Use this URL to cite or link to this record in EThOS:
Title: Understanding the activation mechanism of the Chk1 kinase
Author: Zhang, Yue
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
Faithful replication of DNA and correct segregation of duplicated chromosomes into two daughter cells are essential to ensure genome integrity and cell survival. Genome integrity is constantly threatened by endogenous and exogenous sources of DNA damage. Consequently, eukaryotic cells have evolved the DNA damage response (DDR), a signalling network that monitors and repairs DNA lesions promptly and efficiently. The ATR-Chk1 pathway is an essential component of the DDR, which is activated by single-strand DNA (ssDNA) generated as a result of DNA replication stress in S phase. Chk1 is a threonine/serine kinase that transduces the DNA damage signal and promotes cell cycle arrest to allow time for DNA repair. The Chk1 structure consists of conserved N-terminal kinase (Chk1KD) and C-terminal regulatory domains (Chk1RD). Available evidence has shown that the isolated Chk1KD is constitutively active, but how the enzymatic activity is regulated in the context of the full length (Chk1FL) kinase is unknown. In this thesis, the relative enzyme efficiency of purified, recombinant Chk1KD and Chk1FL was quantitatively studied using kinase assays. It was found that the enzyme efficiency of Chk1FL was up to two orders of magnitude lower than that of Chk1KD. Biophysical and biochemical characterisation of Chk1FL provided evidence of a compact shape and an intramolecular association of the Chk1RD with the Chk1KD. A putative Chk1RD-binding site on the Chk1KD surface was identified and disrupted by mutagenesis, resulting in partial increase of Chk1FL kinase activity, thus supporting an inhibitory role of the Chk1RD in controlling kinase activity. Activation of Chk1 requires its phosphorylation by the PI3K-like ATR kinase and its recruitment to the replisome via a direct interaction with the core replisome component, Claspin. The Chk1-binding domain of Claspin contains a tandem repeat of three phosphothreonine/serine motifs, which need to be phosphorylated for interaction. However, the structural basis of Chk1 binding remains presently unclear. The Chk1-Claspin interaction was analysed by alanine scanning of Claspin's Chk1-binding motif and Chk1 binding to the mono-phosphorylated Chk1-binding domain of Claspin prepared using the amber codon suppression method. Furthermore, the affinity of Chk1KD and Chk1FL for a phosphorylated Claspin peptide spanning a single Chk1-binding motif or the three motifs was determined using fluorescence polarization and bio-layer interferometry. Chk1FL kinase activity was increased by the presence of a Claspin peptide corresponding to a single phosphorylated Chk1-binding motif. The results of my thesis provide new insights into the mechanism of Chk1 function. They support a model of Chk1 auto-inhibition mediated by the intramolecular interaction of its kinase and regulatory domains, and of Chk1 enhanced activity promoted by interaction with its replisome partner Claspin.
Supervisor: Pellegrini, Luca Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Cell-cycle checkpoint ; DNA damage response ; Chk1