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
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 ensur...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+DNA+lesions+associated+with+triplex-forming+oligonucleotides.">Repair of DNA lesions associated with triplex-forming oligonucleotides.</a> Mol Carcinog. 48, 389-399 (2009).</li><li>Seidman, M. M., Glazer, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+for+gene+repair+via+triple+helix+formation.">The potential for gene repair via triple helix formation.</a> J Clin Invest. 112, 487-494 (2003).</li><li>Vasquez, K. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagenesis+in+mammalian+cells+induced+by+triple+helix+formation+and+transcription-coupled+repair.">Mutagenesis in mammalian cells induced by triple helix formation and transcription-coupled repair.</a> Science. 271, 802-805 (1996).</li><li>Chen, Z., Xu, X. S., Yang, J., Wang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+function+of+XPC+protein+in+psoralen+and+cisplatin-mediated+DNA+repair+and+mutagenesis.">Defining the function of XPC protein in psoralen and cisplatin-mediated DNA repair and mutagenesis.</a> Carcinogenesis. 24, 1111-1121 (2003).</li><li>Derheimer, F. A., Hicks, J. K., Paulsen, M. T., Canman, C. E., Ljungman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psoralen-induced+DNA+interstrand+cross-links+block+transcription+and+induce+p53+in+an+ataxia-telangiectasia+and+rad3-related-dependent+manner.">Psoralen-induced DNA interstrand cross-links block transcription and induce p53 in an ataxia-telangiectasia and rad3-related-dependent manner.</a> Mol Pharmacol. 75, 599-607 (2009).</li><li>Vare, D., 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=DNA+interstrand+crosslinks+induce+a+potent+replication+block+followed+by+formation+and+repair+of+double+strand+breaks+in+intact+mammalian+cells.">DNA interstrand crosslinks induce a potent replication block followed by formation and repair of double strand breaks in intact mammalian cells.</a> DNA repair. 11, 976-985 (2012).</li><li>Huang, Y., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+crosslinking+damage+and+cancer+-+a+tale+of+friend+and+foe.">DNA crosslinking damage and cancer - a tale of friend and foe.</a> Transl Cancer Res. 2, 144-154 (2013).</li><li>Mukherjee, A., Vasquez, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HMGB1+interacts+with+XPA+to+facilitate+the+processing+of+DNA+interstrand+crosslinks+in+human+cells.">HMGB1 interacts with XPA to facilitate the processing of DNA interstrand crosslinks in human cells.</a> Nucleic Acids Res. 44, 1151-1160 (2016).</li><li>Bidney, D. L., Reeck, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+from+cultured+hepatoma+cells+of+two+nonhistone+chromatin+proteins+with+preferential+affinity+for+single-stranded+DNA:+apparent+analogy+with+calf+thymus+HMG+proteins.">Purification from cultured hepatoma cells of two nonhistone chromatin proteins with preferential affinity for single-stranded DNA: apparent analogy with calf thymus HMG proteins.</a> Biochem Biophys Res Commun. 85, 1211-1218 (1978).</li><li>Bianchi, M. E., Beltrame, M., Paonessa, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+recognition+of+cruciform+DNA+by+nuclear+protein+HMG1.">Specific recognition of cruciform DNA by nuclear protein HMG1.</a> Science. 243, 1056-1059 (1989).</li><li>Jung, Y., Lippard, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+of+full-length+HMGB1+binding+to+cisplatin-modified+DNA.">Nature of full-length HMGB1 binding to cisplatin-modified DNA.</a> Biochemistry. 42, 2664-2671 (2003).</li><li>Reddy, M. C., Christensen, J., Vasquez, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+human+high+mobility+group+protein+1+and+replication+protein+A+on+psoralen-cross-linked+DNA.">Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA.</a> Biochemistry. 44, 4188-4195 (2005).</li><li>Das, D., Scovell, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+binding+interaction+of+HMG-1+with+the+TATA-binding+protein/TATA+complex.">The binding interaction of HMG-1 with the TATA-binding protein/TATA complex.</a> J Biol Chem. 276, 32597-32605 (2001).</li><li>Little, A. J., Corbett, E., Ortega, F., Schatz, D. 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Y., Ahn, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+DNA+topology+on+plasmid+DNA+repair+in+vivo.">Effect of DNA topology on plasmid DNA repair in vivo.</a> FEBS letters. , 174-178 (2000).</li><li>Wu, Q., 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=High-affinity+triplex-forming+oligonucleotide+target+sequences+in+mammalian+genomes.">High-affinity triplex-forming oligonucleotide target sequences in mammalian genomes.</a> Mol Carcinog. 46, 15-23 (2007).</li><li>Sheflin, L. G., Spaulding, S. 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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 ...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5&#39;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&#160;</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&#160;</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>Cell lines</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&#8217;s recommendation</td>
		</tr>
		<tr>
			<td>Human cervical adenocarcinoma HeLa</td>
			<td>ATCC</td>
			<td>ATCC-CCL-2</td>
			<td>Cultured according to supplier&#8217;s recommendation</td>
		</tr>
		<tr>
			<td>Proximal forward primer</td>
			<td>5&#8242;-gcc ccc ctg acg agc atc ac</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Proximal reverse primer</td>
			<td>5&#8242;-tag tta ccg gat aag gcg cag cgg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Distal forward primer</td>
			<td>5&#8242;-aat acc gcg cca cat agc ag</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Distal reverse primer</td>
			<td>5&#8242;-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 (F...

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, ...

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