工具来研究蛋白质建筑HMGB1的作用，螺旋窜改，站点特定的DNA间交联处理
Summary
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.
Abstract
高迁移率族蛋白1（HMGB1）蛋白是参与调节基因组中的许多重要的功能，如转录，DNA复制和DNA修复的非组蛋白的建筑蛋白。 HMGB1结合具有较高的亲和力结构扭曲的DNA，而不是典型的B-DNA。例如，我们发现，HMGB1结合DNA链间交联（ICLS），其共价连接的DNA的两条链，导致螺旋的畸变，并且如果左未修理可引起细胞死亡。由于它们的细胞毒性潜力，几个ICL诱导剂目前用作在临床化疗剂。而ICL形成剂显示某些碱基序列（如 5'-TA-3“是首选的交联网站补骨脂）的喜好，他们在很大程度上引起不加区别的时尚DNA损伤。然而，由ICL诱导剂共价偶联到三链形成性寡核苷酸（TFO），其结合的DNA序列中的特异性方式，可以实现目标DNA损伤。这里，我们使用一个TFO共价结合于5'末端到4'-羟甲基4,5'，8三甲基补骨脂（HMT）补骨脂上产生一个突变报告质粒一个位点特异性的ICL作为工具使用通过在人类细胞HMGB1研究建筑修改，处理和复杂的DNA损伤的修复。我们描述的实验技术编写有关报告质粒TFO定向ICLS，并使用染色质免疫测定法来询问蜂窝背景与TFO定向ICLS HMGB1的关联。此外，我们描述的DNA超螺旋测定法通过测量由HMGB1补骨脂交联的质粒引入超螺旋匝的数量，以评估受损的DNA的特定的架构的修改。这些技术可以用于研究参与TFO定向ICLS或在任何感兴趣的细胞系其他靶向DNA损伤的处理和修复的其它蛋白质的作用。
Introduction
通过Hoogsteen碱基-氢键形成三寡核苷酸（TFOs）结合双链DNA的序列特异性的方式，以形成三重螺旋结构1-5。三缸技术已被用于询问各种生物分子机制，如转录，DNA损伤修复和基因定位（在参考文献6-8中综述）。 TFOs已广泛用于诱导对报告质粒9,10-位点特异性破坏。我们的实验室和其他人以前用过的一个TFO，AG30，拴补骨脂素分子诱导特异性位点DNA间交联（ICLS）在质粒pSupFG1 5,10-12的supF基因。 ICLS是高度细胞毒性，因为这些病变共价交联的两条DNA链，并且如果左未修理，可阻断基因转录和阻碍DNA复制机械13,14。因为它们的细胞毒性潜力，ICL诱导剂已经被用作化学治疗药在治疗的癌症和其他疾病15。然而，ICLS的人类细胞中的处理和修复还不是很清楚。因此，更好地理解在人类细胞参与ICLS的处理的机制可能有助于改善基于ICL-化疗方案的效力。 TFO诱导ICLS及其修复中间体具有引起显著结构扭曲到DNA螺旋的潜力。这种失真是用于建筑的蛋白质，其结合到扭曲的DNA比对规范B型双螺旋DNA 16-20更高的亲和力可能的目标。这里，我们通过染色质免疫沉淀（ChIP）上补骨脂交联的质粒分析研究了高丰度的建筑蛋白的关联，HMGB1与人体细胞ICLS和在调节人体补骨脂交联的质粒DNA的拓扑确定为HMGB1作用肿瘤细胞裂解物。
HMGB1是一种高度丰富和广泛表达非他音建筑蛋白结合到受损的DNA和可替换的结构的DNA底物，比典型的B-型DNA 17-20更高的亲和力。 HMGB1参与几个DNA的代谢过程，如转录，DNA复制和DNA修复16,21-23。我们以前表明，HMGB1结合的TFO定向ICLS 体外以高亲和性20。此外，我们已经证明，缺乏的HMGB1增加TFO定向ICLS的诱变处理和识别的HMGB1的核苷酸切除修复（NER）辅因子23,24。最近，我们已发现，HMGB1是在人细胞中具有TFO定向ICLS相关联，并且其招募这种病变取决于NER蛋白质，XPA 16。的DNA的负超螺旋已经显示由NER 25以促进高效去除DNA损伤的，我们已经发现，HMGB1优先诱导负超螺旋上的TFO定向含ICL-纤溶酶中间基板（相对于非破坏质粒基板）16，提供了一个更好的理解的HMGB1作为NER辅因子的潜在作用（多个）。 ICLS的处理未完全在人类细胞中理解;因此，根据本文所描述的分子工具的技术和开发的实验可能导致涉及ICL修理额外的蛋白质，这反过来又可以起到可以被利用来改善癌症化疗方案的效力作为药理学靶的鉴定。
这里，一个有效的方法，以通过变性琼脂糖凝胶电泳进行了讨论评估TFO定向的ICL形成的质粒DNA的效率。另外，使用含有TFO定向ICLS质粒，技术来使用改性ChIP实验确定在蜂窝上下文HMGB1和ICL-损坏质粒的关联已经描述。此外，一个浅显的方法来研究由日推出拓扑修改Ë建筑HMGB1蛋白质，特别是对人的细胞裂解物ICL-损坏质粒基材已被通过二维凝胶电泳进行超螺旋化验确定。所描述的技术可用于促进DNA修复和建筑的蛋白质中的目标DNA损伤处理的参与对人体细胞的质粒的理解。
我们描述了TFO定向的位点特异性补骨脂ICLS对质粒DNA，和随后的质粒沉淀和超螺旋测定法分别以确定与病变相关联该改变的DNA拓扑蛋白，和蛋白，形成详细协议。这些测定法可以修改与其他DNA损伤剂，TFOs，质粒底物，和感兴趣的哺乳动物细胞系进行。事实上，我们已经表明，至少有一个潜在的独特和高亲和力的TFO结合位点在人基因组26每注释基因内。怎么样以往，为清楚起见，我们在人U2OS细胞的特定突变报道质粒（pSupFG1）描述了这些技术的使用特定的补骨脂素-缀合的TFO（pAG30），因为我们在慕克吉＆Vasquez的，2016年16已经利用。
Protocol
Representative Results
Discussion
TFO定向ICLS的有效形成取决于两个关键因素：第一，适当的缓冲液成分（ 如 ， 氯化镁 ），并用其靶DNA底物的TFO的孵育时间（取决于TFO的与其靶的结合亲和力双工和浓度使用）;第二，UVA的适当剂量（365 nm）的照射下有效地形成补骨脂交联。最佳三缸形成可以通过使用HPLC或凝胶纯化TFOs来实现。杂质污染的TFO可能会导致不希望的交联产物，其可以在实验结果进一步复杂化。以增加TFOs?…
Declarações
The authors have nothing to disclose.
Acknowledgements
笔者想感谢瓦斯奎兹实验室的成员，有益的讨论。这项工作是由健康/美国国家癌症研究所的国家机构[CA097175，CA093279到KMV]的支持;与癌症预防和得克萨斯研究院[RP101501。资助为开放式接入费：健康/美国国家研究院国家癌症研究所[CA093279到KMV。
Materials
| 5'HMT Psoralen-AG30 | Midland Certified Reagent Co., Midland, TX | Custom order | HPLC (or gel) purified |
| Simple ChIP Enzymatic Chromatin IP Kit | Cell signaling Inc. | 9003 | 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. |
| GoTaq Green | Promega | M7122 | PCR master mix |
| HMGB1 antibody | Abcam | Ab18256 | |
| Wizard Gel and PCR cleanup kit | Promega | A9281 | |
| Chloroquine-diphosphate salt | Sigma | C6628-50G | For two-dimensional agarose gel electrophoresis |
| EcoRI | New England BioLabs | R0101S | |
| GenePORTER transfection reagent | Genlantis | T201015 | |
| Vaccinia Topoisomerase I | Invitrogen | 38042-024 | |
| Blak-Ray long wave ultraviolet lamp | Upland, CA | B 100 AP | UVA lamp |
| EpiSonic Multi-Functional Bioprocessor 1100 | Epigentek | EQC-1100 | Water bath sonicator |
| Mylar filter | GE Healthcare Life Sciences | 80112939 | |
| Agarose | Sigma-Aldrich | A6111 | |
| BIO-RAD Chemidoc | BIO-RAD | XRS+ system | DNA imaging system |
| Glycine | Fisher Scientific | BP381-500 | |
| 37% Formaldehyde | Sigma-Aldrich | F8775-25ML | Used for crosslinking |
| Proteinase K | New England Biolabs | P8107S | |
| DMEM | ThermoFisher | 11965092 | Cell culture media |
| PBS | ThermoFisher | 70013073 | Cell culture |
| Trypsin-EDTA | ThermoFisher | 25200056 | Cell culture |
| Bromocresol green | Sigma-Aldrich | 114-359 | DNA loading dye |
| Siliconized tubes | Fisher Scientific | 02-681-320 | Low retention microfuge tube |
| Tris base | Fisher Scientific | BP152-1 | |
| Boric Acid | Fisher Scientific | A74-1 | |
| EDTA | Fisher Scientific | BP24731 | |
| Photometer | InternationalLight Technologies | ILT1400-A | UVA dose measurement |
| Cell lines | |||
| Human U2OS osteosarcoma | ATCC | ATCC HTB-96 | Cultured according to supplier’s recommendation |
| Human cervical adenocarcinoma HeLa | ATCC | ATCC-CCL-2 | Cultured according to supplier’s recommendation |
| Proximal forward primer | 5′-gcc ccc ctg acg agc atc ac | ||
| Proximal reverse primer | 5′-tag tta ccg gat aag gcg cag cgg | ||
| Distal forward primer | 5′-aat acc gcg cca cat agc ag | ||
| Distal reverse primer | 5′-agt att caa cat ttc cgt gtc gcc |
Referências
- Moser, H. E., Dervan, P. B. Sequence-specific cleavage of double helical DNA by triple helix formation. Science. 238, 645-650 (1987).
- Cooney, M., Czernuszewicz, G., Postel, E. H., Flint, S. J., Hogan, M. E. Site-specific oligonucleotide binding represses transcription of the human c-myc gene in vitro. Science. 241, 456-459 (1988).
- Beal, P. A., Dervan, P. B. Second structural motif for recognition of DNA by oligonucleotide-directed triple-helix formation. Science. 251, 1360-1363 (1991).
- Vasquez, K. M., Dagle, J. M., Weeks, D. L., Glazer, P. M. Chromosome targeting at short polypurine sites by cationic triplex-forming oligonucleotides. J Biol Chem. 276, 38536-38541 (2001).
- Vasquez, K. M., Christensen, J., Li, L., Finch, R. A., Glazer, P. M. Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions. Proc Natl Acad Sci U S A. 99, 5848-5853 (2002).
- Mukherjee, A., Vasquez, K. M. Triplex technology in studies of DNA damage, DNA repair, and mutagenesis. Biochimie. 93, 1197-1208 (2011).
- Chin, J. Y., Glazer, P. M. Repair of DNA lesions associated with triplex-forming oligonucleotides. Mol Carcinog. 48, 389-399 (2009).
- Seidman, M. M., Glazer, P. M. The potential for gene repair via triple helix formation. J Clin Invest. 112, 487-494 (2003).
- Vasquez, K. M. Targeting and processing of site-specific DNA interstrand crosslinks. Environ Mol Mutagen. 51, 527-539 (2010).
- Wang, G., Levy, D. D., Seidman, M. M., Glazer, P. M. Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation. Mol Cell Biol. 15, 1759-1768 (1995).
- Wang, G., Seidman, M. M., Glazer, P. M. Mutagenesis in mammalian cells induced by triple helix formation and transcription-coupled repair. Science. 271, 802-805 (1996).
- Chen, Z., Xu, X. S., Yang, J., Wang, G. Defining the function of XPC protein in psoralen and cisplatin-mediated DNA repair and mutagenesis. Carcinogenesis. 24, 1111-1121 (2003).
- Derheimer, F. A., Hicks, J. K., Paulsen, M. T., Canman, C. E., Ljungman, M. Psoralen-induced DNA interstrand cross-links block transcription and induce p53 in an ataxia-telangiectasia and rad3-related-dependent manner. Mol Pharmacol. 75, 599-607 (2009).
- Vare, D., et al. DNA interstrand crosslinks induce a potent replication block followed by formation and repair of double strand breaks in intact mammalian cells. DNA repair. 11, 976-985 (2012).
- Huang, Y., Li, L. DNA crosslinking damage and cancer – a tale of friend and foe. Transl Cancer Res. 2, 144-154 (2013).
- Mukherjee, A., Vasquez, K. M. HMGB1 interacts with XPA to facilitate the processing of DNA interstrand crosslinks in human cells. Nucleic Acids Res. 44, 1151-1160 (2016).
- Bidney, D. L., Reeck, G. R. Purification from cultured hepatoma cells of two nonhistone chromatin proteins with preferential affinity for single-stranded DNA: apparent analogy with calf thymus HMG proteins. Biochem Biophys Res Commun. 85, 1211-1218 (1978).
- Bianchi, M. E., Beltrame, M., Paonessa, G. Specific recognition of cruciform DNA by nuclear protein HMG1. Science. 243, 1056-1059 (1989).
- Jung, Y., Lippard, S. J. Nature of full-length HMGB1 binding to cisplatin-modified DNA. Bioquímica. 42, 2664-2671 (2003).
- Reddy, M. C., Christensen, J., Vasquez, K. M. Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA. Bioquímica. 44, 4188-4195 (2005).
- Das, D., Scovell, W. M. The binding interaction of HMG-1 with the TATA-binding protein/TATA complex. J Biol Chem. 276, 32597-32605 (2001).
- Little, A. J., Corbett, E., Ortega, F., Schatz, D. G. Cooperative recruitment of HMGB1 during V(D)J recombination through interactions with RAG1 and DNA. Nucleic Acids Res. 41, 3289-3301 (2013).
- Lange, S. S., Mitchell, D. L., Vasquez, K. M. High mobility group protein B1 enhances DNA repair and chromatin modification after DNA damage. Proc Natl Acad Sci U S A. 105, 10320-10325 (2008).
- Lange, S. S., Reddy, M. C., Vasquez, K. M. Human HMGB1 directly facilitates interactions between nucleotide excision repair proteins on triplex-directed psoralen interstrand crosslinks. DNA repair. 8, 865-872 (2009).
- Park, J. Y., Ahn, B. Effect of DNA topology on plasmid DNA repair in vivo. FEBS letters. , 174-178 (2000).
- Wu, Q., et al. High-affinity triplex-forming oligonucleotide target sequences in mammalian genomes. Mol Carcinog. 46, 15-23 (2007).
- Sheflin, L. G., Spaulding, S. W. High mobility group protein 1 preferentially conserves torsion in negatively supercoiled DNA. Bioquímica. 28, 5658-5664 (1989).
- Betous, R., et al. Role of TLS DNA polymerases eta and kappa in processing naturally occurring structured DNA in human cells. Mol Carcinog. 48, 369-378 (2009).
- Luo, Z., Macris, M. A., Faruqi, A. F., Glazer, P. M. High-frequency intrachromosomal gene conversion induced by triplex-forming oligonucleotides microinjected into mouse cells. Proc Natl Acad Sci U S A. 97, 9003-9008 (2000).
- Vasquez, K. M., Wang, G., Havre, P. A., Glazer, P. M. Chromosomal mutations induced by triplex-forming oligonucleotides in mammalian cells. Nucleic Acids Res. 27, 1176-1181 (1999).
- Puri, N., et al. Minimum number of 2′-O-(2-aminoethyl) residues required for gene knockout activity by triple helix forming oligonucleotides. Bioquímica. 41, 7716-7724 (2002).
- Christensen, L. A., Finch, R. A., Booker, A. J., Vasquez, K. M. Targeting oncogenes to improve breast cancer chemotherapy. Cancer Res. 66, 4089-4094 (2006).
- Boulware, S. B., et al. Triplex-forming oligonucleotides targeting c-MYC potentiate the anti-tumor activity of gemcitabine in a mouse model of human cancer. Mol Carcinog. 53, 744-752 (2014).
