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 aberrations. 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 in). 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
histones, forming foci within 5-15 minutes. 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 repair. 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 proteins. 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 DSBs.
DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis. 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 DSBs. 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 mitosis. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of
DDR proteins in eukaryotic cells.