Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC Irvine Electronic Theses and Dissertations (2018)

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.

    Cover page: Analyses of the Cellular Signaling Responses to DNA Damage and DNA Repair Factor Recruitment Using Fluorescence Lifetimes and FluctuationsCreative Commons 'BY-NC-ND' version 4.0 license
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2018)
    Lung cancer is one of the deadliest cancers in the world because of chemo-resistance to the commonly used cisplatin-based treatments. The use of low fidelity DNA polymerases in the translesional synthesis (TLS) DNA damage response pathway that repairs lesions caused by cisplatin also presents a mutational carcinogenic burden on cells that needs to be regulated by the tumor suppressor protein p53. However, there is much debate over the roles of the reversionless 3-like (REV3L) protein responsible for TLS and p53 in regulating cancer cell metabolism. In this study, the fluorescence lifetime of the metabolic coenzyme NADH reveals that the absence of REV3L can promote the p53-mediated upregulation of oxidative phosphorylation in cisplatin-treated H1299 lung carcinoma cells and increases cancer cell sensitivity to this platinum-based chemotherapy. These results demonstrate a previously unrecognized relationship between p53 and REV3L in cancer cell metabolism and may lead to improvements in chemotherapy treatment plans that reduce cisplatin resistance in lung cancer.
      Cover page: Absence of REV3L promotes p53-regulated cancer cell metabolism in cisplatin-treated lung carcinoma cells
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2017)
      A correction to this article has been published and is linked from the HTML version of this paper. The error has not been fixed in the paper.
        Cover page: Publisher Correction: Synthesis method of asymmetric gold particles
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2017)
        Asymmetric particles can exhibit unique properties. However, reported synthesis methods for asymmetric particles hinder their application because these methods have a limited scale and lack the ability to afford particles of varied shapes. Herein, we report a novel synthetic method which has the potential to produce large quantities of asymmetric particles. Asymmetric rose-shaped gold particles were fabricated as a proof of concept experiment. First, silica nanoparticles (NPs) were bound to a hydrophobic micro-sized polymer containing 2-chlorotritylchloride linkers (2-CTC resin). Then, half-planar gold particles with rose-shaped and polyhedral structures were prepared on the silica particles on the 2-CTC resin. Particle size was controlled by the concentration of the gold source. The asymmetric particles were easily cleaved from the resin without aggregation. We confirmed that gold was grown on the silica NPs. This facile method for synthesizing asymmetric particles has great potential for materials science.
          Cover page: Synthesis method of asymmetric gold particles
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2019)
          DNA damage signaling is critical for the maintenance of genome integrity and cell fate decision. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor rapidly activated in a damage dose- and complexity-dependent manner playing a critical role in the initial chromatin organization and DNA repair pathway choice at damage sites. However, our understanding of a cell-wide consequence of its activation in damaged cells is still limited. Using the phasor approach to fluorescence lifetime imaging microscopy and fluorescence-based biosensors in combination with laser microirradiation, we found a rapid cell-wide increase of the bound NADH fraction in response to nuclear DNA damage, which is triggered by PARP-dependent NAD+ depletion. This change is linked to the metabolic balance shift to oxidative phosphorylation (oxphos) over glycolysis. Inhibition of oxphos, but not glycolysis, resulted in parthanatos due to rapid PARP-dependent ATP deprivation, indicating that oxphos becomes critical for damaged cell survival. The results reveal the novel prosurvival response to PARP activation through a change in cellular metabolism and demonstrate how unique applications of advanced fluorescence imaging and laser microirradiation-induced DNA damage can be a powerful tool to interrogate damage-induced metabolic changes at high spatiotemporal resolution in a live cell.
            Cover page: NAD+ consumption by PARP1 in response to DNA damage triggers metabolic shift critical for damaged cell survival
            Top