Telomeres are specialised nucleoprotein structures that are essential for genome
stability and cell survival. Despite their protective role, telomeres pose a challenge to
the replisome machinery and may present gaps or breaks in mitosis characteristic of
fragile sites. Here, I assess the role of the structure specific endonucleases (SSEs)
MUS81 and GEN1 in the resolution of DNA replication intermediates at telomeres.
Preliminary data reveals the importance of both nucleases in telomere replication
and that GEN1 might act as a backup upon MUS81 depletion. To gain an overview
on the factors recruited to fragile telomeres, an unbiased proteomic approach (PICh)
during telomeric replicative stress was performed. Although several DNA repair
proteins were shown to be enriched at replication stressed telomeres, this analysis
conducted in an asynchronous population seemed to dilute the intrinsic power of
such an approach at identifying specific telomeric stress-response proteins. To
acquire information regarding the source, maintenance and resolution of telomeric
fragile structures, a cell cycle synchronisation protocol was optimised to achieve the
first cell cycle proteomic analysis of telomeres by PICh. This revealed the permanent
binding of shelterin to telomeres through the cell cycle and provided a database of
previously unknown telomere-binding proteins. The novel hSKI complex is found to
be recruited to telomeres in G2 and bind several shelterin components. Depletion of
SKIV2L, which bears the RNA helicase activity and has a role in RNA surveillance,
Abstract reveals high levels of telomere fragility, loss and the activation of the ATM DDR
pathway at telomeres. In addition, telomeric-repeat containing RNAs (TERRAs),
appear to be upregulated in its absence. Collectively, the data identifies SKIV2L and
the hSKI complex as key players in the maintenance of telomere physiology. Overall,
this PhD thesis examines the telomeric proteome remodeling necessary to
safeguard the integrity of the genome and specifically identify and study factors
involved in telomere maintenance.