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Updated: May 23, 2025
Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method
Published on: May 2, 2025
Structural basis for intrinsic strand displacement activity of mitochondrial DNA polymerase
Ashok R Nayak1, Viktoriia Sokolova1, Sirelin Sillamaa2
1Department of Biochemistry and Molecular Biology, Thomas Jefferson University; 1020 Locust St, Philadelphia, USA.
Abstract:
Members of the Pol A family of DNA polymerases, found across all domains of life, utilize various strategies for DNA strand separation during replication. In higher eukaryotes, mitochondrial DNA polymerase γ relies on the replicative helicase TWINKLE, whereas the yeast ortholog, Mip1, can unwind DNA independently. Using Mip1 as a model, we present a series of high-resolution cryo-EM structures that capture the process of DNA strand displacement. Our data reveal previously unidentified structural elements that facilitate the unwinding of the downstream DNA duplex. Yeast cells harboring Mip1 variants defective in strand displacement exhibit impaired oxidative phosphorylation and loss of mtDNA, corroborating the structural observations. This study provides a molecular basis for the intrinsic strand displacement activity of Mip1 and illuminates the distinct unwinding mechanisms utilized by Pol A family DNA polymerases.
Insights
Yeast DNA polymerase Mip1 unwinds DNA independently using novel structural elements. This intrinsic strand displacement activity is crucial for mitochondrial function and DNA maintenance.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA polymerases are essential for replication across all life domains.
- Pol A family polymerases use diverse DNA strand separation strategies.
- Mitochondrial DNA polymerase gamma in higher eukaryotes requires TWINKLE helicase, unlike yeast Mip1.
Purpose of the Study:
- To elucidate the molecular mechanism of DNA strand displacement by yeast Mip1.
- To identify structural elements responsible for Mip1's independent unwinding activity.
Main Methods:
- High-resolution cryo-electron microscopy (cryo-EM) to capture DNA strand displacement.
- Structural analysis of Mip1 variants.
- Functional studies in yeast cells.
Main Results:
- Novel structural elements facilitating downstream DNA duplex unwinding by Mip1 were identified.
- Cryo-EM structures captured the DNA strand displacement process.
- Mip1 variants defective in strand displacement showed impaired oxidative phosphorylation and mtDNA loss.
Conclusions:
- Mip1 possesses intrinsic DNA strand displacement activity, distinct from other Pol A polymerases.
- This activity is mediated by specific structural features.
- Mip1's unwinding mechanism is vital for mitochondrial DNA stability and function.
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