Investigations of genome instability utilizing quantitative proteomics
Loading...
Date issued
Authors
Editors
Journal Title
Journal ISSN
Volume Title
Publisher
Reuse License
Abstract
The field of mass spectrometry-based proteomics has had a profound impact on discoveries in almost every field of science. Specifically, bottom-up proteomics enables the exploration of scientific questions at a high-throughput and proteome-wide level. In this thesis, mass spectrometry-based proteomics was employed to understand aspects of genome instability related to: 1) telomere biology in Caenorhabditis elegans , 2) phylogenetic diversity of recognition and repair of in vitro DNA damage lesions, and 3) DNA damage kinetics in Tetrahymena thermophila.
Article I (Dietz et al. 2021) describes the extensive characterization of the first novel double-stranded telomere binders in C. elegans , TEBP-1 and TEBP-2. These proteins were discovered using in vitro telomere pulldown assays coupled with label-free and dimethyl quantitative mass spectrometry. TEBP-1 and TEBP-2 bind directly and specifically to double-stranded telomeric DNA. Both proteins are critical to the negative and positive regulation of telomere homeostasis. The double knockout strain of tebp-1;tebp-2 exhibits severe germline arm atrophy and synthetic sterility, suggesting their critical role in fertility. TEBP-1 and TEBP-2dimerize and directly interact with the known single-stranded binder POT-1, thereby connecting them to the known telomere complex in C. elegans.
Article II (Nischwitz and Schoonenberg et al., 2023) explores the conservation of recognition and repair of DNA damage lesions. Due to the imperative need for accurate maintenance of the genome, DNA repair has been highly conserved across all domains of life. To study both the shared and unique elements of the DNA damage response, we conducted a phylointeractomic study to identify enriched binders in 11 different species at the 8-oxoG and abasic lesions, as well as a uracil base incorporated into DNA. While numerous binders were canonical DNA damage factors, we also observed enrichment of proteins not previously associated with DNA repair. Through orthology, network, and domain analysis, we linked 44 proteins that were previously unassociated to DNA repair.
Article III (unpublished, Nischwitz and Schoonenberg et al., xxxx) delves into the kinetics of the DNA damage response (DDR) in the ciliate Tetrahymena thermophila (Tetrahymena) . To date, there have been limited studies that combine the power of proteomics and transcriptomics to investigate DNA damage kinetics across various treatments. Our screen monitored the dynamic DNA damage response over eight hours after exposure to six different mutagens. We observed upregulation of previously associated DNA damage repair pathways, as well as unexpected DDR crosstalk. All treatments elicited a dynamic response at both the transcript and protein level. Through unsupervised machine learning clustering, we examined expression profile trends to gain a more comprehensive understanding of the DDR, as many of these proteins exhibited damage-specific responses. Currently, we are employing a knockdown system to target a subset of these PARP-related proteins to further characterize their specific roles in Tetrahymena.