Year: 2021
Type: dissertation
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.
DOI: 10.17077/etd.006021
Authors: Melissa Shelby Gildenberg, Marc Wold, Brandon Davies, Maria Spies, Michael Schnieders, Jon Houtman
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funders: U.S. Department of Energy, Iowa State University, National Institutes of Health, Office of Science, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, National Institute of General Medical Sciences, National Institute of Environmental Health Sciences, Argonne National Laboratory
Awards: G1017699072, G102163711, G107546177, G1313983767, G1555076680, G1664757510, G2008846556, G2287611682, G262962599, G2853440140, G3075337988, Investigation of Rad5 Structure and Function in Error-Free Template Switching, G3961716793, G409777406, G498139845, G5499275266, G5607048534, Mechanisms of Damaged DNA Replication in Eukaryotes, G6918803902, G7532037738, G7701581323, G7758485239, G9000275877, G993149817
OpenAlex ID: W4226017136