Nitric oxide induced S-nitrosation causes base excision repair imbalance

Area of Science:

• Molecular Biology
• Genetics
• Toxicology

Background:

• Inflammation produces DNA-damaging chemicals, including reactive oxygen and nitrogen species.
• Nitric oxide forms S-nitrosoglutathione (GSNO), which modifies proteins and impacts DNA repair.
• The base excision repair (BER) pathway, initiated by alkyl-adenine DNA glycosylase (AAG), repairs damaged DNA, particularly methylated bases.

Purpose of the Study:

• To investigate the effect of GSNO on BER pathway enzymes and DNA repair.
• To determine if GSNO causes imbalanced BER when cells are exposed to methylating agents.
• To explore the link between GSNO, BER imbalance, and cytotoxicity.

Main Methods:

• Fluorescence-based Multiplexed Host Cell Reactivation Assay (FM-HCR) to assess AAG activity.
• Analysis of abasic (AP) sites and BER intermediates using CometChip technology.
• Cellular assays to measure methylation-induced cytotoxicity.

Main Results:

• GSNO enhances AAG activity, promoting DNA repair initiation.
• GSNO induces translocation of AP endonuclease from the nucleus to the cytoplasm.
• GSNO increases the levels of AP sites and BER intermediates, leading to higher methylation-induced cytotoxicity.

Conclusions:

• GSNO causes an imbalance in the BER pathway by increasing initiation while decreasing AP site processing.
• This BER imbalance results in a toxic accumulation of DNA repair intermediates.
• Inflammatory chemicals like GSNO can drive cytotoxicity and mutagenesis through BER pathway disruption.