Identifying a role for Ciz1 in the DNA damage response

In proliferating cells, cell cycle arrest and DNA repair are induced upon detection of DNA damage. The DNA damage response (DDR) to double strand DNA breaks is mediated by phosphatidylinositol 3-kinase-like protein kinases (PIKKs), which propagate DDR signalling by phosphorylating SQ/TQ motifs in substrate proteins. Ciz1 is a vertebrate protein previously shown to stimulate DNA replication initiation and facilitate cyclin exchange at pre-replication complexes. Ciz1 contains several SQ/TQ motif clusters, suggesting it is a PIKK substrate. This thesis investigates the potential role of Ciz1 in the DDR. A cell free system cell free system previously used to study DNA replication initiation was adapted to permit study of events resulting from etoposide-induced DNA damage. The system was characterised, and its potential as a screening tool to assess inhibitors of the DDR demonstrated, before being used to confirm PIKK-dependent phosphorylation of SQ/TQ motifs in both full length Ciz1 and a C-terminal domain fragment, Ciz1-C274. This phosphorylation is inhibited by both wortmannin and LY294002, implicating DNA-PK as the responsible kinase. More traditional methods including immunofluorescence and RT-PCR were used to characterise the effects of DDR signalling upon endogenous and overexpressed Ciz1. The resulting data show that Ciz1 does not localise to foci of phosphorylated histone H2AX, unlike many known DDR signalling proteins. However etoposide treatment affected levels of C-terminal mRNA in intact cells, increasing transcription of exon 16 in WI38 cells, but decreasing overall mRNA levels in a cancer cell line. Instead the potential of Ciz1 to act as a modulator of DNA replication initiation through its established role in pre-replication complex assembly is discussed, and three models by which it may act are identified. I hypothesise that phosphorylation of Ciz1 stabilises cyclin E upon the pre-replication complex, preventing the CDK2-cyclin A complex from forming, and preventing origin licensing.

Name dc:type.qualificationname

Ph.D

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of York

Year dc:date.issued

2011

Author dc:creator

• Roper, Katherine

Advisor dc:contributor.advisor

• Coverley, Dawn

Identifier

uk.bl.ethos.547339

OAI identifier oai:identifier

oai:etheses.whiterose.ac.uk:1714