Dissertation
Visualizing DNA polymerase iota catalyze Hoogsteen-directed DNA synthesis
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2025
DOI: 10.25820/etd.008023
Abstract
DNA is constantly damaged by both exogenous and endogenous sources. This damage interferes with normal DNA replication and leads to genome instability. When the replication fork encounters DNA damage in the template strand, the replicative polymerases stall. To overcome these replication blocks, cells are equipped with multiple pathways to bypass the damage. One pathway is Translesion Synthesis, which utilizes polymerases that have evolved to specifically incorporate nucleotides across DNA lesions. The research presented in this thesis focuses on DNA polymerase iota. Vertebrates evolved DNA polymerase iota to accurately incorporate across minor groove and exocyclic purine adducts. Conventional X-ray crystallography showed that this enzyme accommodates damaged adenines and guanines by rotating the template base from the anti to the syn configuration forming Hoogsteen base pairs with the incoming nucleotide. In contrast, polymerase iota is highly inaccurate when incorporating across template pyrimidines. Thus, to prevent mutations, it is vital that the cell utilizes polymerase iota only for its cognate lesions.
The goal of this research is to understand the entirety of the nucleotide incorporation reaction utilized by polymerase iota, so that we can better comprehend how the cell selects the correct polymerase during DNA damage bypass. I show that polymerase iota maintains Hoogsteen base pairing throughout the entirety of the reaction in crystalo, but that the nascent base pair becomes less stable at each step, thereby promoting the flip from syn to anti configuration necessary for the polymerase to translocate along the DNA. Additionally, I demonstrate that polymerase iota has a previously uncharacterized pyrophosphatase activity. These discoveries have advanced the DNA polymerase field by providing insight into the mechanisms of DNA synthesis across lesions and highlighting the unique ability of polymerase iota during bypass.
Details
- Title: Subtitle
- Visualizing DNA polymerase iota catalyze Hoogsteen-directed DNA synthesis
- Creators
- Zachary B. Frevert
- Contributors
- M. Todd Washington (Advisor)Maria Spies (Committee Member)Adrian Elcock (Committee Member)Brandon Davies (Committee Member)Kris DeMali (Committee Member)Robert Piper (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biochemistry
- Date degree season
- Spring 2025
- DOI
- 10.25820/etd.008023
- Publisher
- University of Iowa
- Number of pages
- xiv, 124 pages
- Copyright
- Copyright 2025 Zach Frevert
- Language
- English
- Date submitted
- 04/23/2025
- Description illustrations
- illustrations (some color)
- Description bibliographic
- Includes bibliographical references (page 115-124).
- Public Abstract (ETD)
DNA contains the blueprints for life. It tells cells how to grow and how to function properly. In order for an organism to grow, cells must replicate often – a crucial component of which is copying their DNA. But DNA is constantly being attacked by sunlight, pollution, and even natural bodily processes. These attacks can damage DNA, and if it is not fixed, it can lead to a roadblock for the enzymes that replicate DNA – this leads to cells accumulating mutations or dying from the complications caused by stalled DNA replication.
This research explores how one enzyme called DNA polymerase iota helps cells deal with DNA damage. Unlike the usual polymerases that copy DNA, polymerase iota serves as a backup when the usual process hits a roadblock. Polymerase iota functions to band-aid over the damaged DNA. This allows the usual polymerases to continue, preventing the problems caused by stalled replication.
The goal of my research was to understand how polymerase iota works in detail, including the unique way it interacts with DNA and breaks down some of the byproducts of making DNA. These discoveries help us understand how cells protect themselves from DNA damage and the harmful mutations it can cause.
- Academic Unit
- Biochemistry and Molecular Biology
- Record Identifier
- 9984831124202771
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