दो और तीन आयामी डीएनए क्षति प्रतिक्रिया प्रोटीन की लाइव सेल इमेजिंग
Summary
इस प्रोटोकॉल एक डीएनए डबल कतरा तोड़ संकेतन प्रोटीन डीएनए के रूप में के रूप में अच्छी तरह से पिंजरे का बँटवारा दौरान अपने स्थानीयकरण क्षति प्रतिक्रिया में सक्रिय visualizing के लिए एक विधि का वर्णन है.
Abstract
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.
Protocol
Discussion
जीनोमिक अखंडता के रखरखाव सेल अस्तित्व के लिए महत्वपूर्ण है. समय से पहले बूढ़ा, carcinogenesis, या 8 मौत में जीनोम परिणाम के संरक्षण में विफलता. समझदार में गहन रुचि है कैसे DDR कार्यों, दोनों बुनियादी और नैदानिक ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
R01NS064593 और R21ES016636 (केवी) के हिस्से में समर्थन. माइक्रोस्कोपी VCU पर प्रदर्शन किया गया था – तंत्रिका जीव विज्ञान और एनाटॉमी माइक्रोस्कोपी सुविधा का विभाग, NIH NINDS केंद्र कोर अनुदान 5P30NS047463 से धन के साथ भाग में, समर्थित. कताई डिस्क confocal खुर्दबीन एक पुरस्कार NIH-NCRR साथ (1S10RR027957) खरीदा गया था.
Materials
| Product | Company |
| CellStar culture dishes | Greiner Bio-one |
| FluroDish glass bottom dishes | World Precision Instruments, Inc. |
| MEM media | GIBCO |
| Non-essential amino acids | GIBCO |
| Amino acids | GIBCO |
| Vitamins | GIBCO |
| Sodium Pyruvate | Invitrogen |
| Penicillin/Streptomycin | HyClone |
| Fetal Bovine Serum | GIBCO |
| N-Myc-53BP1 WT pLPC-Puro; plasmid 19836 |
Addgene |
| pCLNR-H2BG; plasmid 17735 | Addgene |
| SuperFect | Qiagen |
| Zeiss Cell Observer SD Imaging system | Zeiss |
| AxioVision (release 4.8.2) | Zeiss |
| Zeiss Immersol W Oil | Zeiss |
| Volocity software (version 6.0) | PerkinElmer |
References
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