Related Experiment Video
Updated: Jun 10, 2026
Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
Published on: August 21, 2021
The dynamic stress-induced "O-GlcNAc-ome" highlights functions for O-GlcNAc in regulating DNA damage/repair and other
Natasha E Zachara1, Henrik Molina, Ker Yi Wong
1The Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205-2185, USA. nzachara@jhmi.edu
Abstract:
The modification of nuclear, mitochondrial, and cytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is a dynamic and essential post-translational modification of metazoans. Numerous forms of cellular injury lead to elevated levels of O-GlcNAc in both in vivo and in vitro models, and elevation of O-GlcNAc levels before, or immediately after, the induction of cellular injury is protective in models of heat stress, oxidative stress, endoplasmic reticulum (ER) stress, hypoxia, ischemia reperfusion injury, and trauma hemorrhage. Together, these data suggest that O-GlcNAc is a regulator of the cellular stress response. However, the molecular mechanism(s) by which O-GlcNAc regulates protein function leading to enhanced cell survival have not been identified. In order to determine how O-GlcNAc modulates stress tolerance in these models we have used stable isotope labeling with amino acids in cell culture to determine the identity of proteins that undergo O-GlcNAcylation in response to heat shock. Numerous proteins with diverse functions were identified, including NF-90, RuvB-like 1 (Tip49α), RuvB-like 2 (Tip49β), and several COPII vesicle transport proteins. Many of these proteins bind double-stranded DNA-dependent protein kinase (PK), or double-stranded DNA breaks, suggesting a role for O-GlcNAc in regulating DNA damage signaling or repair. Supporting this hypothesis, we have shown that DNA-PK is O-GlcNAc modified in response to numerous forms of cellular stress.
Insights
O-linked β-N-acetylglucosamine (O-GlcNAc) modification protects cells from injury by regulating the stress response. This study identified proteins, including DNA-PK, modified by O-GlcNAc during cellular stress, revealing mechanisms of cell survival.
Area of Science:
- Biochemistry
- Cellular Biology
- Molecular Biology
Background:
- O-linked β-N-acetylglucosamine (O-GlcNAc) is a crucial post-translational modification in metazoans.
- Elevated O-GlcNAc levels are observed following various cellular injuries and confer protection against stress.
- O-GlcNAc is implicated as a regulator of the cellular stress response, but its precise mechanisms remain unclear.
Purpose of the Study:
- To elucidate the molecular mechanisms by which O-GlcNAc modulates protein function and enhances cell survival under stress.
- To identify specific proteins that undergo O-GlcNAcylation in response to cellular stress, particularly heat shock.
- To investigate the potential role of O-GlcNAc in regulating DNA damage signaling and repair pathways.
Main Methods:
- Utilized stable isotope labeling with amino acids in cell culture (SILAC) to identify O-GlcNAcylated proteins after heat shock.
- Analyzed proteins identified for their known functions, including DNA binding and interactions with DNA-PK.
- Performed experiments to confirm O-GlcNAc modification of DNA-PK in response to various cellular stresses.
Main Results:
- Identified numerous proteins, including NF-90, RuvB-like 1 (Tip49α), RuvB-like 2 (Tip49β), and COPII vesicle transport proteins, as targets of O-GlcNAcylation during heat shock.
- Observed that many identified proteins interact with double-stranded DNA-dependent protein kinase (DNA-PK) or double-stranded DNA breaks.
- Demonstrated that DNA-PK itself is modified by O-GlcNAc in response to diverse cellular stress conditions.
Conclusions:
- O-GlcNAc modification plays a significant role in cellular stress tolerance by regulating proteins involved in DNA damage signaling and repair.
- The O-GlcNAcylation of DNA-PK suggests a direct link between this modification and the cellular response to DNA damage.
- These findings provide novel insights into the molecular mechanisms underlying O-GlcNAc-mediated cytoprotection during cellular stress.
Related Concept Videos
02:36DNA Damage can Stall the Cell Cycle
02:36DNA Damage Can Stall the Cell Cycle
02:25Overview of DNA Repair
Chemically...
02:25Overview of DNA Repair
Chemically...
01:38Nucleotide Excision Repair
Cells are regularly exposed to mutagens—factors in the environment that can damage DNA and generate mutations. UV radiation is one of the most common mutagens and is estimated to introduce a significant number of changes in DNA. These include bends or kinks in the structure, which can block DNA replication or transcription. If these errors are not fixed, the damage can cause mutations, which in turn can result in cancer or disease depending on which sequences are...
01:08Nucleotide Excision Repair