title: Imaging of the DNA damage-induced dynamics of nuclear proteins via nonlinear photoperturbation.
abstract: Understanding the cellular response to DNA strand breaks is crucial to decipher the mechanisms maintaining the integrity of our genome. We present a novel method to visualize how the mobility of nuclear proteins changes in response to localized DNA damage. DNA strand breaks are induced via nonlinear excitation with femtosecond laser pulses at λ = 1050 nm in a 3D-confined subnuclear volume. After a time delay of choice, protein mobility within this volume is analysed by two-photon photoactivation of PA-GFP fusion proteins at λ = 775 nm. By changing the position of the photoactivation spot with respect to the zone of lesion the influence of chromatin structure and of the distance from damage are investigated. As first applications we demonstrate a locally confined, time-dependent mobility increase of histone H1.2, and a progressive retardation of the DNA repair factor XRCC1 at damaged sites. This assay can be used to map the response of nuclear proteins to DNA damage in time and space.
authors: Department of Physics, Modern Optics and Quantum Electronics and Center for Applied Photonics, University of Konstanz, Konstanz, 78457, Germany.
authors: Martin
authors: M
authors: Tomas
authors: Philipp
authors: P
authors: Blumhardt
authors: Anja
authors: A
authors: Deutzmann
authors: Tobias
authors: T
authors: Schwarz
authors: Dimitri
authors: D
authors: Kromm
authors: Alfred
authors: A
authors: Leitenstorfer
authors: Elisa
authors: E
authors: Ferrando-May
mesh_terms: D002843:Chromatin / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D007259:Infrared Rays; D007834:Lasers; D036641:Microscopy, Fluorescence, Multiphoton; D057054:Molecular Imaging; D017711:Nonlinear Dynamics; D009687:Nuclear Proteins / Q000378:metabolism*; D017785:Photons; D021381:Protein Transport / Q000528:radiation effects; D000076105:X-ray Repair Cross Complementing Protein 1
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D049452:Green Fluorescent Proteins
keywords: FRAP; chromatin; femtosecond pulses; fiber laser; two-photon photoactivation