Visualizing DNA Repair Proteins with the FV3000 Confocal Microscope

The FV3000 microscope from Olympus has allowed Dr. JaeJin Kim and others in the Miller lab to generate DNA damage using laser-microirradiation and study DNA damage response factor behavior both in fixed and live cells. The availability of sensitive detectors, automatic focus capabilities, and super-corrected objectives has made the FV3000 microscope an effective instrument in conducting studies on DNA damage response pathways in human cancer cells.

Fluorescence imaging is a widely used technique in DNA damage signaling and repair studies to analyze the localization and kinetics of DNA damage response factors to DNA damage sites. Obtaining this information has been critical in identifying how these factors detect and repair DNA lesions at single cell resolution.

Double-stranded DNA breaks are one of the most deleterious forms of DNA damage. In response to the damage, DNA damage response (DDR) pathways in the cell are triggered that lead to the recruitment of DDR factors to the break site, as well as cell cycle checkpoint signaling and the regulation of DNA repair activities.

Immediate and faithful signaling and repair of the break site are crucial for the viability of the cell and to prevent mutations that can lead to the development of cancer. Thus, understanding the mechanisms involved in the DNA repair process is of vital importance. In this application, we used human osteosarcoma epithelial cancer cells (U2OS) to study the recruitment of DNA repair proteins to laser-induced damage, including double-strand DNA break sites, using the FV3000 confocal microscope.

The resulting images enabled us to determine the kinetics and accumulation levels of repair proteins recruited to break sites and (2) characterize the colocalization of endogenous transcriptional regulators and DDR pathway factors at DNA break sites.