View/Open - Università degli Studi di Milano-Bicocca

Introduction
overhangs serve as substrate to telomerase, they are important to maintain
telomere length homeostasis.
Yeast cells acquire telomeric TG1–3 ss overhang in cell cycle regulated manner
[4,199]. The formation of the telomeric G tail and elongation requires the
passage of the replication fork at telomeres [14,200]. Cdk1 activity is required
for the generation of the long telomeric 3′ overhang in late S phase [70].
Passage of replication fork at telomeres, might require the release of telomere
bound proteins and lead to a transient state of telomeric deprotection. At this
stage, CDK1-dependent 5′ resection might take place to generate telomeric
overhangs.
Telomeres must be protected from uncontrolled nucleolytic activities.
Telomere shortening caused by telomerase deletion increases the amount of
telomeric ssDNA in predominantly Exo1 nuclease dependent manner [201]
and triggers a DNA damage and other stress related responses [202]. In yku
null cells, telomeres are shorter with accumulation of telomeric ssDNA and
checkpoint-mediated cell cycle arrest at elevated temperatures. The
generation of telomeric ssDNA in yku70Δ occurs in cell cycle independent
manner and due to action of Exo1 nuclease at telomeres [124,131,203]. In
cdc13-1 mutant, telomeres undergo Exo1 dependent 5’ C strand degradation
and cell cycle arrest at restrictive temperature [31,204]. In contrast to yku
lacking cells, cdc13-1 mutant or cells lacking CDC13 or STN1, the overhang
generation is cell cycle dependent occurring only in G2/M, but not in G1 of the
cell cycle and requires the completion of S phase and Cdk1 kinase activity
[205]. This observation supports the hypothesis that requirement of
replication fork passage might lead to transient unprotected state at
telomeres. Also, normal human telomeres are recognized as DNA damage in
G2 phase of cell cycle [206].
At de novo telomeres, MRX complex is involved in telomeric ss Gtail
generation at telomeres [138]. MRX is required for generation of proper
constitutive telomeric G-tails in linear plasmids [135]. Previous work from our
lab discovered that similar to DSB processing, Sae2, Exo1, Sgs1, Dna2 are all
involved in generation of telomeric overhangs (See figure 12) [207]. Again,
extensive resection of DSB requiring Cdk1 phosphorylation of Sae2, it was also
important for telomere overhang formation and telomere elongation. A very
interesting finding from this work came from Sae2-S267D variant mimicking
25