| dc.description.abstract | The six minichromosome maintenance proteins (Mcm2‐7), which presumably constitute the
core of the replicative DNA helicase, are present on chromatin in large excess relative to the numer of
active replication forks. The function of this apparent surplus is not well understood, as most of them
are displaced from the DNA during S‐phase, apparently without having played an active role in DNA
replication. At least in yeast, Xenopus and Drosophila the concentration of MCM proteins can be
reduced without affecting the DNA replication. This is known as the “MCM paradox”. In the first part of
this work we tried to solve this classic controversy of eucharyotic cells. To evaluate the importance of
MCM concentration in human cells, we have used RNA interference (RNAi) to modulate the expression
of Mcm2‐7 genes and effectively reduce the concentration of Mcm2‐7 proteins on chromatin. We found
conditions in which cells are capable of apparently normal replication with a very reduced concentration
of Mcm2‐7 complexes. However, under these conditions of limited licensing, cells progressively
accumulated DNA lesions and displayed chromosomal fragility. An analysis of origin density revealed
that “excess” MCM proteins, although not necessarily active during an unperturbed S‐phase, might
activate a reservoir of backup origins that are required to recover from DNA replication stress. Our data
show that the chromatin‐bound “excess” Mcm2‐7 complexes are essential to maintain genomic integrity
in human cells.
Different lines of evidence show that Mcm2‐7 complex or its individual subunits could be
involved in other processes apart from DNA replication. The systematic Knock‐Down of these proteins
led us to identify an essential role for MCM proteins during the checkpoint activation. Our data indicate
that this role would be limited to the replication checkpoint, where the Mcm2 and Mcm5 proteins
appear to be essential to promote Chk1 activation upon diverse treatments that cause fork stalling. We
show that after checkpoint induction these proteins interacted with the ATR allosteric activator TopBP1,
which mails to relocate to affected replication forks in Mcm2 Knocked‐down cells. These data propose
the participation of these two single proteins in the activation of the replication checkpoint by
locating/estabilizing TopBP1 to stalled forks.
Finally, we tried to identify new proteins associated with the MCM complex that could help to
understand their different functions. This led us to identify Cohesin, a protein complex that mediates
sister chromatid cohesion, as an MCM partner. To our surprise, MCM was not involved in cohesion
establishment but Cohesin appeared to be essential for DNA replication. Cohesin is enriched at
replication origins and its downregulation led to a decreased density of active origins, delaying S‐phase
progression. Taking into account the ability of Cohesin to constrain the DNA topology and its presence in
the nucleoskeleton, we demonstrate that Cohesin is an architectural element of the nucleus
participating in the generation of chromatin loops. This function affects DNA replication, as some of the
matrix‐bound regions are replication origins. This study contributes to our understanding of the roles of
Cohesin in a variety of processes and adds new clues to the higher order chromatin organization in the
nucleus. | en |
