Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Reactive oxygen species (ROS) cause oxidative damage to cellular nucleotide pools, leading to DNA double-strand breaks (DSBs).
• Non-homologous end-joining (NHEJ) is a critical pathway in mammalian cells for repairing DSBs by directly ligating broken chromosomal ends.
• DNA polymerase mu (pol μ) plays a role in NHEJ by inserting ribonucleotides, preparing breaks for ligation, and its fidelity is crucial for genomic stability.

Purpose of the Study:

• To elucidate the structural mechanisms underlying the proficient incorporation of oxidized ribonucleotides (8-oxo-rGTP) by DNA polymerase mu during DSB repair.
• To understand how pol μ bypasses its discrimination checkpoints, allowing the incorporation of oxidized nucleotides that can lead to mutagenesis.

Main Methods:

• Time-lapse crystallography was employed to capture structural snapshots of DNA polymerase mu during nucleotide insertion.
• Computational simulations were utilized to analyze substrate, metal ion, and side chain dynamics during the incorporation process.
• Previous findings on pol μ's lack of discrimination against oxidized deoxyguanosine triphosphate (8-oxo-dGTP) were considered.

Main Results:

• Structural intermediates reveal dynamic processes allowing oxidized ribonucleotides (8-oxo-rGTP) to evade polymerase discrimination checkpoints.
• Pol μ exhibits a structural basis for proficient incorporation of oxidized ribonucleotides during the DSB repair process.
• Inefficient cellular sanitization and repair mechanisms, coupled with abundant nucleotide pools, exacerbate the issue.

Conclusions:

• Pol μ-mediated insertion of oxidized ribonucleotides represents an emerging source of widespread, persistent mutagenesis.
• This process contributes significantly to genomic instability, potentially driving aging and human diseases.
• Understanding these mechanisms is crucial for addressing mutations linked to cancer and other pathologies.