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Action of mitochondrial DNA polymerase gamma at sites of base loss or oxidative damage
K G Pinz1, S Shibutani, D F Bogenhagen
1Department of Pharmacological Sciences, State University of New York, Stony Brook 11794-8651, USA.
Abstract:
Mitochondrial DNA is subject to oxidative damage generating 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) residues and to spontaneous or induced base loss generating abasic sites. Synthetic oligonucleotides containing these lesions were prepared and used as templates to determine their effects on the action of Xenopus laevis DNA polymerase gamma. An analogue of an abasic site in DNA, tetrahydrofuran, was found to inhibit elongation by DNA polymerase gamma. When the DNA polymerase was able to complete translesional synthesis, a dA residue was incorporated opposite the abasic site. In contrast, elongation by DNA polymerase gamma was not inhibited by an 8-oxo-dG residue in the template strand. The polymerase inserted dA opposite 8-oxo-dG in approximately 27% of the extended products. The effects of these lesions on the 3'-->5' exonuclease proofreading activity of DNA polymerase gamma were also investigated. The 3'-->5' exonuclease activity excised any of the four normal bases positioned opposite either a tetrahydrofuran residue or 8-oxo-dG, suggesting that proofreading may not play a major role in avoiding misincorporation at abasic sites or 8-oxo-dG residues in the template. Thus, both of these lesions have the prospect of causing high rates of mutation during mtDNA replication.
Insights
Mitochondrial DNA lesions like abasic sites and 8-oxo-dG can cause mutations. DNA polymerase gamma struggles with abasic sites, inserting adenine, but tolerates 8-oxo-dG, potentially leading to replication errors.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Mitochondrial DNA (mtDNA) is vulnerable to oxidative damage, forming lesions like 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) and abasic sites.
- These DNA lesions can impede DNA replication and lead to mutations, impacting mitochondrial function.
Purpose of the Study:
- To investigate the effects of abasic sites and 8-oxo-dG on the activity of Xenopus laevis DNA polymerase gamma.
- To determine how these lesions affect DNA elongation, translesional synthesis, and proofreading by mitochondrial DNA polymerase.
Main Methods:
- Synthesis of DNA oligonucleotides containing abasic site analogues (tetrahydrofuran) and 8-oxo-dG.
- Assessing the activity of Xenopus laevis DNA polymerase gamma using these damaged DNA templates.
- Analyzing the incorporation of nucleotides opposite lesions and the 3'-->5' exonuclease proofreading activity.
Main Results:
- Tetrahydrofuran, an abasic site analogue, inhibited DNA polymerase gamma elongation, with dA incorporation opposite the lesion during translesional synthesis.
- Elongation by DNA polymerase gamma was not inhibited by 8-oxo-dG, and dA was inserted opposite 8-oxo-dG in approximately 27% of products.
- The 3'-->5' exonuclease activity excised normal bases opposite both tetrahydrofuran and 8-oxo-dG, indicating limited proofreading at these sites.
Conclusions:
- Abasic sites and 8-oxo-dG lesions pose a significant risk for inducing mutations during mtDNA replication.
- The limited inhibition and proofreading by DNA polymerase gamma at these lesions suggest potential for high mutation rates in mitochondrial DNA.