The kinetics of most fluorescently tagged DNA repair proteins subsequent to exposure to ionizing radiation, or radiomimetic chemical agents, cannot be quantified in the living cell without serious perturbation of the system under study. With the exception of only few proteins that attach in large numbers near DNA damage sites (e.g. 3-H2AX, 53BP1), most other proteins attach in fewer copies near the DNA damage sites and cannot be visualized by fluorescence microscopy. This because of the high background from freely moving, or immobile, fluorescent proteins that mask the weak aggregation of DNA repair proteins at damage sites. As a result, DNA damage is usually visualized by inducing clustered DNA damage by illumination with a laser beam. This illumination induces very high accumulation of repair proteins in one or more large spots in the nucleus. Nevertheless, the laser-induced damage is complicated in nature and may not be a good surrogate to ionizing radiation or radiomimetic agents for studying DNA repair in vivo. In this work we propose to develop quantitative microscopy methods that can overcome the current major limitation of not being able to quantify the kinetics of fluorescently tagged DNA repair proteins at sparse DNA damage sites. More specifically we will apply raster image correlation spectroscopy (RICS), a technique that analyzes the spatio-temporal fluorescence intensity fluctuations in image pixels, to quantify the repair kinetics of proteins with sparse accumulation in the cell nucleus. We will use RICS to quantify the repair kinetics of the DNA-dependent protein kinase catalytic subunit (DNA-PKCS), in its wild type and repair-deficient 7A forms after exposure to 3-rays and bleomycin that are both double strand break forming agents. We will also test hydrogen peroxide, a single strand break (DSB) forming agent, as a negative control. We will show that the DNA-PKCS kinetics after formation of DSBs can be quantified by RICS. Furthermore we propose to develop two specialized forms of RICS to further enhance the quantification of DNA repair kinetics of these proteins: (i) Photo-Activation RICS (PA-RICS) will offer control of the fluorescently tagged repair protein concentration, and (ii) Coherent Control RICS (CC-RICS) will optimize laser pulse characteristics to enhance fluorescence emission by up to an order of magnitude without increasing excitation power. PA-RICS and CC-RICS will enable improved quantification of the binding kinetic constants of DNA-PKCS variants at DNA damage sites. Importantly, the proposed RICS techniques can potentially be used to quantify the kinetics of a wide range of DNA damage sensing, signaling, and repair proteins with sparse accumulation patterns in the nucleus. Therefore, the proposed methods are very generally applicable to the DNA repair field and beyond.

Public Health Relevance

We propose to develop fluorescence microscopy techniques that will enable researchers to monitor if and how fast cells can fix DNA after this is damaged by X-rays or chemical agents. By looking at how cancer cells respond to treatment-induced DNA damage when their repair proteins work properly, or when they don't due to a genetic mutation, we can understand the mechanisms of treatment resistance and design better therapies.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZCA1-SRLB-R (O1))
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Knowlton, John R
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University of Texas Arlington
Biomedical Engineering
Schools of Engineering
United States
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