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Proceedings Paper

Quantification of DNA repair protein kinetics after gamma-irradiation using number and brightness analysis
Author(s): Salim Abdisalaam; Milan Poudel; David J. Chen; George Alexandrakis
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Paper 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-PKCS), 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.

Paper Details

Date Published: 23 February 2011
PDF: 10 pages
Proc. SPIE 7905, Single Molecule Spectroscopy and Imaging IV, 790517 (23 February 2011); doi: 10.1117/12.873853
Show Author Affiliations
Salim Abdisalaam, The Univ. of Texas at Arlington (United States)
Milan Poudel, The Univ. of Texas Southwestern Medical Ctr. at Dallas (United States)
David J. Chen, The Univ. of Texas Southwestern Medical Ctr. at Dallas (United States)
George Alexandrakis, The Univ. of Texas at Arlington (United States)

Published in SPIE Proceedings Vol. 7905:
Single Molecule Spectroscopy and Imaging IV
Jörg Enderlein; Zygmunt Karol Gryczynski; Rainer Erdmann, Editor(s)

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