Analisi dei meccanismi di mantenimento dei telomeri in Saccharomyces cerevisiae
Author(s)
Di Domenico, Enea Gino
Date Issued
March 16, 2010
Type
Doctoral Thesis
Abstract
Telomeres protect the ends of eukaryotic chromosomes from undesired processes such as
deletions, loss of genomic information due to end replication problem and degradation.
Telomerase is an essential enzyme required to maintain the appropriate number of telomere
repeats counteracting the loss of terminal DNA due to conventional DNA replication. In fact,
loss of a critical number of telomeric DNA repeats leads to cell-cycle arrest, mediated by the DNA damage checkpoint pathways, with important implications in many eukaryotes including human. Telomere structure is basically conserved between human and yeast, opening the
possibility to study human telomeres in a more amenable genetic system such as budding
yeast. Moreover, since some of the telomeric proteins, belong to essential gene in
Saccharomyces cerevisiae, such as Rap1, the role of such genes can be studied by altering the
telomere sequence thereby blocking the binding of proteins normally associated with telomere.
We have proposed the use yeast with humanized telomeres in, obtained by reprogramming the
RNA component of the telomerase complex. These telomeres, which are not bound by Rap1,
are regulated by an alternative length regulation pathway that allow cell survival with
apparently no telomeric defects. Herein we report the presence of telomeric fusions in yeast
cells harbouring human telomeres (HY strains) and we investigate on the possible role of G-tail
on telomere maintenance.
In the first part of this work we report that budding yeasts carrying human-type
telomeric repeats at their chromosome termini show a chronic activation of the Rad53-
dependent DNA damage checkpoint pathway and a G2/M cell cycle delay. Furthermore, in the
absence of either TEL1/ATM or MEC1/ATR genes, which encoder phosphatidylinositol 3-kinaserelated
kinases (PIKKs), we detected telomere fusions, whose appearance correlates with a
reduced cell viability and a high rate of genome instability. Based on sequence analysis,
telomere fusions occurred primarily between ultrashort telomeres. Microcolony formation
assays argue against the possibility that fusion-containing cells are eliminated by PIKKdependent
signaling. These findings reveal that humanized telomeres in yeast cells are sensed
as a chronically damaged DNA but do not greatly impair cell viability as long as the cells have
a functional DNA damage checkpoint. These results suggest that PIKK kinases are important
for the ability of HY cells to tolerate a certain amount of chronic telomere damage, even
though in other contexts, they prevent the proliferation of cells with acute telomere damage.
In the second part of the work we have investigated the length of the 3’protruding
chromosome end which, in yeast telomeres, is very short (at the limit of detection) in nonsynchronized cells, whereas they are longer (50-100nt) in late S-phase. In our study we have
detected G-tails in non-synchronized yeast cells with human telomeres suggesting that they
are longer and/or maintained through the cell cycle with respect to canonical yeast telomeres.
Analysis of G-tails modulation during cell cycle showed that the length of the telomeric 3’-
overhang is regulated in the course of the cell cycle. Short G-tails are present in G1,
progressively increases in S phase and is maximum in G2. Additionally we have shown that
cells lacking either Tel1 or Rad50 have shot G-tail and this phenotype is also associated with
short telomere. This evidence suggest that either Tel1 and Rad50 pathway promotes telomere
elongation through the regulation of the G-tail. Importantly we found that the HY rad51
mutant, with a 3’-overhang similar in length to the reference strain HY, shows extremely long
telomeres. This result let us to speculate on a possible role Rad51 in the telomere regulation,
probably through a competition with Cdc13 for the telomeric single strand binding. This
hypothesis has been corroborated by the evidence that HY strains undergo Y’ amplification in a
Rad51-dependent manner, without hallmark of senescence and in the presence of a functional
telomerase. To confirm our hypothesis of a competition between Rad51 and Cdc13 for the
telomeric single strand access, we are setting ChIP experiments to measure the levels of
Rad51 binding to humanized telomere.
An added layer of complexity is provided by the analysis of double mutants HY tel1
rad51, which showed an increased level of Y’ amplification. This strain have short G-tail and
short telomere, according to the Tel1 involvement in the modulation of the 3’ overhang and
telomerase engagement, however the presence of a Rad51-indipendent Y’ amplification
mechanism, suggest that Tel1 hold a central role, also in the control of the ALT mechanisms.
Another clue of the importance of Tel1 is provided by ChIP analysis. Actually we have reported, a significative higher level of Tel1 binding in humanized telomere in G2, with respect to the wild type strain. This considerations suggests that the Tel1 pathway for promoting telomere elongation is the result of a multilevel control of this checkpoint protein, on different process such as the G-tail modulation, telomerase loading and activation of ALT mechanisms.
Additional information
Dottorato di ricerca in Genetica e biologia cellulare
Subjects
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