期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 131, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/56213
关键词
Genetics; Issue 131; Laser microirradiation; lasers in medicine; DNA damage; DNA damage response; DNA repair; poly (ADP-ribose) polymerase (PARP); complex damage; strand breaks; base damage
资金
- Air Force Office of Scientific Research [FA9550-04-1-0101]
- Beckman Laser Institute Inc. Foundation
- Ford Foundation Fellowship from Academy of Sciences
- NSF [MCB-1615701]
- CRCC [CRR-17-426665]
DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.
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