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The replicative mitochondrial DNA polymerase, Polgamma, and its protein regulation are essential for the integrity of the mitochondrial genome. The intricacies of Polgamma regulation and its interactions with regulatory proteins, which are essential for fine-tuning polymerase function, remain poorly understood. Misregulation of the Polgamma heterotrimer, consisting of (i) PolG, the polymerase catalytic subunit and (ii) PolG2, the accessory subunit, ultimately results in mitochondrial diseases. Here, we used single particle cryo-electron microscopy to resolve the structure of PolG in its apoprotein state and we captured Polgamma at three intermediates within the catalytic cycle: DNA bound, engaged, and an active polymerization state. Chemical crosslinking mass spectrometry, and site-directed mutagenesis uncovered the region of LonP1 engagement of PolG, which promoted proteolysis and regulation of PolG protein levels. PolG2 clinical variants, which disrupted a stable Polgamma complex, led to enhanced LonP1-mediated PolG degradation. Overall, this insight into Polgamma aids in an understanding of mitochondrial DNA replication and characterizes how machinery of the replication fork may be targeted for proteolytic degradation when improperly functioning.
The replicative mitochondrial DNA polymerase, Polgamma, and its protein regulation are essential for the integrity of the mitochondrial genome. The intricacies of Polgamma regulation and its interactions with regulatory proteins, which are essential for fine-tuning polymerase function, remain poorly understood. Misregulation of the Polgamma heterotrimer, consisting of (i) PolG, the polymerase catalytic subunit and (ii) PolG2, the accessory subunit, ultimately results in mitochondrial diseases. Here, we used single particle cryo-electron microscopy to resolve the structure of PolG in its apoprotein state and we captured Polgamma at three intermediates within the catalytic cycle: DNA bound, engaged, and an active polymerization state. Chemical crosslinking mass spectrometry, and site-directed mutagenesis uncovered the region of LonP1 engagement of PolG, which promoted proteolysis and regulation of PolG protein levels. PolG2 clinical variants, which disrupted a stable Polgamma complex, led to enhanced LonP1-mediated PolG degradation. Overall, this insight into Polgamma aids in an understanding of mitochondrial DNA replication and characterizes how machinery of the replication fork may be targeted for proteolytic degradation when improperly functioning.


Coordinated DNA polymerization by Polgamma and the region of LonP1 regulated proteolysis.,Riccio AA, Brannon AJ, Krahn JM, Bouvette J, Williams JG, Borgnia MJ, Copeland WC Nucleic Acids Res. 2024 Jun 27:gkae539. doi: 10.1093/nar/gkae539. PMID:38932681<ref>PMID:38932681</ref>
, PMID:38932681<ref>PMID:38932681</ref>


From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>

Latest revision as of 09:12, 4 June 2025

Engaged conformation of the human mitochondrial DNA polymerase gamma bound to DNA

8v54, resolution 4.10Å

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