Inhibition of short patch and long patch base excision repair by an oxidized abasic site

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• 5'-(2-Phosphoryl-1,4-dioxobutane) (DOB) is an oxidized abasic lesion.
• DOB is generated by DNA damaging agents, including antitumor antibiotics.
• DNA polymerase β is crucial for base excision repair in mammalian cells.

Purpose of the Study:

• To investigate the inhibitory mechanism of DOB on DNA polymerase β.
• To explore the role of DOB in DNA repair pathways.
• To understand the cytotoxicity associated with DOB-inducing DNA damage.

Main Methods:

• Enzyme inhibition assays using DNA polymerase β and λ.
• Mass spectrometry-based protein digests to identify modification sites.
• Synthesis and testing of DOB analogues.

Main Results:

• DOB irreversibly inhibits DNA polymerase β and inactivates DNA polymerase λ.
• Lys72 and Lys84 in the DNA polymerase β active site are modified by DOB.
• The 1,4-dicarbonyl moiety of DOB is essential for its inhibitory activity.
• DOB blocks both lyase and polymerase functions of DNA polymerase β, preventing repair.
• DOB is resistant to both short and long patch base excision repair.

Conclusions:

• DOB is a potent inhibitor of DNA polymerase β, impacting base excision repair.
• The inability to repair DOB contributes to the cytotoxicity of DNA damaging agents.
• DOB's mechanism highlights the importance of DNA polymerase β in maintaining genomic integrity.