View/Open - Università degli Studi di Milano-Bicocca

Abstract
Eukaryotic cells distinguish their chromosome ends from accidental DNA
double-strand breaks (DSBs) by packaging them into protective structures
called telomeres that prevent DNA repair/recombination activities. In this
work, we investigated the role of key telomeric proteins in protecting
Saccharomyces cerevisiae telomeres from degradation. We show that the
shelterin-like proteins Rif1, Rif2, and Rap1 inhibit nucleolytic processing at
both de novo and native telomeres during G1 and G2 cell cycle phases, with
Rif2 and Rap1 showing the strongest effects. Also Yku prevents telomere
resection in G1, independently of its role in non-homologous end joining. Yku
and the shelterin-like proteins have additive effects in inhibiting DNA
degradation at G1 de novo telomeres. In particular, while Yku plays the major
role in preventing initiation, Rif2 and Rap1 act primarily by limiting extensive
resection. Finally, Rap1 and Rif2 prevent telomere degradation by inhibiting
MRX access to telomeres, which are also protected from the Exo1 nuclease by
Yku. Thus, chromosome end degradation is controlled by telomeric proteins
that specifically inhibit the action of different nucleases.
Since Rif1 plays a very minor role in protecting wild type telomeres from
degradation, we further investigated whether Rif1 participates in telomere
protection in combination with other capping activities, like those exerted by
the CST complex (Cdc13-Stn1-Ten1). We found that, unlike RIF2 deletion, the
lack of RIF1 is lethal for stn1ΔC cells and causes a dramatic reduction in
viability of cdc13-1 and cdc13-5 mutants. Both cdc13-1 rif1Δ and cdc13-5 rif1Δ
cells display very high amounts of telomeric single-stranded DNA and DNA
damage checkpoint activation, indicating that severe defects in telomere
integrity cause their loss of viability. In agreement with this hypothesis,
lethality in cdc13 rif1Δ cells is partially counteracted by the lack of the Exo1
nuclease, which is involved in telomeric single-stranded DNA generation. Like
CDC13, RIF1 also genetically interacts with the Polα-primase complex, which is
involved in the fill-in of the telomeric complementary strand. Thus, these data
highlight a novel role for Rif1 in assisting the essential telomere protection
function of the CST complex.
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