Fluorescence Lifetime Mapping of NADH Reveals DNA Repair Activity in Live Cells

Abstract

Reduced nicotinamide adenine dinucleotide (NADH) is an important coenzyme that catalyzes many cellular functions including energy production and DNA damage repair. The ratio of free and bound NADH has recently emerged as a regulator of transcriptional activity and age-associated diseases. The DNA damage response, which requires NADH to gain full functionality, senses and repairs any breaks and errors in the genome. Uncovering the role of this ubiquitous molecule in response to DNA damage will further our understanding of how DNA protects itself and provide insight on how cancer cells evade these processes. In laser-induced damage experiments, HeLa cells were damaged with a femto-second pulsed two-photon laser at a wavelength of 780nm. Fluorescence lifetime imaging microscopy (FLIM) data was acquired and processed using the FLIM-phasor method. Induction of DNA damage and initiation of DNA repair were confirmed via immunostaining for γH2AX and PAR. The lifetime of NADH species in each pixel of FLIM images were transformed into polar coordinates on the phasor plot. The relative fraction of bound NADH was then calculated from the phasor plot and plotted over time. Low laser power damage resulted in a transient change toward a high fraction of bound NADH that was restored in approximately 8 hours. High laser power damage resulted in an immediate (∼5 minutes) and persistent (after 12 hours) shift toward a high fraction of bound NADH. The data show that the extent of DNA damage correlates to increases in the fraction of bound NADH in the nucleus owing to increased binding to DNA repair-associated substrates. Thus, monitoring NADH activity can be an intrinsic biosensor for DNA repair activity. Future studies will explore the role of NADH in chromatin remodeling to facilitate transcriptional regulation of DNA repair.