DNA damage causes replication forks to stall, which can ultimately lead to double strand breaks, chromosomal rearrangements, and cell death. Proliferating cell nuclear antigen is a clamp protein which encircles DNA and acts as a sliding platform for proteins that will act on the DNA, including polymerases. Replication fork stalling causes PCNA to be mono-ubiquitylated, resulting in a polymerase switch from the classical polymerases that perform replication to the Y-family polymerases which conduct translesion synthesis (TLS) to bypass the DNA damage and allow replication to continue. Y-family polymerases include DNA polymerases eta, kappa, iota, and Rev1. The mechanism by which these polymerases are assembled into multi-protein complexes which include PCNA has previously not been well-understood. The studies outlined below demonstrate that ternary complexes involving PCNA, Rev1, and pol η can adopt multiple conformations which can rapidly interconvert. Additionally, I have demonstrated that ubiquitin stimulates the catalytic activity of DNA polymerase eta through a novel interaction with the catalytic core. These studies represent significant progress towards understand the regulation of TLS polymerase activity through both recruitment to and activation by ubiquitylated PCNA.
Dissertation
The regulation of translesion synthesis through the binding and activation of polymerases by PCNA
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2016
DOI: 10.17077/etd.zy8rz6sv
Abstract
Details
- Title: Subtitle
- The regulation of translesion synthesis through the binding and activation of polymerases by PCNA
- Creators
- Elizabeth Marie Boehm - University of Iowa
- Contributors
- M. Todd Washington (Advisor)Maria Spies (Committee Member)Kris DeMali (Committee Member)Ernesto Fuentes (Committee Member)Madeline Shea (Committee Member)Jon Houtman (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biochemistry
- Date degree season
- Summer 2016
- DOI
- 10.17077/etd.zy8rz6sv
- Publisher
- University of Iowa
- Number of pages
- ix, 195 pages
- Copyright
- Copyright © 2016 Elizabeth Marie Boehm
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 171-195).
- Public Abstract (ETD)
DNA is the genetic information of the cell and is composed of building blocks known as bases. DNA is duplicated into two new DNA molecules in a process known as replication. Polymerases are the proteins which act in DNA replication to add bases to the growing DNA strand. They are anchored to the DNA molecule through interactions with proliferating cell nuclear antigen (PCNA), a ring-shaped protein which encircles the DNA.
DNA is damaged through numerous cellular and extracellular mechanisms. This damage stalls replication because the classical polymerases which perform the bulk of replication are unable to bypass the roadblock of DNA damage. This causes the polymerases to switch from classical polymerases to those that can bypass the DNA damage and allow the replication fork to continue. The mechanism by which these damage-tolerating polymerases are recruited to PCNA at stalled replication forks and switch with classical polymerases is not well understood.
In this thesis, I have conducted the first direct studies of two damage-tolerating polymerases binding to PCNA. This knowledge will allow for more focused future studies into controlling the action of these polymerases because, although their action is necessary, they are also associated with increased resistance to chemotherapeutics and persistent cancer.
- Academic Unit
- Biochemistry and Molecular Biology
- Record Identifier
- 9983776925902771
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