Dissertation
Structures and non-canonical functions of E3 ubiquitin ligases in DNA damage bypass
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2023
DOI: 10.25820/etd.007038
Abstract
DNA damage is caused by several different sources, such as ultraviolet and ionizing radiation and many chemicals found in the environment. DNA damage is extremely problematic because it interferes with DNA replication, thus leading to mutations, genomic instability, and ultimately cell death. To prevent DNA damage from interfering with DNA replication, eukaryotes possess multiple DNA damage bypass pathways, such as translesion synthesis and template switching. Both of these DNA damage bypass pathways are regulated through the ubiquitylation of the homo-trimeric sliding clamp, proliferating cell nuclear antigen (PCNA). In translesion synthesis and template switching, there are two E3 ubiquitin ligases that are responsible for the ubiquitylation of PCNA: Rad18 and Rad5. Rad18 and Rad5 possess both a canonical role (ubiquitylating PCNA) and a non-canonical role. However, the non-canonical roles of Rad18 and Rad5 are unknown.
First, Rad5 is a fork-reversal helicase. Little is known mechanistically of Rad5’s helicase activity, so I wanted to determine the optimal substrate for Rad5. I found that Rad5 preferentially reverses fork DNA substrates with short gaps (10 to 30 nt.) in the leading strand. Thus, Rad5 preferentially reverses fork DNA substrates that form chicken foot intermediates with 5’ overhangs that can be extended by replicative DNA polymerases during the subsequent steps of template switching. I also showed that Rad5 is both a DNA-DNA helicase and an RNA-DNA helicase. Furthermore, I found that Rad5 binds fork DNA substrates in a 1:1 ratio. Lastly, I found that RPA reduces the rate of fork reversal in some, but not all substrates. In conclusion, my studies have led us to having a greater understand of how Rad5 reverses fork DNA substrates to promote DNA damage bypass.
Second, Rad18 is a scaffolding protein. Little is known about the structural basis of Rad18 scaffold function, so I wanted to model the Rad18 protein. To model the Rad18 protein, I used small-angle X-ray scattering (SAXS) and mass photometry with molecular simulations to examine the structure and conformational flexibility of Rad18. I found a that Rad18 forms a complex with Rad6 in a 2:2 oligomeric state. It has been reported that Rad18 constitutively binds pol η, but its binding to Rev1 is controlled by auto-inhibition. I found that the release of this auto-inhibition is associated with the dissociation of the Rad6-Rad18 complex to free Rad18 monomers and Rad6-Rad18 dimers. In conclusion, I have generated a model of the Rad18 protein complex and given insight into the structural basis of Rad18 scaffold function. Taken together, my studies have led to a greater understanding of the non-canonical roles of E3 proteins in regulating DNA damage bypass.
Details
- Title: Subtitle
- Structures and non-canonical functions of E3 ubiquitin ligases in DNA damage bypass
- Creators
- Justin A. Ling
- Contributors
- M Todd Washington (Advisor)Sheila Baker (Advisor)Maria Spies (Committee Member)Lori Wallrath (Committee Member)Daniel Weeks (Committee Member)John Koland (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biochemistry and Molecular Biology
- Date degree season
- Summer 2023
- DOI
- 10.25820/etd.007038
- Publisher
- University of Iowa
- Number of pages
- xviii, 157 pages
- Copyright
- Copyright 2023 Justin A. Ling
- Language
- English
- Date submitted
- 06/29/2023
- Description illustrations
- illustrations, tables, graphs
- Description bibliographic
- Includes bibliographical references (pages 116-138).
- Public Abstract (ETD)
- DNA damage causes DNA replication to stop in cells. This has been linked to many diseases and cancers. Cells can bypass the DNA damage to prevent DNA replication from stopping. Thus, the bypass of DNA damage prevents disease. Eukaryotes have two DNA damage bypass pathways called translesion synthesis and template switching. Both pathways are controlled by two proteins called Rad18 and Rad5. However, Rad18 and Rad5 do more than control translesion synthesis and template switching. Both proteins have other functions. Rad5 is a helicase that remodels the structure of DNA. Rad18 is a scaffolding protein that recruits other translesion synthesis proteins to the site of DNA damage.
First, in this thesis, I study Rad5’s DNA remodeling activity. I discovered that Rad5 has a preferred DNA structure that allows for the DNA damage to be bypasses. Second, I studied Rad18’s structure. I discovered that structure of Rad18 changes and controls its protein-protein interactions. Overall, I found that both Rad18 and Rad5 do much more than regulate the DNA damage bypass pathways.
- Academic Unit
- Biochemistry and Molecular Biology
- Record Identifier
- 9984454742602771
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