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DNA Damage and Repair - Quantification of Sub-Cellular Events Using Automated Confocal Imaging

Introduction

Oxidative stress, radiation and other external insults have been shown to damage DNA molecules in cells derived from organisms as diverse as bacteria, yeast, drosophila, rodents and man (Friedberg et al. 2005). The presence of DNA damage may lead to cell cycle checkpoint arrest to allow time for DNA repair processes to occur. If however, the system becomes overwhelmed or the DNA repair mechanisms are impaired, the cell either enters the apoptosis pathway or become cancerous due to the accumulation of mutations resulting from replication of damaged DNA. Therefore, a complete understanding of DNA repair mechanisms is of great interest in the study of cancer prevention and treatment. In addition, this process has been implicated in cellular senescence and aging (Sedelnikova et al. 2004). Following induction of DNA double strand breaks, the specialized histone protein H2AX becomes phosphorylated and rapidly (within minutes) accumulates at the sites of DNA damage forming distinct foci (Paull et al. 2000).

H2AX foci formation is followed by recruitment of many other proteins involved in the DNA repair process including the p53 binding protein 53BP1 (Schulz et al ., 2000). Over long time periods (hours), the number of foci declines with the progression of DNA repair. Detection and quantification of foci development as an indicator of DNA damage and repair is of great interest to groups investigating these pathways (Kim et al . 2005). The assay is generally performed using coverslips and chamber slides and imaged on fluorescence microscopes (Sedelnikova et al . 2004). The demands for high spatial resolution and sophisticated image analysis, however, have hindered the transfer of this assay into a multi-well plate format and thus into the drug screening arena. Our goal was to establish image acquisition and analysis methods allowing quantification of foci develop
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