Quantification of DNA repair protein kinetics after gamma-irradiation using number and brightness analysis

Salim Abdisalaam; Milan Poudel; David J. Chen; George Alexandrakis

doi:10.1117/12.873853

23 February 2011 Quantification of DNA repair protein kinetics after γ-irradiation using number and brightness analysis

Salim Abdisalaam, Milan Poudel, David J. Chen, George Alexandrakis

Proceedings Volume 7905, Single Molecule Spectroscopy and Imaging IV; 790517 (2011) https://doi.org/10.1117/12.873853
Event: SPIE BiOS, 2011, San Francisco, California, United States

Abstract

The kinetics of most proteins involved in DNA damage sensing, signaling and repair following ionizing radiation exposure cannot be quantified by current live cell fluorescence microscopy methods. This is because most of these proteins, with only few notable exceptions, do not attach in large numbers at DNA damage sites to form easily detectable foci in microscopy images. As a result a high fluorescence background from freely moving and immobile fluorescent proteins in the nucleus masks the aggregation of proteins at sparse DNA damage sites. Currently, the kinetics of these repair proteins are studied by laser-induced damage and Fluorescence Recovery After Photobleaching that rely on the detectability of high fluorescence intensity spots of clustered DNA damage. We report on the use of Number and Brightness (N&B) analysis methods as a means to monitor kinetics of DNA repair proteins during sparse DNA damage created by γ-irradiation, which is more relevant to cancer treatment than laser-induced clustered damage. We use two key double strand break repair proteins, namely Ku 70/80 and the DNA-dependent protein kinase catalytic subunit (DNA-PK_CS), as specific examples to showcase the feasibility of the proposed methods to quantify dose-dependent kinetics for DNA repair proteins after exposure to γ-rays.

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Salim Abdisalaam, Milan Poudel, David J. Chen, and George Alexandrakis "Quantification of DNA repair protein kinetics after γ-irradiation using number and brightness analysis", Proc. SPIE 7905, Single Molecule Spectroscopy and Imaging IV, 790517 (23 February 2011); https://doi.org/10.1117/12.873853

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KEYWORDS

Proteins

Luminescence

Double sideband modulation

Microscopy

Molecules

Fluorescence correlation spectroscopy

Laser induced damage

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