Two- and three-dimensional live cell imaging of DNA damage response proteins.

摘要: 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 detect DSBs, activate damage response (DDR), repair DNA. When stimulated, DDR works preserve integrity by triggering cycle arrest allow take place or force undergo apoptosis. The predominant mechanisms of DSB occur through nonhomologous end-joining (NHEJ) homologous recombination (HRR) (reviewed in). There many proteins whose activities must be precisely orchestrated function properly. Herein, we describe method 2- 3-dimensional (D) visualization one these proteins, 53BP1. p53-binding protein 1 (53BP1) localizes areas binding modified histones, forming foci within 5-15 minutes. histone modifications recruitment 53BP1 other sites believed facilitate structural rearrangement chromatin around contribute repair. Beyond direct participation in repair, additional roles have been described DDR, such as regulating an intra-S checkpoint, G2/M activating downstream proteins. Recently, was discovered that does not form induced during mitosis, instead waiting enter G1 before localizing vicinity DSBs. found associate with mitotic structures (such kinetochores) progression mitosis. In protocol use 3-D live imaging visualize formation damaging agent camptothecin (CPT), well 53BP1's behavior Camptothecin topoisomerase I inhibitor primarily causes replication. accomplish this, used previously 53BP1-mCherry fluorescent fusion construct consisting domain able bind addition, H2B-GFP monitor dynamics throughout but particular Live multiple dimensions excellent tool deepen our understanding eukaryotic cells.