In situ analysis of DNA damage response and repair using laser microirradiation.
A proper response to DNA damage is critical for the maintenance of genome integrity. However, it is difficult to study the in vivo kinetics and factor requirements of the damage recognition process in mammalian cells. In order to address how the cell reacts to DNA damage, we utilized a second harmonic (532 nm) pulsed Nd:YAG laser to induce highly concentrated damage in a small area in interphase cell nuclei and cytologically analyzed both protein recruitment and modification. Our results revealed for the first time the sequential recruitment of factors involved in two major DNA double-strand break (DSB) repair pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR), and the cell cycle-specific recruitment of the sister chromatid cohesion complex cohesin to the damage site. In this chapter, the strategy developed to study the DNA damage response using the 532-nm Nd:YAG laser will be summarized.
Kim
Jong-Soo
JS
Department of Biological Chemistry, School of Medicine, University of California, Irvine, California 92697, USA.
Heale
Jason T
JT
Zeng
Weihua
W
Kong
Xiangduo
X
Krasieva
Tatiana B
TB
Ball
Alexander R
AR
Yokomori
Kyoko
K
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D047369:Chromatin Immunoprecipitation; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional; D007834:Lasers; D011839:Radiation, Ionizing
D002110:Caffeine