starting point) with psoralen-conjugated TFO AG30 in 8 &#181;l of triplex binding buffer [50% glycerol, 10 mM Tris (pH 7.6), 10 mM MgCl2] and dH2O to a final volume of 40 &#181;l in an amber tube. Mix thoroughly by pipetting up and down. Incubate the reaction at 37 &#176;C water bath for 12 hr.</li>
	<li>Warm up the UVA lamp to ensure full power output. Place the triplex reaction on paraffin film, and place the paraffin film on ice under the UVA (365 nM) lamp. Place a Mylar filter between the lamp and the reaction.</li>
	<li>UVA irradiate for a total dose of 1.8 J/cm2. Store the samples at 4 &#176;C for further use. 
	Caution: Wear proper protective eyewear and clothing with UVA irradiation.</li>
	<li>Linearize 200 ng of the above prepared plasmid with the restriction enzyme EcoR1 in a 20 &#181;l total volume using the manufacturer supplied 10x buffer. Heat denature the enzyme for 20 min at 65 &#176;C. Spin the samples for 10 min at 10,000 x g and store at 4 &#176;C.</li>
	<li>Prepare 50x alkaline buffer [1.5 M NaOH, 50 mM Ethylenediaminetetraacetic acid (EDTA)]. Add 0.5 mg of agarose to 50 ml dH2O. Boil to dissolve the agarose and place it in a 50 &#176;C water bath.</li>
	<li>After the temperature of the dissolved agarose is down to 50 &#176;C, add 1 ml of the 50x alkaline buffer, mix it and then pour the gel into the gel tray. Allow time for the gel to solidify at room temperature prior to use.</li>
	<li>Add 4 &#181;l of 0.5 M EDTA to the 20 &#181;l of linearized DNA sample and incubate for 10 min at room temperature.</li>
	<li>Add 6x alkaline gel loading buffer (300 mM NaOH, 6 mM EDTA, 18% (w/v) high molecular weight polysaccharide, 0.06% Bromocresol Green in dH2O) to the samples and to a 1 kb DNA ladder. 
	NOTE: It is important to use 6x alkaline gel loading buffer, please see discussion.</li>
	<li>Load samples onto the gel in the cold room and run overnight in 1x alkaline buffer (12-16 hr) at 3.2 volts per cm. Once the samples have entered the gel, place a glass plate on top of the gel to prevent it from floating.</li>
	<li>Neutralize the samples by soaking the gel in neutralization buffer [1 M Tris-Cl (pH 7.6), 1.5 M NaCl, 3 M sodium acetate (pH 5.2)] for 45 min at room temperature.</li>
	<li>Stain the gel for DNA with 4 &#181;l of 1% ethidium bromide (EtBr) in 50 ml water for 1 hr at room temperature. Destain with water for 15 min. Visualize the DNA by using an imaging system. 
	Caution: EtBr is a potent mutagen and can be absorbed through the skin. Avoid direct contact with EtBr.</li>
</ol>
2. Transfection and Immunoprecipitation of TFO-directed ICL-containing Plasmids in Human Cells
<ol>
	<li>Plate 400,000 mammalian cells (e.g., U2OS osteosarcoma cells) per 60 mm dish in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) without antibiotics 24 hr before transfection. Incubate cells overnight at 37 &#176;C with 5% CO2.</li>
	<li>Prior to transfection, warm growth media and phosphate buffered saline (PBS) to 37 &#176;C in a water bath. Warm the transfection reagent to room temperature.</li>
	<li>Incubate 30 &#181;l of transfection reagent with 500 &#181;l of 1x growth media (mix 1) and 2 &#181;g of TFO-directed ICL-containing plasmids in 500 &#181;l of 1x growth media (mix 2) in separate 14 ml round bottom tubes for 10 min at room temperature.</li>
	<li>Add mix 1 to mix 2, mix well by pipetting up and down. Incubate for 25-30 min at room temperature.</li>
	<li>Wash cells twice with warm PBS. Add the transfection mix in a drop-wise fashion distributing evenly throughout the plate of cells. Add 1 ml of growth media to the plate and bring the final volume to 2 ml. Mix well. Place the culture plates in the 37 &#176;C incubator with 5% CO2 for 4 hr.</li>
	<li>Replace the media with 3 ml growth media supplemented with 10% FBS, and incubate further for 16 hr. Do not use antibiotics.</li>
	<li>Treat cells with 80 &#181;l of 37% fresh formaldehyde per plate to a final concentration of approximately 1% and incubate for 10 min at room temperature in the absence of light. 
	Caution: Formaldehyde is toxic and should be handled according to its safety instructions. Formaldehyde exposure can cause harm to skin, eye and respiratory tract.</li>
	<li>Quench formaldehyde crosslinking by adding 300 &#181;l of chilled glycine (supplied in commercially available kits) per plate and incubating for 5 min. Remove the media and wash twice with chilled PBS, and then collect cells by scraping in 1 ml chilled (4 &#176;C) PBS into a microcentrifuge tube. Keep the samples on ice at all times.</li>
	<li>Prepare cell pellets by centrifuging at 4 &#176;C for 5 min at 13,400 x g using a tabletop refrigerated centrifuge. Pipet out the supernatant and place the cell pellet on ice.</li>
	<li>Homogeneously resuspend the pellet in 1 ml chilled (4 &#176;C) Buffer A (supplied in commercially available kits) and incubate on ice for 10 min. Mix occasionally by inverting the tube. Pellet cells as before (see step 2.9 above).</li>
	<li>Homogeneously resuspend the pellet in 1 ml chilled (4 &#176;C) Buffer B (supplied in commercially available kits) and incubate on ice for 10 min with occasional mixing by inverting. Pellet cells by centrifugation as before (see step 2.9 above).</li>
	<li>Resuspend the cells again in 200 &#181;l chilled Buffer B. Add 2 &#181;l of Micrococcal nuclease (MNase) and incubate at room temperature for 10 min. Mix reaction occasionally by flicking the tube. Stop the MNase reaction by placing the tube on ice and adding 40 mM EDTA. Pellet cells as before (see step 2.9 above).</li>
	<li>Resuspend cells in 100 &#181;l 1x chromatin immunoprecipitation buffer (ChIP) buffer (supplied in commercially available kits) supplemented with protease inhibitor cocktail.</li>
	<li>Place the resuspended cells in a thin-walled microfuge tube. Sonicate the samples by floating them on water bath sonicator for 20 sec followed by 20 sec incubation on ice. Repeat sonication steps 9 times to generate ~800-1,200 bp fragments.
	<ol>
		<li>To 20 &#181;l of the sonicated samples add 3 &#181;l 5 M NaCl and 1 &#181;l RNase A. Mix well by vortexing and incubate at 37 &#176;C for 30 min. Subsequently, add 2 &#181;l Proteinase K and incubate for 2 hr at 65 &#176;C. Purify the samples using a PCR purification kit. Run samples on 1% agarose gel and visualize with EtBr. 
		NOTE: Maximum intensity of the resulting DNA should be at or under 1,000 bp.</li>
	</ol>
	</li>
	<li>In three separate tubes, add 30 &#181;l of lysate in a pre-chilled siliconized tube and then add 70 &#181;l of chilled 1x ChIP buffer with added protease inhibitors. Add 1 &#181;g of anti-immunoglobulin G (IgG), anti-histone H3 (as a positive control), or anti-HMGB1 antibodies to the respective tubes. Incubate overnight in a cold room with continuous rotation.</li>
	<li>Resuspend protein G beads homogeneously in the cold room. Add 4 &#181;l of protein G beads per tube and incubate for another 2-4 hr in the cold room with rotation.</li>
	<li>Using a magnetic rack, pellet the beads and remove the supernatant. Add 200 &#181;l 1x ChIP buffer mixed with protease inhibitor cocktail to the pellet and wash for 5 min in the cold room with rotation. Repeat wash twice.</li>
	<li>Perform one additional wash with high salt 1x ChIP buffer (containing 70 mM NaCl).</li>
	<li>Resuspend the pellets with 160 &#181;l elution buffer (supplied in commercially available kits) and incubate at 37 &#176;C with rotation for 30 min.</li>
	<li>Pellet the beads and collect the supernatant in a fresh tube. Add 6 &#181;l of 5 M NaCl and 2 &#181;l of proteinase K, and incubate overnight at 65 &#176;C.</li>
	<li>Purify the DNA using a PCR product purification kit and elute the DNA using 50 &#181;l dH2O.</li>
	<li>Perform PCR16 reactions using primer sets to the region(s) of interest, and resolve the products on a 1% agarose gel stained with EtBr.</li>
</ol>
3. Supercoiling Assay and 2-dimensional Agarose Gel Electrophoresis
<ol>
	<li>Prepare DNA repair synthesis buffer (45 mM Hepes-KOH, pH 7.8; 70 mM KCl; 7.4 mM MgCl2; 0.9 mM DTT; 0.4 mM EDTA; 2 mM ATP, 20 &#181;M dNTPs, 3.4% glycerol and 18 &#181;g bovine serum albumin). Store at -80 &#176;C. Thaw on ice and prior to use add 40 mM phosphocreatine and 2.5 &#181;g creatine phosphokinase.</li>
	<li>Prepare 10x supercoiling buffer [500 mM Tris (pH 8.0), 500 mM NaCl, 25 mM MgCl2, 1 mM EDTA], and store at room temperature.</li>
	<li>Linearize 300 ng of supercoiled plasmid pSupFG1 DNA completely using 1 &#181;l Vaccinia Topoisomerase I (5-15 U/&#181;l) with 1x supercoiling buffer in a 10 &#181;l reaction. Incubate the mixture at 37 &#176;C for 1 hr.</li>
	<li>Thaw HeLa cell-free extract24 on ice. To the completely relaxed plasmids, add 25 &#181;l HeLa cell-free extract and DNA repair synthesis buffer. Mix well. Add additional 1 &#181;l of Vaccinia Topoisomerase I to the mix, and incubate for 1 hr at 37 &#176;C.</li>
	<li>Terminate the reactions by adding sodium dodecyl sulfate (SDS) (1% final concentration) and proteinase K (0.25 mg/ml final concentration). Incubate at 65 &#176;C for 2 hr.</li>
	<li>Cast a 1% agarose gel with 1x Tris Borate EDTA (TBE) buffer. Add DNA loading dye and load the reactions directly onto the gel at least 4 lanes apart. Run the gel for 8-10 hr at 2 V/cm in 1x TBE (dimension 1) at room temperature to resolve the topoisomers. 
	NOTE: Prepare a 5x TBE stock solution in 1 L of dH2O by adding 54 g of Tris base. 27.5 g of boric acid and 20 ml of 0.5 M EDTA (pH 8.0).</li>
	<li>Prepare 2 L of 1x TBE with 3 &#181;g/ml chloroquine-phosphate. Soak the gel in 200 ml of this solution for 20 min. Cover the gel tray with aluminum foil.</li>
	<li>To electrophorese the gel in the second dimension, turn the gel 90&#176; in a clockwise fashion and run it for 4-6 hr at 2 V/cm in chloroquine containing 1x TBE to resolve the positive and negative supercoils.</li>
	<li>Stain the gel with EtBr for 1 hr on a rocker. Destain the gel with dH2O for 15 min and subsequently visualize the thermal distribution of the topoisomers using an imager.</li>
</ol>

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The authors declare no conflict of interest.

The efficient formation of TFO-directed ICLs is dependent upon two crucial factors: First, proper buffer components (e.g., MgCl2) and time of incubation of the TFO with its target DNA substrate (dependent upon the binding affinity of the TFO to its target duplex, and the concentrations used); and second, the proper dose of UVA (365 nm) irradiation to form psoralen crosslinks efficiently. Optimal triplex formation can be achieved by using HPLC or gel purified TFOs. Impurities contaminating the TFO may lead to unwanted crosslinked products, which can further complicate the experimental outcome. To increase the stability of the TFOs covalently attached to a 5&#39;-HMT psoralen, the TFOs should be stored in aliquots at -80 &#176;C, as multiple freeze thaw of the TFOs may result in degradation of the oligonucleotides. After triplex formation to target the psoralen to a specific site (e.g., 5&#39;-TA-3&#39; and 5&#39;-AT-3&#39; are the preferred psoralen crosslinking sites), psoralen ICL formation requires irradiation with UVA. A dose of 1.8 J/cm2 UVA is sufficient for optimal formation of ICLs on plasmids in vitro. The exposure time should be determined by using a photometer. Depending on the UVA lamp source, in addition to emitting UV at 365 nm, the lamp may also emit shorter wavelengths, which may lead to formation of unintended DNA damage throughout the DNA backbone. Because the TFO-directed ICL-containing plasmids will be used for studies related to DNA repair of an ICL at a specific site, such unintended photoproducts may confound the outcome of the DNA repair studies. Therefore, a Mylar filter should be used to prevent exposure of the samples to shorter wavelengths. Successful ICL formation also depends on the stability of the triplex structure during UVA irradiation. The time of UV irradiation required depends on the wattage of the UVA source. In our case, 20-30 min is required to generate the desired dose of UVA. During this time, heat generated by the lamp may cause evaporative water loss from the samples. Such water loss may alter the buffer concentration and may cause destabilization of the triplex structures. Placing the reactions on ice during UVA irradiation will help to prevent evaporation of the samples.
In order to determine the efficiency of psoralen crosslink formation on the plasmid substrates, the samples were resolved in 1% alkaline agarose gels (Figure 1). Most protocols for alkaline gel electrophoresis suggest a 2x alkaline gel loading dye, although some protocols suggest that alkaline loading dye is not necessary, as the denaturing buffer condition of the gel is sufficient to denature the samples. While a 2x dye works well with concentrated DNA samples, this protocol requires loading a large sample volume. Therefore, use a 6x alkaline gel loading dye. To ensure proper denaturation of the psoralen crosslinked plasmid substrates, incubate the samples in the loading buffer for 10 min at room temperature. In addition, Mg2+ can form Mg(OH)2 under alkaline conditions, which entraps the DNA and can cause it to precipitate. The triplex-forming reaction buffer contains Mg2+, and therefore it is important to incubate the samples with EDTA to ensure that the Mg2+ is chelated prior to adding the alkaline loading dye to the samples. After electrophoresis, it is important to neutralize the gel, as EtBr does not intercalate with the DNA under alkaline conditions.
An alternative approach to assess the efficiency of the TFO-directed crosslink formation is the use of denaturing polyacrylamide gel electrophoresis, but this technique is more laborious, and requires restriction digestion of the plasmids followed by radioactive isotope labeling of the fragments. This current approach demonstrates a relatively simple, yet effective method to determine the efficiency of TFO-directed ICL formation on plasmid substrates. However, this technique can be applied to assess the formation of ICLs by other crosslink-forming agents as well.
Successful outcome of the plasmid ChIP assay depends on a number of factors. The critical steps within the protocol and troubleshooting suggestions are discussed here. Critical steps involved in this assay, include formaldehyde crosslinking, micrococcal nuclease digestion, sonication, sample washes, and reversal of the formaldehyde protein-DNA crosslinks in the samples. For efficient protein-DNA crosslinking, it is important to use fresh formaldehyde. Stock bottles of 37% formaldehyde should not be used for more than six months after opening, as exposure to light and oxygen degrades formaldehyde. Therefore, it is also beneficial to perform the formaldehyde crosslinking without light in the cell culture hood. Another important step is micrococcal nuclease (MNase) digestion of the samples. MNase digestion not only cleaves the genomic DNA into smaller fragments but also removes any lingering plasmids that were not delivered into the cells during the transfection method, which can dramatically reduce the background. The incubation time of MNase digestion should be empirically determined. Longer digestion may result in loss of samples, whereas shorter incubation periods may lead to inefficient removal of unwanted DNA. Excellent results can be seen after 10 min incubation at room temperature with occasional mixing of the reactions by inverting the tubes. For efficient immunoprecipitation, the DNA should be fragmented to sizes between 800-1,200 bp. Such fragmentation of DNA can be achieved by thorough sonication of the samples. Efficient sonication of samples can be achieved by re-suspending the pellets in thin-walled PCR tubes and subsequently placing them in a water bath sonicator. Sonic pulses are more effective compared to continuous sonication. In addition, it is important to place the samples on ice between pulses to prevent protein degradation, and it is also important to supplement the 1x ChIP buffer with protease inhibitors, as stable proteins are necessary for the antibody recognition and subsequent precipitation steps. However, during the immunoprecipitation process, due to the hydrophobic nature of the magnetic beads, non-specific DNA will be pulled down. To reduce signals from such non-specific templates, rigorous washing is absolutely necessary. Lastly, proper reversal of the protein-DNA crosslinks of the samples is necessary for PCR amplification of the immunoprecipitated templates. To achieve optimal results, samples should be incubated for at least 12 hr with SDS and proteinase K. There are benefits of using a plasmid-based ChIP assay over genomic ChIP assays. For example, targeted DNA damage can be easily achieved using a plasmid DNA substrate and a TFO, compared to TFO-targeted lesion formation in a genomic locus. The amount of substrate can also be controlled to modulate the intensity of signals when using a TFO-targeted damaged plasmid. Further, such substrates can be used in any cell line of choice and tested for association with various candidate proteins, thus extending the scope of studies on processing and repair of ICLs.
The supercoiling assay is based on the difference in shape between linear and supercoiled plasmid DNA. Supercoiled DNA plasmids are more compact and migrate faster through the gel matrix compared to relaxed plasmids. The critical steps within the protocol and troubleshooting suggestions are discussed here. Because architectural proteins facilitate topological modifications on damaged DNA substrates, measured through induction of supercoiling of the relaxed plasmids, it is critical to completely relax the plasmids with Topoisomerase I. It is necessary to examine the extent of DNA relaxation caused by Topoisomerase I via agarose gel electrophoresis. The MgCl2 present in the supercoiling buffer can precipitate over time, and the presence of MgCl2 affects the thermal distribution of the positive and negative supercoils by facilitating the formation of positive supercoils. Therefore, it is beneficial to add MgCl2 separately to the mix or prepare fresh buffer each time. To separate the topoisomers of the plasmid populations, it is important to perform the gel electrophoresis at a very low voltage (~2 V/cm) so that migration of the DNA is predominantly based on the structure/shape of the molecules. If relaxation of the plasmids by Topoisomerase I is not complete, then the incubation time and/or the amount of enzyme used should be increased. Complete relaxation has to be ensured before moving on to the subsequent DNA supercoiling step because use of the supercoiling assay with incompletely relaxed plasmids may lead to misinterpretation of the data and the results may be difficult to replicate. Once complete relaxation has been achieved, the supercoiling assay is relatively easy to perform. One important component of this assay is the use of a DNA repair synthesis buffer. The phosphocreatine and the creatine phosphokinase are required to be added to the mix each time to generate ATP. Subsequent to the supercoiling step, it is important to remove proteins from the plasmid DNA by incubating with SDS and proteinase K. SDS assists in dissociating the proteins from the DNA and proteinase K degrades the protein, which leaves the DNA intact with various levels of supercoiling. If the protein is not removed completely, the thermal distribution of the topoisomers will not be clearly visible. The DNA cannot be purified by phenol:chloroform:isoamyl alcohol and ethanol precipitated because this method (and others) can nick the DNA, resulting in the loss of supercoiling. Another important step is addition of chloroquine to the buffer. Chloroquine is an intercalating agent and unwinds the DNA. Upon intercalation, it relaxes the negatively supercoiled DNA and introduces positive supercoiling to relaxed DNA which facilitates separation of the topoisomers containing a high density of supercoiling. Thus positive and negative supercoils can be differentiated. This is generally a facile experiment and is highly reproducible when all points above are considered.
However, if no supercoiling is obvious, then it is necessary to determine the activity of the protein of interest. The 2D supercoiling assay has been used to study topological modifications facilitated by architectural proteins27. This assay has been used here in the context of DNA damage processing of TFO-directed ICLs in human cells. HMGB1 binds to TFO-directed ICLs with high affinity and specificity, as previously demonstrated via in vitro gel electrophoretic mobility-shift assays20 and in human cells using the plasmid ChIP assay described within16. Here, using the 2D supercoiling assay it has been demonstrated that upon binding to the TFO-directed ICLs in HeLa cell-free extracts, HMGB1 assists in the formation of negative supercoiling on the substrates (Figure 3), which may be an important step in repair of the lesion because negative supercoiling is known to facilitate the removal of DNA damage via the NER pathway25. There are many architectural proteins that have been implicated in DNA damage processing that could be studied using this assay. This approach is limited to in vitro studies, nevertheless, it is a simple and useful assay to interrogate the structure-function relationship of architectural proteins in the context of DNA damage repair. However, the ChIP protocol can be easily modified to study DNA-protein interactions in the context of the genome, while assessing the effects on DNA supercoiling by such interactions may prove to be quite difficult. We and other groups have indeed performed triplex-based studies 1) by stable transfection of the plasmid DNA into the genome28-30 2) by targeting a genomic locus31-33 and 3) by incorporating the psoralen-modified TFO (and UVA irradiation) into cells after plasmid transfection10.

Triplex-forming oligonucleotides (TFOs) bind duplex DNA in a sequence-specific fashion via Hoogsteen-hydrogen bonding to form triple-helical structures1-5. Triplex technology has been used to interrogate a variety of biomolecular mechanisms, such as transcription, DNA damage repair, and gene targeting (reviewed in references6-8). TFOs have been used extensively to induce site-specific damage on reporter plasmids9,10. Our lab and others have previously used a TFO, AG30, tethered to a psoralen molecule to induce site-specific DNA interstrand crosslinks (ICLs) in the supF gene on the plasmid pSupFG15,10-12. ICLs are highly cytotoxic as these lesions covalently crosslink the two DNA strands, and if left unrepaired, can block gene transcription and impede the DNA replication machinery13,14. Because of their cytotoxic potential, ICL-inducing agents have been used as chemotherapeutic drugs in the treatment of cancer and other diseases15. However, the processing and repair of ICLs in human cells is not well understood. Thus, a better understanding of the mechanisms involved in the processing of ICLs in human cells may help to improve the efficacy of ICL-based chemotherapeutic regimens. TFO-induced ICLs and their repair intermediates have the potential to cause significant structural distortions to the DNA helix. Such distortions are probable targets for architectural proteins, which bind to distorted DNA with higher affinity than to canonical B-form duplex DNA16-20. Here, we studied the association of a highly abundant architectural protein, HMGB1 with ICLs in human cells via chromatin immunoprecipitation (ChIP) assays on psoralen-crosslinked plasmids and identified a role for HMGB1 in modulating the topology of the psoralen-crosslinked plasmid DNA in human cancer cell lysates.
HMGB1 is a highly abundant and ubiquitously expressed non-histone architectural protein that binds to damaged DNA and alternatively structured DNA substrates with higher affinity than canonical B-form DNA17-20. HMGB1 is involved in several DNA metabolic processes, such as transcription, DNA replication, and DNA repair16,21-23. We have previously demonstrated that HMGB1 binds to TFO-directed ICLs in vitro with high affinity20. Further, we have demonstrated that lack of HMGB1 increased the mutagenic processing of TFO-directed ICLs and identified HMGB1 as a nucleotide excision repair (NER) co-factor23,24. Recently, we have found that HMGB1 is associated with TFO-directed ICLs in human cells and its recruitment to such lesions is dependent upon the NER protein, XPA16. Negative supercoiling of DNA has been shown to promote the efficient removal of DNA lesions by NER25, and we have found that HMGB1 induces negative supercoiling preferentially on TFO-directed ICL-containing plasmid substrates (relative to non-damaged plasmids substrates)16, providing a better understanding of the potential role(s) of HMGB1 as an NER co-factor. The processing of ICLs is not fully understood in human cells; thus, the techniques and assays developed based on the molecular tools described herein could lead to the identification of additional proteins involved in ICL repair, which in turn may serve as pharmacological targets that can be exploited to improve the efficacy of cancer chemotherapy regimens.
Here, an effective approach to assess the efficiency of TFO-directed ICL formation in plasmid DNA by denaturing agarose gel electrophoresis has been discussed. Further, using the plasmids containing the TFO-directed ICLs, techniques to determine the association of HMGB1 with ICL-damaged plasmids in a cellular context using modified ChIP assays have been described. Additionally, a facile method to study topological modifications introduced by the architectural protein HMGB1, specifically on ICL-damaged plasmid substrates in human cell lysates has been determined by performing supercoiling assays via two-dimensional agarose gel electrophoresis. The techniques described can be used to further the understanding of the involvement of DNA repair and architectural proteins in the processing of targeted DNA damage on plasmids in human cells.
We describe detailed protocols for the formation of TFO-directed site-specific psoralen ICLs on plasmid DNA, and subsequent plasmid ChIP and supercoiling assays to identify proteins that associate with the lesions, and proteins that alter the DNA topology, respectively. These assays can be modified to perform with other DNA damaging agents, TFOs, plasmid substrates, and mammalian cell lines of interest. In fact, we have shown that there is at least one potential unique and high affinity TFO-binding site within every annotated gene in the human genome26. However, for clarity, we described these techniques for the use of a specific psoralen-conjugated TFO (pAG30) on a specific mutation-reporter plasmid (pSupFG1) in human U2OS cells as we have utilized in Mukherjee &#38; Vasquez, 201616.

<table><tbody><tr><td>5&#039;HMT Psoralen-AG30</td><td>Midland Certified Reagent Co., Midland, TX</td><td>Custom order</td><td>HPLC (or gel) purified</td></tr><tr><td>Simple ChIP Enzymatic Chromatin IP Kit</td><td>Cell signaling Inc.</td><td>9003</td><td>Manufacturer suggested protocol is optimized for chromatin IP and not for plasmid IP. The control IgG and H3 antibodies as well as the Protein G beads and micrococcal nuclease are supplied by the manufacturer.</td></tr><tr><td>GoTaq Green</td><td>Promega</td><td>M7122</td><td>PCR master mix</td></tr><tr><td>HMGB1 antibody</td><td>Abcam</td><td>Ab18256</td><td></td></tr><tr><td>Wizard Gel and PCR cleanup kit</td><td>Promega</td><td>A9281</td><td></td></tr><tr><td>Chloroquine-diphosphate salt</td><td>Sigma</td><td>C6628-50G</td><td>For two-dimensional agarose gel electrophoresis</td></tr><tr><td>EcoRI</td><td>New England BioLabs</td><td>R0101S</td><td></td></tr><tr><td>GenePORTER transfection reagent</td><td>Genlantis</td><td>T201015</td><td></td></tr><tr><td>Vaccinia Topoisomerase I</td><td>Invitrogen</td><td>38042-024</td><td></td></tr><tr><td>Blak-Ray long wave ultraviolet lamp</td><td>Upland, CA</td><td>B 100 AP</td><td>UVA lamp</td></tr><tr><td>EpiSonic Multi-Functional Bioprocessor 1100</td><td>Epigentek</td><td>EQC-1100</td><td>Water bath sonicator</td></tr><tr><td>Mylar filter</td><td>GE Healthcare Life Sciences</td><td>80112939</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A6111</td><td></td></tr><tr><td>BIO-RAD Chemidoc</td><td>BIO-RAD</td><td>XRS+ system</td><td>DNA imaging system</td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP381-500</td><td></td></tr><tr><td>37% Formaldehyde</td><td>Sigma-Aldrich</td><td>F8775-25ML</td><td>Used for crosslinking&amp;nbsp;</td></tr><tr><td>Proteinase K</td><td>New England Biolabs</td><td>P8107S</td><td></td></tr><tr><td>DMEM</td><td>ThermoFisher</td><td>11965092</td><td>Cell culture media</td></tr><tr><td>PBS</td><td>ThermoFisher</td><td>70013073</td><td>Cell culture</td></tr><tr><td>Trypsin-EDTA</td><td>ThermoFisher</td><td>25200056</td><td>Cell culture</td></tr><tr><td>Bromocresol green</td><td>Sigma-Aldrich</td><td>114-359</td><td>DNA loading dye</td></tr><tr><td>Siliconized tubes</td><td>Fisher Scientific&amp;nbsp;</td><td>02-681-320</td><td>Low retention microfuge tube</td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP152-1</td><td></td></tr><tr><td>Boric Acid</td><td>Fisher Scientific</td><td>A74-1</td><td></td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>BP24731</td><td></td></tr><tr><td>Photometer</td><td>InternationalLight Technologies</td><td>ILT1400-A</td><td>UVA dose measurement</td></tr><tr><td>&lt;strong&gt;Cell lines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Human U2OS osteosarcoma</td><td>ATCC</td><td>ATCC HTB-96</td><td>Cultured according to supplier&amp;rsquo;s recommendation</td></tr><tr><td>Human cervical adenocarcinoma HeLa</td><td>ATCC</td><td>ATCC-CCL-2</td><td>Cultured according to supplier&amp;rsquo;s recommendation</td></tr><tr><td>Proximal forward primer</td><td>5&amp;prime;-gcc ccc ctg acg agc atc ac</td><td></td><td></td></tr><tr><td>Proximal reverse primer</td><td>5&amp;prime;-tag tta ccg gat aag gcg cag cgg</td><td></td><td></td></tr><tr><td>Distal forward primer</td><td>5&amp;prime;-aat acc gcg cca cat agc ag</td><td></td><td></td></tr><tr><td>Distal reverse primer</td><td>5&amp;prime;-agt att caa cat ttc cgt gtc gcc</td><td></td><td></td></tr></tbody></table>

tools to study the role of architectural protein hmgb1 in the processing of helix distorting, site-specific dna interstrand crosslinks

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5&#39;-TA-3&#39; is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5&#39; end to a 4&#39;-hydroxymethyl-4,5&#39;,8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.

Formation of the TFO-directed ICL is critical for the plasmid based assays, which are used to interrogate the roles of architectural proteins in ICL processing in human cells. Denaturing agarose gel electrophoresis is a facile way to determine the efficiency of TFO-directed ICL formation. The plasmids harboring TFO-directed ICLs migrate with a slower mobility through the agarose gel matrix (Figure 1A, lane 3) when compared to the un-crosslinked control plasmids (Figure 1A, lane 2). Densitometric quantification of the bands in lanes 2 and 3 (Figure 1A) reveals approximately 70% of the plasmid population migrates through the gel with slowed mobility (identified by a black arrow), indicating TFO-directed ICL formation on those plasmids.
The chromatin immunoprecipitation assays performed on extracts from human U2OS cells transfected with either control plasmid (P) or plasmids containing TFO-directed ICLs (P-ICL) show enrichment of HMGB1 at the P-ICL region compared to the control (P) region, when immunoprecipitated with an anti-HMGB1 antibody (Figure 2, top panel, lanes 2 and 3). These results indicate that HMGB1 associates with the ICLs in human cells. When HMGB1 was depleted from the cells via siRNA treatment, the P-ICL-specific enrichment was diminished (Figure 2, top panel, lanes 4 and 5), as expected. When the same samples were immunoprecipitated using an anti-IgG antibody as an antibody specificity control, no PCR amplification was detected, as expected (Figure 2, top panel, Lanes 6-10). Lanes 10-14 (top panel) in Figure 2 were input samples used for normalization. The top panel shows PCR amplification by the primers near the TFO-directed ICL site and the bottom panel shows PCR amplification when primers distal to the TFO-directed ICL site were used.
HMGB1 is an architectural protein, which binds to distorted DNA with higher affinity than it binds undamaged DNA. The DNA supercoiling assays resolved through two-dimensional agarose gel electrophoresis demonstrate architectural modification of the TFO-directed ICL-containing plasmids (P-ICL) compared to control plasmids (P) by HMGB1 in HeLa cell extracts (Figure 3). The architectural modification induced by HMGB1 preferably on P-ICL-containing substrates is detected as increase in negative supercoiling. Supercoiled plasmids migrate faster through agarose gels relative to relaxed or linear plasmids. The distribution of the topoisomers indicates more supercoiling of the P-ICLs compared to P in the presence of HMBG1 (Figure 3). Negatively supercoiled DNA runs as left-handed arc in the second dimension in the presence of chloroquine. The supercoiling induced by HMGB1 on P-ICL seems to consist mostly of topoisomers that form a left-handed arc (~14 topoisomers were identified on the TFO-directed ICL containing plasmids compared to ~7 topoisomers from the control plasmid), indicating formation of negative supercoils. Altogether, these data suggest that plasmids with TFO-directed ICLs can be used as a tool to study the high-affinity association of the architectural protein HMGB1 with DNA lesions in human cells through chromatin immunoprecipitation assays. In addition, specific architectural modifications of the P-ICL substrates by HMGB1 can also be studied using supercoiling assays and two-dimensional agarose gels.
<img alt="Figure 1" src="/files/ftp_upload/54678/54678fig1.jpg" /> 
Figure 1: Alkaline agarose gel electrophoresis assay to quantify TFO-directed ICL formation efficiency on the plasmid pSupFG1. (A) Lane 1, 1 kb DNA ladder; lane 2, plasmid pSupFG1 (P) used as a control; and lane 3, TFO-directed ICL-containing pSupFG1 (P-ICL). The plasmids harboring ICLs migrate more slowly in the gel as demonstrated in lane 3 (indicated by the black arrow on the right hand side of the gel). (B) Densitometric quantification of the bands in lane 2 and lane 3 indicate that approximately 70% of the plasmids are crosslinked. This figure has been modified from reference16. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54678/54678fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54678/54678fig2.jpg" /> 
Figure 2: ChIP assay demonstrating enrichment of HMGB1 on the TFO-directed ICL-containing region of the crosslinked plasmid. (A) PCR amplification of the immunoprecipitated fragments were resolved on a 1% agarose gel using a set of proximal primers near the TFO-directed ICL site (B; P1 and P2, top panel) and a second set of primers used as a specificity control that were further (approximately 2,000 bp) from the site-directed ICL (B; P3 and P4, bottom panel). Lanes 2 and 3 indicate enrichment of HMGB1 near the TFO-directed ICL (lane 3) relative to undamaged DNA (lane 2). Lanes 4 and 5 are antibody specificity controls using an IgG antibody. Lanes 6 and 7 are the input samples. Lane 8 is a negative control for the PCR reaction and contains no DNA template. P, plasmid pSupFG1. P-ICL, TFO-directed ICL-containing plasmid pSupFG1. This figure has been modified from reference 16. (B) A schematic diagram of the plasmid pSupFG1 showing the primer sites for P1 and P2 as well as P3 and P4. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54678/54678fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54678/54678fig3.jpg" /> 
Figure 3: 2D agarose gel electrophoresis showing formation of negative supercoils in TFO-directed ICL-containing plasmids, facilitated by HMGB1. The 1x TBE agarose gel reveals the thermal distribution of the topoisomers generated by the plasmid alone (P) or the psoralen-crosslinked plasmid (P-ICL). The mobility of the supercoiled species is faster compared to the relaxed plasmids. Each band represents a topoisomer that differs from the band below or above by one less or more superhelical turn, respectively. The left-handed arc represents negatively supercoiled (-ve) species and the right-handed arc represents positively supercoiled (+ve) species. The psoralen ICL-containing plasmid population (P-ICL) contains more supercoiled species than the control plasmids (P). R, relaxed plasmids; S, supercoiled plasmids; 1D, first dimension of the gel electrophoresis; 2D, second dimension of the gel electrophoresis. This figure has been modified from16. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54678/54678fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks at JoVE.com

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein.

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, ...

tools-to-study-role-architectural-protein-hmgb1-processing-helix

Nanyang Technological University

Research

JoVE Journal

Genetics

8.2K Views.  The University of Texas at Austin. Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein. 

Video: Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

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 ...

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology. Instead, it relies on standard recombinant DNA procedures and widely available real-time, quantitative polymerase chain reaction (qPCR) instrumentation. Given the inherent flexibility of the strategy utilized, the size of the crosslinked protein, as well as the nature of the chemical linkage and the precise DNA sequence context of the attachment site can be varied to address the respective contributions of these parameters to the overall efficiency of DPC repair. Using this method, plasmids containing a site-specific DPC were transfected into cells and low molecular weight DNA recovered at various times post-transfection. Recovered DNA is then subjected to strand-specific primer extension (SSPE) using a primer complementary to the damaged strand of the plasmid. Since the DPC lesion blocks Taq DNA polymerase, the ratio of repaired to un-repaired DNA can be quantitatively assessed using qPCR. Cycle threshold (CT) values are used to calculate percent repair at various time points in the respective cell lines. This SSPE-qPCR method can also be used to quantitatively assess the repair kinetics of any DNA adduct that blocks Taq polymerase.

The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR.

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Biology

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, ...

Biochemistry

CMG-Mediated DNA Unwinding Observed by ssDNA-RPA Tract Growth

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

Author Spotlight: Unraveling the Dynamics of Eukaryotic DNA Replication Through Single-Molecule Visualization

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...

Capturing Common Fragile Site Breaks by Native &#947;H2A.X ChIP

Replication stress induced by exposure to extrinsic agents can lead to DNA breaks at common fragile sites, which are regions in the genome known to be prone to structural instability. The &#947;H2A.X chromatin immunoprecipitation (ChIP) assay serves as a powerful tool in genotoxicity studies, as &#947;H2A.X phosphorylation is a well-established marker for DNA double-strand breaks. Traditional &#947;H2A.X ChIP assays, however, are often labor-intensive and involve multiple, time-consuming steps. In this study, we present a simplified yet effective method that combines subcellular fractionation with native ChIP to isolate &#947;H2A.X-associated complexes. This approach is particularly suitable for analyzing &#947;H2A.X-chromatin interactions with enhanced specificity and efficiency. Using subcellular fractionation, chromatin-unbound materials are effectively removed, resulting in a purified chromatin fraction. Subsequent micrococcal nuclease (MNase) digestion under mild conditions allows chromatin fragmentation while preserving physiological interactions between &#947;H2A.X and its associated protein complexes. This preservation is essential for studying native interaction partners involved in DNA damage response pathways. This optimized native ChIP protocol substantially reduces the time and labor associated with conventional &#947;H2A.X ChIP assays. The streamlined procedure not only simplifies the workflow but also yields highly reproducible results, making it particularly advantageous in settings where high-throughput processing of multiple samples is required. This method has broad applicability in studies focused on genome stability, DNA repair, and chromatin biology, where accurate and efficient detection of DNA damage sites is critical. By employing optimized protocols and streamlined steps, this method enables the detection of DNA damage at fragile sites with improved sensitivity and minimal sample handling, making it a valuable tool for studies on genome stability and DNA damage response.

We present a rapid and efficient method to detect common fragile site breaks through native &#947;H2A.X chromatin immunoprecipitation (ChIP). This approach significantly reduces both the time and labor associated with traditional &#947;H2A.X ChIP assays while maintaining high reproducibility and reliability of results.

Replication stress induced by exposure to extrinsic agents can lead to DNA breaks at common fragile sites, which are regions in the genome known to be ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

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 ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

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