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 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.
Maintenance of genomic integrity is crucial for cell survival. Failure to preserve the genome results in premature aging, carcinogenesis, or death 8. There is intense interest in discerning how the DDR functions, stemming from its importance to both basic and clinical research. Many techniques have been developed over the years to aid in the study of how cells detect and repair DNA damage. Traditional methods such as immunocytochemistry and western blotting have been mainstays of the field, though recen…
The authors have nothing to disclose.
Supported in part by R01NS064593 and R21ES016636 (K.V.). Microscopy was performed at the VCU – Department of Neurobiology & Anatomy Microscopy Facility, supported, in part, with funding from NIH-NINDS Center core grant 5P30NS047463. The spinning disc confocal microscope was purchased with an NIH-NCRR award (1S10RR027957).
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 |