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Date of Award

Summer 2025

Document Type

Masters Thesis

Department or Program Affiliation

Chemistry

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Cullati, Sierra

Second Advisor

Antos, John M.

Third Advisor

Amacher, Jeanine

Abstract

CK1 enzymes are conserved acidophilic serine/threonine kinases involved in DNA repair and damage response pathways, contributing to homologous recombination and non-homologous end joining. CK1 activity is regulated by autophosphorylation in cis at two regions, the C-terminal tail as well as the kinase domain. Schizosaccharomyces pombe CK1 orthologs, Hhp1 and Hhp2, are known to phosphorylate numerous substrates, but the full scope of their target proteins is unknown, especially those substrates important for DNA repair. Additionally, for substrates involved in DNA repair, the impact of autophosphorylation state on kinase activity is unknown. These impacts upon activity towards DNA repair substrates, like actin-related protein 8 (Arp8), can be studied by assessing kinetic parameters like Vmax, Km and kcat. Chapter 1 of this thesis compares two methods for measuring Hhp1 kinetics parameters Vmax and kcat in vitro, the traditional Michaelis-Menten method and an alternative which resulted in overestimations of Vmax and kcat. Chapter 2 characterizes changes in autophosphorylation state of Hhp1 and Hhp2 in vivo in response to different sources of DNA damage. Further, in chapter 3 we identified and validated a novel in vitro substrate of Hhp1 and Hhp2, the mediator of the replication checkpoint (Mrc1), involved in DNA damage and replication pathways.

Type

Text

Keywords

Biochemistry, CK1s, Kinases, Enzyme Kinetics, DNA Repair, Autophosphorylation, Quantitative Phosphoproteomics

Publisher

Western Washington University

OCLC Number

1530488950

Subject – LCSH

Schizosaccharomyces pombe--Genetics; Protein kinases--Research; Phosphorylation--Research; DNA repair--Regulation; DNA damage--Physiological effect; Cellular signal transduction--Regulation; Protein-protein interactions--Analysis; Enzyme kinetics--Methodology

Format

application/pdf

Genre/Form

masters theses

Language

English

Rights

Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.

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