Achieving High Replication Fidelity by Intricate Communication between Two Active Sites and Efficient Use of Available Cofactors in Human Mitochondrial DNA Polymerase

Mitochondria, fondly known as the powerhouse of the cell, play an important role of producing ATP for cellular energy through the process of oxidative phosphorylation. Each mitochondrion contains many copies of its own genome. Proper maintenance of mitochondrial genome is important for proper function as mutations and deletions on mitochondrial DNA impairs oxidative phosphorylation and leads to mitochondrial dysfunction, clinically manifesting as cardiovascular, neurodegenerative, and muscular diseases. Yet, many fundamental functions of Pol g, sole replicase in human mitochondria, remain unknown. In this work, we aim to provide structure and function characterization of Pol g with emphasis on replication fidelity and translesion DNA synthesis. First, we provide insight into proofreading pathway in Pol g that can be applied to other replicative DNA polymerases. Using cryo-EM, we have captured three different states of Pol g when bound to error-containing DNA, providing insight into burying wrong nucleotide, initiating primer shuttling, and excising the mismatch. Additionally, we have discovered important residues, coined the fidelity switch, that detect misincorporation in the nascent base pair and initiate proofreading pathway to maintain high fidelity of mitochondrial DNA. Second, we further probe the fidelity gate on Pol g and its additional role in nucleotide incorporation. Through biochemical, structural, and computational studies, we discover that the ribose moiety of the incoming nucleotide, which has been largely ignored in the past, plays an important role in nucleotide incorporation as well as adding to replication fidelity. We show that Watson-Crick base complementarity, which has been long thought to be the sole driving force of the replication fidelity, is necessary, but ribose moiety plays even bigger role. Third, we investigate the role of divalent cations in DNA synthesis ability of Pol g, especially on UV-lesion-containing template. Using biochemical techniques, we discovered that Mg2+ prevents Pol g from replicating over UV-lesion, whereas Mn2+ confer Pol g translesion DNA synthesis ability over UV-lesion. Considering the small active site in Pol g that cannot accommodate bulky UV-lesion, we propose that divalent cations may reshape Pol g’s active site.

Name thesis:degree_name

Molecular Biophysics Educational Track (Doctoral)

Discipline thesis:degree_discipline

Structural Biology and Biophysics

Grantor

The University of Texas Medical Branch at Galveston

Year dc:date.issued

2023

Author dc:creator

• Park, Joon 1995-

Advisors dc:contributor.advisor

• Yin, Yuhui Whitney
• Gagnon, Matthieu

Committee members dc:contributor.committeemember

• Morais, Marc
• Hazra, Tapas
• Patel, Smita

Language dc:language.iso

English

Handle dc:identifier.uri

https://hdl.handle.net/2152.3/12840

OAI identifier oai:identifier

oai:utmb-ir.tdl.org:2152.3/12840