Targeting telomere-containing chromosome ends with a near-infrared femtosecond laser to study the activation of the DNA damage response and DNA damage repair pathways.
Telomeres are at the ends of chromosomes. Previous evidence suggests that laser-induced deoxyribose nucleic acid (DNA) breaks at chromosome ends during anaphase results in delayed cytokinesis. A possible explanation for this delay is that the DNA damage response (DDR) mechanism has been activated. We describe a live imaging method to study the effects of DDR activation following focal point near-infrared femtosecond laser microirradiation either at a single chromosome end or at a chromosome arm in mitotic anaphase cells. Laser microirradiation is used in combination with dual fluorescent labeling to monitor the co-localization of double-strand break marker γH2AX along with the DDR factors in PtK2 (Potorous tridactylus) cells. Laser-induced DNA breaks in chromosome ends as well as in chromosome arms results in recruitment of the following: poly(ADP-ribose) polymerase 1, checkpoint sensors (p-Chk1, p-Chk2), DNA repair protein Ku70/Ku80, and proliferating cell nuclear antigen. However, phosphorylated p53 at serine 15 is detected only at chromosome ends and not at chromosome arms. Full activation of DDR on damaged chromosome ends may explain previously published results that showed the delay of cytokinesis.
Beckman Laser Institute and Medical Clinic, 1002 Health Sciences Road East, Irvine, California 92612bUniversity of California, School of Biological Sciences, Department of Developmental and Cell Biology, Irvine, California 92617.
Bárbara Alcaraz
BA
Silva
Jessica R
JR
Stambaugh
Michael W
MW
Berns
D000818:Animals; D002460:Cell Line; D004249:DNA Damage / Q000502:physiology* / Q000528:radiation effects; D004260:DNA Repair / Q000502:physiology*; D045643:DNA Repair Enzymes / Q000378:metabolism / Q000528:radiation effects; D020287:In Situ Nick-End Labeling; D007259:Infrared Rays; D007834:Lasers; D008297:Male; D008853:Microscopy; D048328:Potoroidae; D016615:Telomere / Q000528:radiation effects*
D045643:DNA Repair Enzymes