A steric gate prevents mutagenic dATP incorporation opposite 8-oxo-deoxyguanosine in mitochondrial DNA polymerases

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Reactive oxygen species (ROS) induce DNA lesions, compromising genome integrity.
• 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) is a mutagenic DNA lesion formed by ROS.
• DNA polymerases (DNAPs) control the accurate replication of DNA, including lesion bypass.

Purpose of the Study:

• To investigate the role of specific amino acid residues in family-A DNAPs in modulating nucleotide incorporation across the 8-oxodG lesion.
• To determine if a steric gate mechanism involving residues Y757 and F763 in yeast mitochondrial DNAP (Mip1) controls 8-oxodG tolerance.
• To explore the potential conservation of this mechanism in human DNA polymerase gamma (HsPolγ).

Main Methods:

• Site-directed mutagenesis was used to create F763 Mip1 mutants.
• Enzymatic assays were performed to measure deoxycytidine monophosphate (dCMP) and deoxyadenosine monophosphate (dAMP) incorporation across 8-oxodG using wild-type and mutant Mip1.
• Comparative analysis of Mip1 and human HsPolγ sequences and structures.

Main Results:

• Both residue F763 and the conserved Y757 in Mip1 form a steric gate.
• This steric gate obstructs the syn conformation of 8-oxodG, thereby reducing dAMP misincorporation.
• The presence of phenylalanine at the corresponding position in human HsPolγ suggests a conserved mechanism.

Conclusions:

• The Y757-F763 steric gate in Mip1 plays a crucial role in maintaining fidelity during 8-oxodG lesion tolerance.
• This mechanism likely prevents the mutagenic incorporation of dAMP opposite 8-oxodG.
• The findings suggest that a similar steric gate mechanism operates in human HsPolγ, contributing to genome stability.