Dissertation
RPA DNA-binding domain dynamics: a target for homologous recombination regulation
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2021
DOI: 10.17077/etd.005974
Abstract
Replication protein A (RPA) is the primary single-stranded DNA (ssDNA) binding protein in eukaryotes and is involved in DNA replication, recombination, and repair. The RPA heterotrimer contains six DNA-binding domains (DBDs) connected by flexible linkers. RPA quickly binds and coats exposed ssDNA due to its subnanomolar affinity for ssDNA and abundance (2μM) in the nucleus. How RPA is displaced by lower affinity DNA processing proteins is a persistent question. In homologous recombination (HR), the DNA repair process by which double strand breaks (DSBs) are repaired, RPA coats long stretches of ssDNA as they are resected. The recombinase, Rad51, must form a nucleoprotein filament on this same ssDNA. The recombination mediator, Rad52, is required for efficient replacement of RPA-coated ssDNA with Rad51 nucleoprotein filament, though the underlying mechanism was unclear. Using yeast RPA labeled with an environmentally sensitive fluorophore on DBD-A or D in combination with single molecule total internal reflection fluorescence microscopy (smTIRFM), I was able to directly observe the conformational dynamics of individual DBDs within the RPA-ssDNA complex for the first time. While the RPA-ssDNA complex remained intact, RPA DBD-A and D both fluctuate rapidly through four conformations with varying degrees of ssDNA engagement. Addition of Rad52 resulted in selective restriction of DBD-D conformational dynamics, reducing DBD-D access to ssDNA observed as loss of the most engaged conformation.
In addition to revealing how Rad52 interaction modulated RPA binding behavior, I also investigated the effect of phosphorylation of RPA at residue S178 of RPA70. Addition of the phosphomimetic mutation (S178D) resulted in RPA binding with positive cooperativity when multiple RPA molecules bind adjacently on long ssDNA. The effect of S178D on DBD dynamics was minor, with DBD-A and D maintaining access to all four conformations. While Rad52 specifically restricted the conformations available to DBD-D of wild-type RPA, this effect of Rad52 was eliminated with S178D phosphomimetic RPA.
Together, these studies have revealed the importance of observing the DBD dynamics of RPA within the RPA-ssDNA complex. The specific modulation DBD-D by Rad52 suggests a mechanism by which Rad52 can recruit Rad51 to RPA-coated ssDNA and allow the lower affinity Rad51 to replace RPA on ssDNA and complete HR. The elimination of this Rad52 effect with the addition of the S178D phosphomimetic mutation suggests that interplay between RPA interacting proteins (RIPs) and posttranslational modifications (PTMs) is relevant to understanding how DNA processing pathway choice is regulated via RPA DBD dynamics.
Details
- Title: Subtitle
- RPA DNA-binding domain dynamics: a target for homologous recombination regulation
- Creators
- Colleen Catherine Caldwell
- Contributors
- Maria Spies (Advisor)Pamela Geyer (Committee Member)Marc Wold (Committee Member)Todd Washington (Committee Member)Ernesto Fuentes (Committee Member)Aloysius Klingelhutz (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biochemistry
- Date degree season
- Summer 2021
- DOI
- 10.17077/etd.005974
- Publisher
- University of Iowa
- Number of pages
- xvi, 145 pages
- Copyright
- Copyright 2021 Colleen Catherine Caldwell
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 130-145).
- Public Abstract (ETD)
Maintaining and replicating DNA, the code by which genetic information is stored, are among the critical functions of cells. A host of molecular machines must be strictly choreographed to properly replicate and repair DNA. Molecular machines that act out of place can result in a loss of genetic information and diseases like cancer. What directs the choreography of these molecular machines is a question that remains to be answered.
One molecular machine, the protein Replication protein A (RPA), functions by binding to single-stranded DNA (ssDNA) very tightly. Because ssDNA is exposed in all DNA replication and repair processes, RPA is also involved in these processes. In this thesis, I have investigated how RPA is able to act in so many different processes and pass ssDNA to weaker binding molecular machines. Observation of RPA bound to ssDNA revealed that, together, they remain flexible. Portions of RPA release and rebind the ssDNA, while RPA as a whole remains bound. Another molecular machine, Rad52, interacts with RPA and restricts flexibility to allow weaker binding molecular machines to replace RPA. Mimicking phosphorylation, which modifies RPA by attaching a phosphate molecule, allowed RPA on ssDNA to help and adjacent RPA bind next to it. Mimicking phosphorylation on RPA also stopped Rad52 from restricting RPA flexibility. These findings suggest that factors like molecular machines and modifications simultaneously target RPA and that one factor can cancel the effect of another. Understanding the interplay between factors may reveal how molecular machines that maintain DNA are choreographed.
Understanding how molecular machines like RPA function reveals a complex set of interacting factors that regulate how DNA is repaired and replicated. Mechanistic understanding of these molecular machines allows for a better understanding of how they relate to disease as well as the targeted design of therapeutic drugs.
- Academic Unit
- Biochemistry and Molecular Biology
- Record Identifier
- 9984124171102771
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