In partial fulfillment of the requirements for the degree of
Master of Science in Biology
in the
School of Biological Sciences
Supreet Singh Randhawa
Will defend his thesis
“Characterizing signatures of ribonucleoside monophosphate incorporation in topoisomerase I deficient Saccharomyces cerevisiae strains”
21st July, 2023
12:00 PM
https://gatech.zoom.us/j/93691155720
Thesis Advisor:
Dr. Francesca Storici
School of Biological Sciences
Georgia Institute of Technology
Committee Members:
Dr. Kirill Lobachev
School of Biological Sciences
Georgia Institute of Technology
Dr. Jie Xu
Chemical and Biological Systems Branch
Georgia Tech Research Institute
Abstract: Ribonucleoside monophosphates (rNMPs) are the most common non-canonical nucleotides incorporated into genomic DNA. The 2′-hydroxyl group on the pentose sugar of rNMPs facilitates self-reactivity, increases genotoxic risk of single-strand breaks, and alters the topological and elastic properties of the DNA duplex, all of which drive intracellular metabolic stress. The highly conserved ribonucleotide excision repair (RER) pathway enables error-free removal of rNMPs from genomes across the tree of life. Failures in the RER pathway have been implicated in promoting type I interferonopathies in humans such as Aicardi-Goutières syndrome, a degenerative neurological encephalopathy. Topoisomerase I (Top1), an enzyme traditionally recognized as relieving torsional stress as a result of genome replication or gene transcription, possesses mutagenic ribonuclease activity in the absence of RER. This thesis investigates Top1-cleavage bias towards rNMPs embedded in nuclear and mitochondrial DNA using Saccharomyces cerevisiae as a model organism. Using ribose-seq, we elucidate distinct dinucleotide rNMP incorporation patterns in top1-null mutants suggesting that Top1 may have a cleavage bias towards ribouridine in nuclear DNA. Our analysis reveals no appreciable rNMP removal bias by Top1 when comparing leading and lagging strands, and no distinct dinucleotide rNMP incorporation patterns in mitochondrial DNA regardless of RER or Top1 activity. Taken together, our analysis provides novel insight regarding Top1-cleavage bias towards rNMPs embedded in nuclear DNA, strengthening the idea that Top1 plays a role in preserving genome stability.