University of Groningen
On the molecular biology of telomeres
Stinus Ruiz de Gauna, Sonia
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Stinus Ruiz de Gauna, S. (2018). On the molecular biology of telomeres: Lessons from budding yeast.
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Chapter 5
Chapter 5
Despite the remarkable advances science has made over the last century, many fundamental
questions remain unanswered. In this thesis, the aim is to contribute to a better understanding
of telomere biology. Specifically, we examine how a cell is able to distinguish between a
double-stranded DNA break (DSB) and a telomere, which resemble each other in structure
and share some proteins, but need to be processed in very different ways. The relevance of
the adequate distinction between DSBs and telomeres becomes apparent when looking at
the consequences of a failure in this process. A DSB is very deleterious for yeast cells, to the
extent that one unrepaired DSB is fatal (Resnick and Martin, 1976; Weiffenbach and Haber,
1981). Therefore, tightly regulated repair mechanisms ensure the proper repair of DSBs.
However, if a DSB is improperly recognized as a chromosome end and it is repaired as
such, telomeric sequence will be added at the broken site. Because genes nearby telomeric
sequence are transcriptionally silenced, addition of telomeric sequence at a DSB will lead to
silencing of genetic information. Moreover, two human diseases have been associated with
truncated chromosomes where telomeric sequence addition occurred, mental retardation
and alpha thalassemia (Wilkie et al., 1990; Wong et al., 1997), highlighting the relevance of
fundamental research for human health. The opposite scenario, where a telomere is treated
as a DSB, also has devastating consequences. Telomere shortening is counteracted by the
specialized reverse transcriptase telomerase (Greider and Blackburn, 1985). It is therefore
very important to keep chromosome ends available for telomerase-dependent extension
and, at the same time, to inhibit the repair mechanisms that act at DSBs. Failure to inhibit
DSB repair results in telomere-telomere fusions that can lead to cell death or
breakage-fusion-bridge cycles, which are thought to be important in the formation of some cancers
(Selvarajah et al., 2006).
A ~40 nt length threshold separates telomeres from DSBs
To answer how a cell discerns between a DSB and a telomere, in chapter 2, we have used
two complementary methods. First, we used artificially constructed DNA ends adjacent to
telomeric sequence of increasing length, and monitored whether such ends are repaired
by telomerase-dependent extension. Second, we developed the iSTEX assay, an inducible
method that allows the
in vivo monitoring of telomerase-dependent telomere extension
events at nucleotide resolution and used it to determine what natural ends were sensed
as telomeres and therefore extended by telomerase. Both methods uncovered a
length-dependent threshold of about 40 nt of telomeric sequence that establishes the difference
between telomeres and DSBs. Below the 40 nt threshold, DNA ends contain too little
telomeric sequence and are treated as DSBs and, above it, DNA ends are extended by
telomerase. We have also found what proteins are important for the establishment of this
length-dependent threshold. Pif1 is important to inhibit telomerase at DNA ends below
the DSB-telomere threshold, as evidenced by the increased telomere addition frequency
at TG
18ends in
pif1-m2 cells. On the other hand, the telomerase recruitment function of
Discussion and future perspectives
93
Chapter 5
DNA ends above the threshold.
An important question that remains unanswered is what is intrinsically different
between the DNA ends below and above the DSB-telomere threshold. One possibility
is that Cdc13 is recruited less efficiently to DNA ends below the threshold.
In vitro, the
minimum binding site of Cdc13 is 11 nt in length (Hughes et al., 2000), so Cdc13 could
have enough space to bind a TG
18end; however, the situation in a living cell might be
different. For example, Cdc13 might not be recruited to such short ends. Alternatively, one
Cdc13 molecule might be recruited, but multiple Cdc13 molecules might be required for
telomere extension. Molecular crowding at such a short end could also affect the binding
or function of Cdc13
in vivo. Another possibility is that Cdc13 recruitment at DNA ends
above the threshold overcomes the telomerase inhibition by Pif1. In this case, when Cdc13
is mutated, telomerase recruitment to the TG
34end might be lost. It is also possible that
Cdc13 recruitment is unaffected at both sides of the threshold, in which case we can
envision at least two options. One is that Pif1 inhibition of telomerase is stronger at DNA
ends below than above the threshold. However, we know that Pif1 inhibits telomerase
both below and above the threshold, indicating that, unless the strength of inhibition is
different below and above the threshold, most likely Cdc13, and not Pif1, is the key player.
Alternatively, it will be important to determine whether the other members of the CST
complex, Stn1 and Ten1, play a role in the establishment of the DSB-telomere threshold.
In short, determining what proteins bind to DNA ends below and above the threshold
would help clarify how telomeres and DSBs are differently recognized and processed.
Development of the inducible STEX assay
Besides defining the length-dependent DSB-telomere threshold, a major contribution
of chapter 2 is the development of the inducible STEX assay (iSTEX). So far, telomere
length has been mostly studied by methods that only provide information about the
average telomere length of a population of cells. However, iSTEX provides information
about telomere extension events on single telomeres at nucleotide resolution. iSTEX
can determine to which telomeres telomerase is recruited and, therefore, whether the
recruitment of telomerase to the shortest telomeres or the recruitment in general is affected.
It also informs about the length of the newly added sequence, and whether the nucleotide
addition processivity or the repeat addition processivity are affected. All these aspects are
informative to understand what is the cause of telomere length change at a molecular level.
Moreover, iSTEX has two improvements with respect to the classic STEX assay
(Teixeira et al., 2004). First, the use of an inducible system largely facilitates the execution
of the experiment because it eliminates the need of the nearly 100% mating efficiency
between the donor (telomerase positive) and the recipient (telomerase negative) strains
required in the original STEX assay (Teixeira et al., 2004). This level of mating efficiency
is rarely observed. Second, the use of the
tlc1-tm template mutant ensures that all telomere
Chapter 5
classic STEX relies on the imperfect nature of the telomere repeats added by the yeast
telomerase. The telomeric sequences are aligned to a reference sequence. The sequence of
the extended telomeres does not align to the non-extended ones, in an otherwise clonal
population. Those misalignments are called divergent events and are the readout for
telomere extension events. However, telomere divergence events can arise from artefacts
generated during PCR amplification, cloning and sequencing (Claussin and Chang, 2016).
To overcome this issue, iSTEX uses a telomerase template different from the wild type,
ensuring that all telomere extension events are dependent on telomerase and, therefore,
avoiding false positives. Moreover, iSTEX excludes telomeric divergence events arising
from recombination. Deletion of
RAD52 is the common way to exclude recombination
events but, when combined with telomerase-negative cells, results in accelerated replicative
senescence (Le et al., 1999), which can render the assay more challenging. Because
tlc1-tm
telomeric sequence is not present in the cell before the start of the experiment, deletion
of
RAD52 is not required to exclude recombination events. It is formally possible that a
recombination event takes place once the telomeres have been extended by the mutant
telomerase. This is however unlikely, since the experiment allows only one cell cycle, and it
would in any case represent a negligible fraction of the total divergent events.
Cdc13-independent telomerase recruitment to chromosome ends
In the course of the studies described in chapter 2 we found that, while
cdc13-2 mutation
inhibits telomere addition at TG
34ends, as expected, further mutation of
PIF1 allows
telomerase-dependent extension of these ends. This is striking because the
cdc13-2 mutant
carries a point mutation in the telomerase recruitment domain, which makes it behave
like a telomerase-null mutant (Nugent et al., 1996). In fact, recruitment of the telomerase
subunits Est1 and Est2 to the telomeres has been recently shown to be largely impaired
in
cdc13-2 cells (Chen et al., 2018). This suggests that telomerase can be recruited in a
Cdc13-independent manner to extend the telomeres, probably through the Yku complex,
which can interact with the telomerase RNA subunit (Stellwagen et al., 2003) and has been
proposed to recruit telomerase (Hass and Zappulla, 2015). Alternatively, the interaction
between Cdc13 and Est1 might not be completely lost in
cdc13-2 cells, and mutation of
PIF1 allows just enough telomerase recruitment to prevent senescence.
Preliminary studies to understand how those
cdc13-2 pif1-m2 telomeres are
maintained show that, contrary to
cdc13-2 cells, cdc13-2 pif1-m2 cells do not senesce and,
although shorter than in
pif1-m2 cells, telomeres are stable in length (data not shown).
Non-essential G-quadruplex-mediated telomere protection
Chapter 4 explores the
in vivo role of yeast telomeric G-quadruplexes. To do so, we
made use of the
tlc1-tm mutant because of the characteristic telomeric sequence that
Discussion and future perspectives
95
Chapter 5
formation of G-quadruplexes. Our
in vitro and in vivo experiments (circular dichroism and
viability rescue experiments of telomere capping-deficient
cdc13-1 strains upon stabilization
of G-quadruplexes) are in line with the hypothesis that these telomeres are impaired in
G-quadruplex formation. However, these experiments do not rule out the possibility that
the
tlc1-tm phenotype is unrelated to G-quadruplex formation. On the one hand, although
widely used in G-quadruplex studies, circular dichroism is an
in vitro method to measure the
ability of oligonucleotides to fold into G-quadruplex structures, which does not necessarily
represent the
in vivo situation. On the other hand, G-quadruplex stabilization was achieved
by deletion of
PIF1. PIF1 deletion was chosen to stabilize G-quadruplexes because it is the
best G-quadruplex unwinding helicase described so far (Paeschke et al., 2013). However,
the many functions that Pif1 carries out complicate the interpretation of the results, as
discussed in chapter 4. Other methods to stabilize G-quadruplexes, like deletion of the
SGS1 helicase (Sun et al., 1999), overexpression of the G-quadruplex binding protein
Stm1 (Hayashi and Murakami, 2002) or addition of the G-quadruplex stabilizing ligand
PhenDC (Piazza et al., 2010) resulted in inconclusive results (data not shown). The use
of a G-quadruplex specific antibody (Biffi et al., 2013) combined with G-quadruplex
stabilizing ligands and chromatin immunoprecipitation could provide more robust data
about the G-quadruplex forming potential of these telomeres. Yet, our data support the
previously reported rudimentary telomere protection role by G-quadruplexes when Cdc13
is affected (Smith et al., 2011). The fitness of
tlc1-tm cells, indistinguishable from wild-type
cells, indicates that the G-quadruplex-mediated telomere capping function is not essential.
The question arises then, why are telomeric G-quadruplexes conserved? If
organisms of different complexities, from ciliates to humans, have telomeric G-quadruplexes,
it is reasonable to think that they have been evolutionary conserved for a reason. Although
G-quadruplex structures do not seem to play an indispensable role when it comes to
telomere protection, they might influence other telomeric aspects. One could also speculate
that G-quadruplexes were ancient chromosome end protecting structures until telomeres
became specialized, then got obsolete and only worked as a backup telomere capping
mechanism. However, it seems unlikely that such complex structures, as G-quadruplexes
are, were only conserved to work when other telomere protecting structures were lacking.
Characterisation of a Rap1-free telomere
tlc1-tm telomeres show a number of interesting characteristics, the most remarkable probably
being that
tlc1-tm telomeres are not bound by the major telomere binding protein Rap1.
Accordingly, telomere length homeostasis of
tlc1-tm telomeres is altered, leading to long
and heterogeneous sized telomeres. However, although longer than wild-type telomeres,
tlc1-tm telomeres are shorter than those obtained in cells harbouring a RAP1 C-terminal
deletion or
RIF1 RIF2 double deletion (Kyrion et al., 1992; Wotton and Shore, 1997),
which might indicate the presence of residual Rap1 at mutant telomeres. A related question
is how
tlc1-tm telomeres remain stable in length. This might be achieved by Rif1, which can
Chapter 5
bind the telomeric DNA in a Rap1-independent manner (Mattarocci et al., 2017), or by
the combination of telomerase-dependent extension, facilitated by the absence of Rap1,
and rapid telomere erosion, due to the lack of telomere protection. This is in line with the
strongly increased telomere extension frequency of
tlc1-tm telomeres and the very rapid
senescence and telomere shortening very early after telomerase depletion, that can be
explained by the absence of Rap1 at these telomeres, rendering them unprotected and very
dependent on telomerase-mediated extension. Interestingly, when
tlc1-tm telomeric repeats
are placed internally, followed by wild-type repeats at the distal ends, the bulk telomere
length increases approximately to the same extent as the length of the internally placed
mutant tract. Therefore, the mutant repeats are not sensed by the protein-counting model
that regulates telomere length (Marcand et al., 1997). Moreover, the fact that Rap1-free
tlc1-tm cells are perfectly viable implies that the Rap1 essential function is not its telomeric
function.
Rap1 binding to
tlc1-tm telomeres was determined by chromatin immunoprecipitation
followed by qPCR. However, because
tlc1-tm telomeres are long, the chromatin shearing
process required prior to immunoprecipitation could affect the result. Generation of
shorter
tlc1-tm telomeres by, for example, further mutation of TEL1, could help overcome
this problem. In addition, electrophoretic mobility shift assays (EMSA) with purified Rap1
would further confirm that Rap1 does not bind
tlc1-tm sequence. Mutagenizing the DNA
binding domain of Rap1 to find a Rap1 mutant able to bind
tlc1-tm sequence would be
useful for a complementation assay, which would confirm whether the absence of Rap1
is responsible for the observed phenotype. Determining the protein composition of
tlc1-tm telomeres would be also important. In the absence of Rap1, the recruitlc1-tment of Rif1
and Rif2 (Hardy et al., 1992; Wotton and Shore, 1997) and the components of the Sir
complex (Moretti et al., 1994; Moretti and Shore, 2001) should be affected. We plan to
use biotinylated oligonucleotides with wild-type or mutant sequence incubated with yeast
protein extracts, followed by mass spectrometry to unbiasedly identify what proteins
are bound to mutant telomeres. Assessing telomere fusion occurrence would also be of
interest, since Rap1 protects chromosome ends from NHEJ events (Marcand et al., 2008).
In short, the finding of a Rap1-free telomere opens many questions. Probably the most
important one that needs to be addressed is how telomeres are regulated in the absence of
their major binding protein.
The general aim of the work presented in this thesis was to determine what
minimally constitutes a telomere in
S. cerevisiae. We propose that Cdc13 and a telomeric
sequence of about 40 nt or longer are essential elements of telomeres. G-quadruplexes, on
the other hand, do not seem to play an important role at the chromosome ends. As often
happens, while trying to find an answer, many new questions arise, leaving plenty of room
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