Consequences of partial chromosome re-replication in mammalian cells

Abstract

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

Date of Award	2013
Original language	English
Awarding Institution	University of Dundee
Supervisor	Julian Blow (Supervisor)