AbstractThe number of replication origins licensed with Mcm2-7 double hexamers is in significant excess over the number required in an unperturbed S phase. These excess dormant origins are required to help the cells overcome replicative stress caused by barriers to the replication fork. Cells also regulate replication factories in a Chk1-dependent manner in response to replicative stress. The combination of dormant origin activation and inhibition of replication factory activation helps ensure that the genome is fully replicated during S phase.
Using human tissue culture cells, this thesis further investigates the regulation of replication factories in response to replicative stress. I show that p53 is important for factory regulation. Knockdown of this protein abolishes factory regulation in response to stress, whilst increasing the levels of p53 enhances the factory phenotype. I provide evidence that the effect of p53 on replication factories is mediated by induction of the CDK inhibitor Cip1p21. This is consistent with previous work suggesting that factories are regulated by CDK activity. However, I show that cells lacking p53 can also reduce factory number in response to replicative stresses, suggesting that other pathways exist. I also show a role for RNF4 in factory regulation. In DT40 cells lacking RNF4, or in U2OS cells treated with RNF4 RNAi, factory regulation is abolished in response to stress. I also show that cells lacking dormant origins are hyper-sensitive to the effect of interstrand crosslinkers suggesting an importance for these excess origins in the repair of these DNA lesions.
Understanding the importance of dormant origins to various types of stress and the regulation of replication factories in response to replicative stress could explain why normal and cancer cells have different sensitivity to anti-cancer drugs and may help in the development of screening methods to identify cancer types which are susceptible to specific agents.
|Date of Award||2014|
|Supervisor||Julian Blow (Supervisor)|
An investigation into the regulation of replication factories and dormant origins in human cells
McIntosh, D. (Author). 2014
Student thesis: Doctoral Thesis › Doctor of Philosophy