Genomic integrity is preserved by checkpoints, which are signal transduction pathways that serve to delay cell cycle progression in the presence of DNA damage or replication stress. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) complex is a proliferating cell nuclear antigen (PCNA)-like clamp that is loaded onto DNA at structures resulting from damage, and is important for initiating and maintaining checkpoint signaling. Rad9 possesses a C-terminal tail unrelated to PCNA that is phosphorylated constitutively and in response to cell cycle position and DNA damage. Previous studies have identified tousled-like kinase 1 (TLK1) as a kinase that may modify Rad9. This thesis establishes that Rad9 is indeed phosphorylated in a TLK-dependent manner in vitro and in vivo, and that T355 within the C-terminal tail is the primary targeted residue. Phosphorylation of Rad9 at T355 is quickly reduced upon exposure to ionizing radiation before returning to baseline later in the damage response. In addition, TLK1 and Rad9 were shown to interact constitutively, and this interaction is enhanced in chromatin-bound Rad9 at later stages of the damage response. Furthermore, this thesis demonstrates that TLK1 is required for progression through S-phase in normally cycling cells, and that depletion of TLK1 results in a prolonged G2/M arrest upon exposure to ionizing radiation, a phenotype that is mimicked by over-expression of a Rad9-T355A mutant. Given that TLK1 is transiently inactivated upon phosphorylation by Chk1 in response to DNA damage, this work proposes that TLK1 and Chk1 act in concert to modulate the phosphorylation status of Rad9, which in turn plays a role in regulating the DNA damage response. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2013-12-24 10:31:57.987
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/8536 |
| Date | 24 December 2013 |
| Creators | Kelly, Ryan |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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