In Time and Space: Laser Microirradiation and the DNA Damage Response

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1999; p. 61
• Main Authors: Kim, Jae Jin, Kumbhar, Ramhari, Gong, Fade, Miller, Kyle M
• Format: Journal Article
• Language: English
• Published: United States 2019
• Subjects: Cell Line, Tumor; DNA Damage - radiation effects; DNA Repair - radiation effects; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; Epigenome - radiation effects; Fluorescent Dyes - chemistry; Genomic Instability - radiation effects; Humans; Intravital Microscopy - instrumentation; Intravital Microscopy - methods; Lasers; Microscopy, Confocal - instrumentation; Microscopy, Confocal - methods; Microscopy, Fluorescence - instrumentation; Microscopy, Fluorescence - methods; Spatio-Temporal Analysis; Transcription, Genetic - radiation effects; Laser microirradiation; Live-cell imaging; Transcription; DNA repair; DNA damage; Fluorescence microscopy
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-9500-4_3

Maintenance of genomic integrity depends on the spatiotemporal recruitment and regulation of DNA damage response and repair proteins at DNA damage sites. These highly dynamic processes have been widely studied using laser microirradiation coupled with fluorescence microscopy. Laser microirradiation has provided a powerful methodology to identify and determine mechanisms of DNA damage response pathways. Here we describe methods used to analyze protein recruitment dynamics of fluorescently tagged or endogenous proteins to laser-induced DNA damage sites using laser scanning and fluorescence microscopy. We further describe multiple applications employing these techniques to study additional processes at DNA damage sites including transcription. Together, we aim to provide robust visualization methods employing laser-microirradiation to detect and determine protein behavior, functions and dynamics in response to DNA damage in mammalian cells.