Dissertation
The structure and function of fork remodeling helicase RAD5
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2021
DOI: 10.17077/etd.006021
Abstract
Humans are regularly exposed to harmful factors that damage DNA. The handling of damaged DNA, particularly during DNA replication, poses a major obstacle for cells. During replication, the replication fork will stall at sites of DNA damage, putting the cell at risk for chromosomal rearrangements and double stranded breaks that often result in cell death. To prevent such devastating outcomes, the cell utilizes damage-bypass pathways that allow for continued DNA replication through regions of damage. This research focuses on one such pathway, error-free template switching.
In yeast, error-free template switching relies upon the activity of the Rad5 protein. Unlike other damage-tolerance pathways, Rad5 rearranges DNA in such a way that regions of damaged DNA are not used as a template during the replication process. As a result, while other damage-bypass pathways inaccurately add nucleotides across from sites of DNA damage (thus introducing disease-causing mutations), error-free template switching always leads to the incorporation of the intended nucleotide, thereby preventing the introduction of mutations.
The goal of this research is to understand the mechanism of Rad5-mediated DNA rearrangement in error-free template switching from structural and biochemical perspectives. I show that Rad5 contains a highly flexible N-terminal tail, and that there is an intramolecular interaction that holds two domains of this protein in close proximity to one another. In addition, I show that Rad5 forms a nonproductive complex with DNA in the absence of ATP, that Rad5 is capable of unwinding various DNA substrates, and that RPA stimulates Rad5-mediated fork reversal. These discoveries have advanced our understanding of the mechanism by which Rad5 carries out error-free DNA damage bypass.
Details
- Title: Subtitle
- The structure and function of fork remodeling helicase RAD5
- Creators
- Melissa Shelby Gildenberg
- Contributors
- Marc Wold (Advisor)Brandon Davies (Committee Member)Maria Spies (Committee Member)Michael Schnieders (Committee Member)Jon Houtman (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biochemistry
- Date degree season
- Spring 2021
- DOI
- 10.17077/etd.006021
- Publisher
- University of Iowa
- Number of pages
- xxi, 83 pages
- Copyright
- Copyright 2019 Melissa Shelby Gildenberg
- Comment
- This thesis has been optimized for improved web viewing. If you require the original version, contact the University Archives at the University of Iowa: https://www.lib.uiowa.edu/sc/contact/
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 76-83).
- Public Abstract (ETD)
All cells contain DNA, the molecule that encodes the instructions for the cell. For an organism to grow, cells must replicate often, and in doing so they copy their DNA. Copying DNA correctly is important for maintaining the correct instructions for the cell, which prevents the development of diseases. However, DNA is frequently damaged by many factors, and damaged DNA cannot be copied easily. This difficulty arises because the molecules that normally copy DNA get stuck when they encounter damaged DNA. If these molecules stay stuck for too long the cell will die. To prevent this, cells have developed two ways of copying damaged DNA.
In the first pathway, another molecule (one that does not get stuck) copies DNA through the damaged area. However, this molecule does not always copy DNA correctly, which leads to mutations and disease. In the second pathway, a molecule called Rad5 rearranges the DNA so the damaged region is not used for the copying process at all. Here the normal molecules that copy DNA correctly are used, thus avoiding the creation of mutations.
The goal of my research was to understand how Rad5 rearranges DNA. I studied the flexibility of Rad5, which can ultimately tell us how frequently this molecule contacts DNA and what roles the different parts of this molecule play. I also tested the ability of Rad5 to rearrange different types of DNA in different contexts. Overall, these results helped us better understand how Rad5 carries out this very important process.
- Academic Unit
- Biochemistry and Molecular Biology
- Record Identifier
- 9984097077802771
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