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

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

Research output: Chapter in Book/Report/Conference proceedingConference contribution

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.

Original languageEnglish (US)
Title of host publicationSingle Molecule Spectroscopy and Imaging IV
DOIs
StatePublished - Apr 20 2011
EventSingle Molecule Spectroscopy and Imaging IV - San Francisco, CA, United States
Duration: Jan 22 2011Jan 23 2011

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
Volume7905
ISSN (Print)1605-7422

Other

OtherSingle Molecule Spectroscopy and Imaging IV
CountryUnited States
CitySan Francisco, CA
Period1/22/111/23/11

Keywords

  • DNA repair
  • Fluorescence Correlation Spectroscopy
  • Number and Brightness analysis
  • double strand break

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Biomaterials
  • Atomic and Molecular Physics, and Optics
  • Radiology Nuclear Medicine and imaging

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  • Cite this

    Abdisalaam, S., Poudel, M., Chen, D. J., & Alexandrakis, G. (2011). Quantification of DNA repair protein kinetics after γ-irradiation using number and brightness analysis. In Single Molecule Spectroscopy and Imaging IV [790517] (Progress in Biomedical Optics and Imaging - Proceedings of SPIE; Vol. 7905). https://doi.org/10.1117/12.873853