Molecular analysis of claspin function in vertebrate cells

QH301 Biology

Larkin, Conor ; Gillespie, David A.

University:University of Glasgow

Department:Institute of Cancer Sciences

关键词: Claspin, Chk1, ATR, DT40 knock-out, Phosphorylation, Gene targeting, DNA Replication, Protein synthesis.;   

Others  :  http://theses.gla.ac.uk/1809/1/2009larkinphd.pdf

来源: University of Glasgow

【 摘 要 】

Claspin is a large, acidic, DNA binding protein required for the ATR mediated activation of Chk1 in response to DNA replication stress.Upon checkpoint activation Claspin is phosphorylated which allows binding to Chk1.This binding in turn promotes the phosphorylation and activation of Chk1 by ATR.Although these findings have led to the designation of Claspin as a mediator protein in the activation of Chk1, the broader biological functions of Claspin have not been extensively explored at the cellular level, in part due to the lack of a genetically tractable system in which to study Claspin function.The purpose of this study was to use reverse genetics in order to generate a Claspin knock-out cell line with which study Claspin function at the cellular level.This was to be achieved by exploiting the recombinogenic vertebrate DT40 cell line, a pre B-cell line of chicken origin.The chicken homologue of Claspin was successfully isolated and characterised.The chicken Claspin orthologue shares 56% identity with human Claspin and the overall domain structure of Claspin appears to be conserved based on sequence comparison.This information facilitated the construction of gene targeting vectors to disrupt Claspin expression in the DT40 cell line.One allele of Claspin was successfully targeted.Despite multiple attempts at disrupting the remaining allele homozygous Claspin knock-out cells were not obtained.This strongly indicates that Claspin is essential for the viability of DT40 cells.In order to circumvent this lethality, efforts were made to conditionally express Claspin in the hemizygous background to facilitate the generation of a conditional knock-out cell line.These efforts proved unsuccessful.Other studies using a polyclonal antibody raised against Claspin investigated phosphorylation of the protein under various stress conditions.Data is presented which shows that phosphorylation of Claspin in response to replication stress is dependent on Chk1 kinase activity.However results from an in vitro kinase assay suggest that Chk1 itself does not directly phosphorylate Claspin.In addition it is also shown that Claspin is phosphorylated following inhibition of protein synthesis.Using specific kinase inhibitors against p38, JNK and mTOR, protein kinases known to be activated during protein synthesis inhibition, the involvement of these kinases in mediating this response was ruled out.The significance of this observation is yet to be determined.Overall the work presented here provides evidence that a conditional DT40 knock-out cell line will be a valuable tool in revealing novel functions of Claspin once a successful rescue of lethality is achieved.