Telomeres are essential for protecting chromosome ends not only from the replication-associated DNA loss, but also from unwanted DNA damage response (DDR) and repair by the damage machinery. Uncapping of telomeres during interphase elicits a DDR mechanism that results in cell cycle arrest. However, it is unclear how chromosome ends are normally protected from the DNA damage machinery, and how DDR is regulated at telomeres during mitosis.
With that in mind, the goal of my thesis is to investigate the consequences of DNA damage occurring at specific chromosomal domains. Laser microirradiation was used in combination with dual fluorescent labeling to monitor the co-localization of DDR factors in PtK2 (Potorous tridactylus) chromosomes.
The results of my thesis show that laser-induced DNA break in chromosome ends as well as in chromosome arms of anaphase cells result in recruitment of the following: poly (ADP-ribose) polymerase 1 (PARP1), checkpoint sensors (p-Chk1, p-Chk2), DNA repair protein Ku70/Ku80, and proliferating cell nuclear antigen (PCNA). However, p53 phosphorylated at serine 15 was detected only at chromosome ends, and not at chromosome arms of anaphase cells.
Furthermore, my experiments show that damage to a single mitotic chromosome end, but not on a chromosome arm, results in specific DDR factor recruitment, damage and spindle checkpoint-dependent mitotic delay, and subsequent micronuclei formation in G1. Together these findings reveal mitosis-specific DDR uniquely associated with chromosome ends.
Furthermore, we found the laser parameters used to induce telomeric TRF2 (repeat-binding factor 2) recruitment. We introduced double-strand breaks (DSBs) and assayed for the recruitment of TRF2. We found that a laser dose of 2.43e+11W/cm2 is sufficient to form DSBs, based on the recruitment of repair factor 53BP1. Nevertheless, at this laser dose, TRF2 fails to accumulate at damage sites. In contrast, at an irradiance of 2.65 e+11 W/cm2 or higher, TRF2 accumulates at damage sites, which is independent of ATM. We also found that phosphorylation of TRF2 on threonine 188 occurs at both low and high irradiance laser-induced DSBs in both interphase and mitotic cells in an ATM-dependent manner. By contrast, Phosphorylated TRF2 on threonine 188 did not form foci by using γ-irradiation.