Analyses of the Cellular Signaling Responses to DNA Damage and DNA Repair Factor Recruitment Using Fluorescence Lifetimes and Fluctuations

Genome integrity is continually challenged by threats including DNA replication errors, toxic metabolic byproducts, and exposure to exogenous genotoxins. Responding to and repairing damaged DNA requires coordinating a number of critical cellular events including activating DNA repair, facilitating chromatin rearrangement, and delaying cell cycle progression. Although the factors important for DNA repair have been identified, how these activities are coordinated in the nucleus and the long-term cellular-wide consequences of DNA repair are not well characterized. Nicotinamide adenine dinucleotide (NAD) is a coenzyme involved in both cellular metabolism and DNA repair. Thus, analyses of NAD species could provide new insight into DNA damage signaling as well as damage-induced metabolic responses to various types of DNA lesions. Here, we use the phasor approach to fluorescence lifetime imaging microscopy in combination with genetically encoded fluorescent biosensors to measure metabolic changes in single cells with high spatiotemporal resolution. We found that the absence of the reversionless 3-like translesional synthesis DNA repair protein promotes p53-mediated upregulation of oxidative phosphorylation (oxphos) in cisplatin-treated H1299 lung carcinoma cells and increases cell sensitivity to this chemotherapeutic treatment. Furthermore, it has been well-documented that depletion of NAD+ leads to an overall metabolic collapse, but it is not clear whether or not regulating metabolism can overcome cell death pathways as a survival mechanism. We observe a PARP-dependent decrease in NAD species and an increased metabolic reliance on oxphos. In all, our analyses revealed a previously unrecognized long-term effect of DNA repair signaling on energy metabolism in DNA damaged cells. Although the patterns of DNA repair protein redistribution following DNA damage have been systematically documented, understanding the complex response to damage requires the characterization of the molecular dynamics of these proteins with high spatiotemporal resolution. By using spatial pair cross-correlation function analysis in two-dimensions, we were able to visualize the barriers to molecular motion of DNA repair proteins in response to laser microirradiation-induced DNA damage.In summary, my project utilized cutting-edge fluorescence dynamics techniques to reveal a connection between the DNA damage response and cellular metabolism and to develop a new method to characterize molecular diffusion in response to DNA damage