Watching a double strand break repair polymerase insert a pro-mutagenic oxidized nucleotide

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Oxidized nucleotides, particularly 8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate (8-oxodGTP), are mutagenic and implicated in cancer and disease.
• The mechanisms by which DNA polymerases discriminate against oxidized nucleotides, especially during error-prone double-strand break (DSB) repair, remain unclear.

Purpose of the Study:

• To elucidate the structural and mechanistic basis of DNA polymerase fidelity, specifically how oxidized nucleotides are handled during DSB repair.
• To investigate the role of active site metals in discriminating against or promoting the insertion of oxidized versus undamaged nucleotides.

Main Methods:

• High-resolution time-lapse X-ray crystallography was employed to capture snapshots of DNA polymerase μ during DNA synthesis.
• Structural analysis focused on the interactions between the polymerase, DNA template, incoming nucleotides (dGTP and 8-oxodGTP), and active site metal ions.

Main Results:

• A third active site metal ion was identified that facilitates the insertion of both oxidized and undamaged dGTP opposite template cytosine.
• This metal ion bridges the O8 atom of 8-oxodGTP with product oxygens in the anti-conformation, a conformation not observed opposite template adenine.
• Rotation of template adenine into the syn-conformation enabled the misinsertion of undamaged dGTP, highlighting the role of nucleotide conformation.

Conclusions:

• DNA polymerase μ utilizes metal and substrate dynamics within its active site to circumvent fidelity safeguards.
• The observed mechanism allows for the circumvention of polymerase fidelity, promoting the mutagenic insertion of 8-oxodGTP during DSB repair.
• This finding provides insights into how oxidative damage can lead to mutations and contribute to disease development.