Oxidized nucleotide insertion by pol β confounds ligation during base excision repair

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Oxidative stress generates reactive oxygen species, leading to DNA precursor oxidation.
• Oxidized purine nucleotides can be incorporated into DNA during replication and repair.
• Base excision repair (BER) is the primary pathway for correcting oxidized DNA bases, with DNA polymerase β (pol β) playing a key role in vertebrates.

Purpose of the Study:

• To investigate the impact of oxidized purine nucleotides on the DNA ligation step of BER.
• To explore the potential for BER-mediated toxic DNA strand breaks under oxidative stress.
• To examine the role of MTH1 in preventing cytotoxicity from oxidized precursor nucleotides.

Main Methods:

• In vitro assays to assess the DNA ligation step of BER after pol β insertion of oxidized purine nucleotides.
• Cytotoxicity assays on mouse fibroblasts expressing pol β treated with oxidizing agents.
• Analysis of cytotoxicity in MTH1 knockout or MTH1 inhibitor-treated cells.

Main Results:

• The DNA ligation step in BER is impaired when pol β inserts oxidized purine nucleotides into the BER intermediate.
• Oxidizing agent treatment of pol β-expressing mouse fibroblasts leads to enhanced cytotoxicity, suggesting DNA strand break formation.
• MTH1 knockout or inhibition increases cytotoxicity, indicating the significance of precursor nucleotide oxidation.

Conclusions:

• Oxidized purine nucleotide incorporation during BER can compromise DNA ligation and potentially lead to toxic strand breaks.
• These findings suggest a mechanism by which oxidative stress contributes to DNA damage and cell death.
• MTH1 plays a crucial role in preventing the accumulation of oxidized precursor nucleotides and subsequent cytotoxicity.