Consequences of partial chromosome re-replication in mammalian cells

To prevent re-replication of DNA in a single cell cycle, the licensing of<br/>replication origins by Mcm2-7 is prevented during S and G2 phases. Metazoans<br/>achieve this by cell cycle regulated proteolysis of the essential licensing factor<br/>Cdt1 and formation of an inhibitory heterohexameric complex of Cdt1 with a<br/>small protein called geminin. The consequences of either stabilising Cdt1 or<br/>ablating geminin in synchronised human U2OS cells are investigated in this<br/>PhD Thesis to elucidate the possible contribution of re-replication in gene<br/>amplifications or rearrangements commonly seen in human tumours. I show that following geminin loss, cells complete an apparently normal<br/>S-phase, but a proportion arrests at the G2/M boundary. When Cdt1 starts to<br/>accumulate in these cells, DNA re-replicates, suggesting that the key role of<br/>geminin is to prevent re-licensing in G2. Inhibition of cell cycle checkpoints in<br/>cells lacking geminin promotes progression through mitosis without detectable<br/>levels of re-replication. Checkpoint kinases thereby amplify re-replication into an<br/>all-or-nothing response by delaying geminin depleted cells in G2 phase.<br/>Comparative Genomic Hybridisation (CGH) array and Solexa Deep DNA<br/>sequencing revealed that re-replication after geminin depletion does not appear<br/>at preferential genomic regions within the human genome. This is consistent<br/>with a recent observation that G2 cells have lost their replication timing<br/>information and reduplicate their genome stochastically. In contrast, when Cdt1<br/>is stabilised by the neddylation inhibitor MLN4924, re-replication starts directly<br/>from within S-phase raising the question whether alternative mechanisms of may cause distinct genomic consequences.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

University of Dundee

Year dc:date.issued

2013

Author dc:creator

• Klotz-Noack, Kathleen

Advisor dc:contributor.advisor

• Blow, John

• Chromosomes
• Mammals
• Cells

Language dc:language

eng

Identifier

oai:discovery.dundee.ac.uk:studenttheses/11c776ae-8192-4993-b317-7eb27d8dbf49

OAI identifier oai:identifier

oai:discovery.dundee.ac.uk:studenttheses/11c776ae-8192-4993-b317-7eb27d8dbf49