Transcript Aucun titre de diapositive - univ

Vincent Geli (Instabilité du Génome
Et Cancérogénèse, IGC, Marseille)
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LES TELOMERES SONT MULTIFONCTIONNELS
ET CONTROLENT LA PROLIFERATION CELLULAIRE
ET LA STABILITE DU GENOME
- PROTECTION DES EXTREMITES
-REPLICATION COMPLETE
-DES CHROMOSOMES
- ORGANISATION DES CHROMOSOMES
DNA damage
ageing
cancer
 b 3’
Ciliate
Ten1
Budding
yeast
Stn1
Sir
Rap1
Sir
Rap1
Rif2
Taz1
Taz1
Rap1
Rap1
Rif1
Rif1
HP1
Mammals
E. Gilson and V. Géli, 2007.
HP1
TRF1
Cdc13
3’
Rif1
Rif1
swi6
Fission
yeast
Rap1
Taz1
Rif2
Pot1
3’
Pot1
Rap1
Rif1
HP1
RAP1
RAP1
TRF2
TRF1
Tin2
TRF2
Pot1
Pot1
Pot1
Pot1
Tin2 TPP1 TPP1 TPP1 TPP1
3’
Titia de Lange, 2004, 2005
• Repeated sequences at the end of linear chromosomes (TTAGGG in humans)
• Protects against environmental damage, recombination with other chromosomes, &
from loss of information during replication
Forms a guaninequadraduplex
Dysfunctional telomeres
Genomic instability
The Eukaryotic Problem of Telomere Replication
RNA primer near end of
the chromosome on
lagging strand can’t be
replaced with DNA
since DNA polymerase
must add to a primer
sequence.
Do chromosomes get
shorter with each
replication?
D. Termination
Replication of the ends of linear DNA
5'
3'
?
Eukaryotes — TELOMERES and
TELOMERASE
Telomeric DNA consists of TANDEM REPEATS of
simple sequences:
Tetrahymena
Humans
TTGGGG
TTAGGG
5-15 kbp
Telomeric DNA is synthesized by the semi-conservative replication machinery
3’
3’
Gradual telomere shortening
3’
Unprotected telomere
3’
Apoptosis Senescence
La télomérase est régulée au cours du développement
Cellules germinales
Taille
des télomères
Télomérase
Cellules somatiques
Taille
des télomères
Télomérase
Naissance
Mort
Structure
des télomères
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Permanent arrest = senescence
Proliferation of primary normal human cells is not indefinite
1. period of rapid proliferation
2. slowing of the proliferation rate
3. cessation of proliferation
= replicative senescence
Senescent cells
permanently arrested Diapositive 23
remains viable for extended periods of time
changes in morphology, nuclear structure
gene expression, metabolism
The role of senescence :
a limit to proliferation
Suggested purpose :
to limit the proliferative capacity of normal cells
-> a mechanism involved in normal development
& tissue maintenance
-> a potent tumor-protection mechanism
-> must be prevented in proliferative cell compartments
(stem cells)
hTERT
+++
P53
RB
Sénescence
(M1)
Crise
(M2)
Immortalité
Telomere dysfunction
can initiate genome instability
End-to-end
fusion
Anaphase
Repeated generation of DNA breaks
through breakage-fusion-bridge cycles
Random
DNA break
Telomere length dynamics during malignant transformation
Telomere
length
Telomerase
activity
Abnormal
proliferation
Extensive proliferation
90 % des cancers surexpriment la télomérase
5 % des cancers ne surexpriment pas la télomérase
mais activent une voie alternative
du maintien de l’ADN télomérique
= ALT
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Most telomeric DNA is synthesized by the semi-conservative replication machinery
3’
3’
Gradual telomere shortening
3’
3’
Telomerase
(only 7% of
cell cycles)
THE TELOMERASE
The telomerase complex
These repeat sequences are found as 3' overhangs:
5'TTGGGGTTGGGGTTGGGG 3'
3'AACCCC 5'
5'TTGGGGTTGGGGTTGGGGT3'
TG
AACCCCAAC
3'AACCCC 5'
3'
RNA
(TTGGGG)n
TELOMERASE
5'
5'TTGGGGTTGGGGTTGGGGTTGGGGTTG
AACCCCAAC
3'AACCCC 5'
3'
RNA
Translocation
5'
How is telomerase
recruited to the
telomere?
How is telomerase action
coupled to DNA
replication?
What tell us the yeast system model
Ten1
Stn1
Budding
yeast
Gilson and V. Géli, 2007.
Rap1
Sir
Rap1
Rap1
Cdc13
Sir
Rif1
Rif1
Rif2
Rif2
3’
5’
3’
Late S-phase
FEN-1
DNA ligase 1
Rnase H1
Okasaki
fragments
Primase
ADN
Pol 
Helicase
Rap1
RFC
PCNA
5’
3’
ADN
Pol d
e
RPA
Rap1
TLC1
Est 2
Ku
3’
5’
Cdc13-Stn1-Ten1
N. Hug and J. Lingner
Chromosoma, june 2006
Est 3
Est1
Cdc13
5’
Est 2
Ku
Fold enrichment
(Tel/Background)
5’
3’
Ku
60
50
40
30
20
10
0
0
15
30
45
Est1-Myc
Est2-Myc
Cdc13-Myc
yKu80-Myc
60
75 Time (min)
3’
Telomerase is acting in S-phase (E. Gilson, D. Gottschling)
Telomerase action requires fork passage (E. Gilson, D. Gottschling)
Generation of ssDNA G-tails is required for telomerase action
(R. Wellinger)
Est 2
Rap1
Lagging strand
Rap1
Rap1
Ku
Stn1
Ten1
Cdc13 3’
Leading strand
Rap1
Late S
Early S
TLC1
Est 2
Sir
Rap1
Rif1
Rif2 Rif1Rif2 Rif1Rif2
Rap1
Rap1
Rap1
Ku
Stn1
Ten 1
Cdc13 3’
Ten1
G1
M
Est 2
Rap1
Stn1
Ten1
Cdc13 3’
Lagging strand
Rap1
Rap1
Ku
Leading strand
Rap1
Late S
Early S
Lagging telomerase
Ku ?
RPA
Leading telomerase
Ku ?
TLC1
TLC1
Est2
Est2
Cdc13
Rap1
Rap1
?
Est1
Est3
Cdk1
Cdc13
Est3
MRX
Exo?
Est1
Tel1
?
Cdk1
G1
S/G2
M
Telomerase activation is linked to the passage of the replication fork
Late S
Early S
Lagging telomerase
Ku ?
RPA
Leading telomerase
Ku ?
TLC1
TLC1
Est2
Est2
Cdc13
Rap1
Cdc13
Rap1
?
Cdk1
Est1
Est3
Est3
MRX
Exo?
Est1
Tel1
RPA
RPA
?
Cdk1
G1
S/G2
TLC1
Rif1
Rif2
M
Pif1
Est2
Rap1 Rap1 Rap1 Rap1 Rap1
Cdc13 Cdc13
Pol /Pol12
Primase
Est1
Est3
RPA
Late S
Early S
TLC1
Est 2
Sir
Rap1
Rif1
Rif2 Rif1Rif2 Rif1Rif2
Rap1
Rap1
Rap1
Ku
Stn1
Ten 1
Cdc13 3’
Ten1
G1
Sir
Rap1
S/G2
Stn1
Ten 1
Rif1
Rif2 Rif1Rif2 Rif1Rif2
Rap1
Rap1
Rap1
Ku
Cdc13 3’
TLC1
Ten1
Rif1
Rif2
M
Pif1
Est2
Rap1 Rap1 Rap1 Rap1 Rap1
Cdc13 Cdc13
Pol /Pol12
Primase
Est1
Est3
RPA
The telomerase cycle
Est 2
Rap1
Stn1
Ten1
Cdc13 3’
Lagging strand
Rap1
Rap1
Ku
Leading strand
Rap1
Late S
Early S
Lagging telomerase
TLC1
Ku ?
Est 2
Sir
Rap1
Rif1
Rif2 Rif1Rif2 Rif1Rif2
Rap1
Rap1
Rap1
Ku
Stn1
Ten 1
Cdc13 3’
Ten1
RPA
Leading telomerase
Ku ?
TLC1
TLC1
Est2
Est2
Cdc13
Rap1
Cdc13
Rap1
?
Est1
Est3
Est3
MRX
Exo?
Est1
Tel1
Cdk1
?
Cdk1
G1
Sir
Rap1
S/G2
Stn1
Ten 1
Rif1
Rif2 Rif1Rif2 Rif1Rif2
Rap1
Rap1
Rap1
Ku
Cdc13 3’
TLC1
Ten1
Rif1
Rif2
M
Pif1
Est2
Rap1 Rap1 Rap1 Rap1 Rap1
Cdc13 Cdc13
Pol /Pol12
Primase
Est1
Est3
RPA
E. Gilson and V. Géli, 2007
Est 2
Rap1
Stn1
Ten1
Cdc13 3’
Lagging strand
Rap1
Rap1
Ku
Leading strand
Rap1
Late S
Early S
Lagging telomerase
TLC1
Ku ?
Est 2
Sir
Rap1
Rif1
Rif2 Rif1Rif2 Rif1Rif2
Rap1
Rap1
Rap1
Ku
Stn1
Ten 1
Cdc13 3’
Ten1
RPA
Leading telomerase
Ku ?
TLC1
TLC1
Est2
Est2
Cdc13
Rap1
Cdc13
Rap1
?
Cdk1
Est1
Est3
Est3
MRX
Exo?
Est1
Tel1
RPA
RPA
?
Cdk1
G1
Sir
Rap1
S/G2
Stn1
Ten 1
Rif1
Rif2 Rif1Rif2 Rif1Rif2
Rap1
Rap1
Rap1
Ku
Cdc13 3’
TLC1
Ten1
Rif1
Rif2
M
Pif1
Est2
Rap1 Rap1 Rap1 Rap1 Rap1
Cdc13 Cdc13
Pol /Pol12
Primase
Est1
Est3
RPA
E. Gilson and V. Géli, 2007