Transcript ter

The genetic interactions between
telomerase and CST proteins in
Kluyveomyces lactis
Binyamin Kaffe
The Telomeres
The end regions of eukaryotic chromosomes.
• These regions are composed of a
repetitive sequence of double stranded
DNA, ending with a single stranded 3’overhang.
• Insure that no gene will get lost during
replication when the chromosomes
shorten.
• Protect the end of the chromosome from
deterioration or from fusion with
neighboring chromosomes.
• Telomeres usually elongate by
telomerase. However In some cases they
elongate via an alternative pathway
(ALT) which involves recombination.
The Telomerase
• A ribonucleoprotein (RNP)
containing an RNA molecule
(TER), which serves as a
template, and the telomerase
reverse transcriptase protein
(TERT), which synthesizes
the DNA upon the RNA
template.
• Active in unicellular
organisms, mammalian germ
cells and most cancer cells.
Not active in most human
somatic cells.
Human template
Model organism
Kluyveromyces lactis
A budding yeast
6 chromosomes – 12 telomeres
Has a long and exact telomeric repeat of 25 repetitive
bases - CGGATTTGATTAGGTATGTGGTGT
The telomerase RNP & accessory
proteins in budding yeast
• Protein subunit TERT – Est2 in
• Double strand binding proteins – Rap1,
•
•
budding yeast
RNA subunit TER – Tlc1 in
Saccharomyces cerevisiae
Rif & Sir family
Single strand binding proteins –the
CST complex (Cdc13-Stn1-Ten1)
and more….
Important domains in K. lactis
TER
Template- a complementary sequence for the telomeres
Pseudoknot- stabilizes the template
The different stems are important for the functionality and
binding of different proteins (ex. TWJ binds the TERT subunit)
CST complex
• Recruits telomerase by its
•
•
•
interaction with the
telomerase protein Est1.
Coordinates the synthesis
of the telomere by its
interaction with DNA
polymerase.
Protects it from
degradation by
exonuclease action.
Functions as a negatively regulator of the
telomere length.
General Objective
To understand the genetic and functional interactions between
Cdc13 & Stn1, and the RNA subunit of telomerase.
Specific objectives
To check the telomere phenotype and viability of K. lactis
strains overexpressing CST proteins in the background
of:
• TER1 (encoding the K. lactis TER) deletion.
• TER1 mutated in Reg2, an element possibly involved in
the recruitment of the telomerase to the telomere.
• TER1 mutated in the pseudoknot, which is known to
affect the activity of telomerase, but it is not assumed to
affect the recruitment of telomerase.
Plasmid insertion by electroporation
Methods
Serial streaks
Morphology observation
Southern blot
The plasmids
TER containing plasmids
• CEN-ARS plasmids with WT, mutated or no TER
•
gene.
All of the introduced TERs have a single
mutation in the template region. (In order to detect
the newly synthesized telomere with a specific probe, and to
set a newly recognition site for the restriction enzyme BclI
which enables to evaluate the difference between the old part
of the telomere and the new one.)
• All the newly introduced plasmids have a HIS1
selectable marker.
The yeast strain contained a WT TER (URA3) plasmid that was negatively
selected by seeding on 5-FOA plates.
Over expression (o.ex.) plasmids
•2µ plasmids with a high copy number.
• All the newly introduced plasmids have a URA3 selective gene.
The mutations used in the project
Previous lab observation
WT
1 telomere
2 telomeres
7 telomeres
Reg2-L2
The telomeres do
not show any BclI
incorporation [no
BclI digestion and
no telomeres were
detected when
hybridized with BclI
probe (not shown)]
which means that
the new template
was not used.
However telomere
length is similar to
WT telomere
length, although the
chromosomes
replicated ~120
times.
The genomic DNA was digested with EcoRI or EcorI+ BclI and analyzed by
Southern blotting and hybridization a WT telomere radioactive probe.
Questions
How could it be that the new telomerase template was not used but the
telomeres maintained their WT length?
•Is the ALT pathway active?
 The telomeric bands do not show a long smear after the hybridization
with a telomeric probe which is typical for the ALT pathway
 Maybe the over expression of Cdc13 or Stn1 regulates the length of the telomeres to
WT?
•Alternatively, is there another mechanism that maintains the telomere
original length even so the telomerase is not active?
1st attempt results
Colony morphology observation
Phenotype scores of colonies from 6 passages. Scores are between 1-4, where 1
indicates a round and smooth colony and 4 indicates the most aberrant looking
colonies. Data in the table are weighted averages of numerous colonies in 2 clones.
Empty Vector
Stn1 o.ex.
Cdc13 o.ex
Streak
#1
#2
#3
#4
#5
#6
#1
#2
#3
#4
#5
#6
#1
#2
#3
#4
#5
#6
WT Ter
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
terD
4
4
3
2.5
1.5
1.5
4
1.5
2.5
3
3
3.5
Reg2-L1
2
1
1.5
2
1.5
1.5
2
2.5
1.5
2.5
2
1.5
1.5
1
2
2
1.5
1.5
Reg2-L2
1.5
1.5
3
1.5
2
1
2.5
2
3
2
2
2
2
2
2.5
2
2.5
1.5
N.A. contamination
Notable is the improvement of the morphology during the passages which is due to the
elongation of the telomeres in the alternative pathway. In the case of terD the improvement
started after the 1st passage, in the Reg2 mutants which have a non-optimal telomerase the
phenotype worsened first due to the telomere shortening and the improvement started in
later stages. In some strains we see the phenotype worsening again after improving, since
the recombination is suppressed when telomeres are long, and activated when they shorten.
There is no distinct difference when the proteins are overexpressed.
telomeric length in
Reg2 ter mutations
is shorter then WT,
however the
overexpression of
the CST proteins
has no significant
effect on the
length.
The smear seen in
terD/EV is typical
for recombinant
pathway survivors
due to the lack of
tight regulation of
telomeric length.
No telomeric
bands were
detected in the
lanes of terD/o.ex
Cdc13, it appeared
to be a
contamination.
Southern blot
(The BclI incorporation enables to evaluate the difference between the old part of
the telomere and the new one by BclI digestion and specific hybridization)
Conclusion
These results disconfirm the previous lab results; the overexpression of the
CST proteins has no significant effect on the length of the telomeres.
However…
The previous lab results were obtained in a somewhat different procedure.
The plasmid shuffling by plating on a 5FOA plate in order to negatively select the cells that
lost the WT plasmid (URA3), was done prior to any of the transformation and not after the
first transformation.
Could the lack of any active telomerase for ~20-30 generations induce an alternative pathway
and lead the cells overexpressing CST proteins to a different pathway? We decided to redo the
experiment with this difference of removing the WT TER prior to transforming a mutant ter.
1st attempt
WT TER1 (URA3),
2nd attempt
Different TER1s (with BclI site) (HIS1),
O.EX. plasmid
2nd attempt results
The WT TER1 was removed by plating on 5FOA
prior to transforming a mutant ter.
• This attempt did not include the L1 Reg2 mutation which leads to significant
similar results as the L2 mutation as we saw in the 1st attempt.
• The pseudoknot (PK) mutation was included in order to check if the CST
proteins have an effect in a case were the catalytic activity of telomerase is
abolished but there is no putative effect on the recruitment of the telomeric
proteins.
Colony morphology observation
‘A’ clones
Streak
Streak
WT
/EV
0
1
2
3
4
5
6
WT
/EV
WTWT
/Stn1
/Stn1
WT
WT/Cdc13
/Cdc13
⌂ ⌂
/EV
/EV
⌂
⌂ / Stn1
/ Stn1
⌂/
⌂/ Cdc13
Cdc13
• The WT strains morphology is round throughout all the streaks.
• In the case of a severe mutation (terD) the morphology is severe in the early passages and it improved
during the passages which is believed to be due to an elongation of the telomeres in a recombination
pathway. The telomeres shorten again after a few passages as we see that in some strains (e.g. passage 6
of D/ Stn1) the phenotype worsens after the improvement, which is due to the fact that the
recombination is suppressed when telomeres are long, and activated when they shorten.
‘A’ clones
Streak
Streak
0
1
2
3
4
5
6
Reg2
Reg2
/EV
/EV
Reg2
Reg2
/Stn1
/Stn1
Reg2
Reg2
/Cdc13
/Cdc13
A
PK
PK
/EV
/EV
PK
PK
/Stn1
/Stn1
PK
PK
/Cdc13
/Cdc13
• In the reg2 mutation which is not as severe, the phenotype worsened first due to the
telomere shortening and the improvement started in later stages. From these photos it
seams like the overexpression of Cdc13 prevents the colonies from fully returning to a
round phenotype (additional photos in next slide).
• In PK mutation which is a severe mutation we see similar behavior to the ter deletion
strain, however it does seam like Cdc13 prevents the colonies from fully returning to a
round phenotype .
Colony morphology of Reg2/Cdc13
Reg
2
/Cdc
13 A
Reg
2
/Cdc
13 B
Reg
2
/Cdc
13 C
Notice a difference between the different clones, which may indicate variability in the
survival pathway:
•In ‘A’ clones the colonies basically maintained the same phenotype throughout the
passages (passage 3 is an exception were we observe two phenotypes).
•In ‘B’ clone we see worsening and improvement in passage 5, and again worsening
in passage 6.
•In ‘C’ clones we could observe a large diversity in colony morphology and two cycles
of worsening and improving.
‘A’ clones
No evidence to BclI
sites incorporation
in PK mutants.
Some of the strains
show smears
indicating the
recombination
pathway.
Southern blot
The different smear patterns
seen in terD is typical for
recombinant pathway
survivors due to the lack of
tight telomeric length control,
and the fact that the
telomeres shorten again after
the recombinant elongation.
In L2 mutants we observe BclI incorporation and
what seems like regulated smears (Smears
maybe more regulated since there is a use of
the new template). Stn1 shows longer telomeres
than EV and Cdc13 which maybe due to the
regulation. Cdc13 shows the same length as EV,
however there is an extra fragment at 4kb which
could be due to excessive recombination.
Not possible
for a terD to
have BclI
incorporatio
n maybe
misplaced
by another
strain.
‘B’ clones
Smear patterns are different than
in clones A. The recombination
undergoes regulation, and
therefor the telomeres shorten
again after the recombinant
elongation. Patterns are different
at different stages
No BclI site incorporation
in Reg2/Cdc13!
However, we do not
observe a telomere length
which is similar to the WT
length, as observed
previously in the lab
Conclusions
• In case where the WT TER1 gene (Ura3) was negatively selected only after the
•
•
transformation of the mutated ter (His1), there was no effect of overexpressing
the CST proteins.
In case were the WT TER1 gene (Ura3) was negatively selected prior to the
transformation of the mutated ter we did observe some variance between the L2
strains and in one case we observed no use of the mutated template when
Cdc13 was overexpressed. However we did not observe the phenotype that was
observed previously in the lab.
There are short smears in the L2 strains, which we believe to be generated by
recombination. Since the elongation in this pathway is induced only when the
telomeres shorten, we have cycles of telomeres shortening-elongating by
recombination-shortening again…. During this cycle the length could be a WT
length as we see in some strains in my results, and that may explain the
previous lab result.
The reason that in one case there was no use of the mutated
template is not clear, but may indicate competition between
telomerase and the recombination pathway. I.e., recombination
was induced by the overexpression of Cdc13 and suppressed
telomerase activity.
Future plans
• To check the different reg2/Cdc13 clones
that show different growth behaviors.
• Check the phenotype in different passages
in order to observe the effect through the
generations.