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Vincent Geli (Instabilité du Génome

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Présentation au sujet: "Vincent Geli (Instabilité du Génome"— Transcription de la présentation:

1 Vincent Geli (Instabilité du Génome
Et Cancérogénèse, IGC, Marseille)

2 LES TELOMERES SONT MULTIFONCTIONNELS
ET CONTROLENT LA PROLIFERATION CELLULAIRE ET LA STABILITE DU GENOME - PROTECTION DES EXTREMITES REPLICATION COMPLETE DES CHROMOSOMES - ORGANISATION DES CHROMOSOMES

3

4 DNA damage ageing cancer

5

6 Ciliate  b Budding yeast Fission yeast Mammals 3’ 3’ 3’ 3’ Taz1 Taz1
Ten1 Stn1 Budding yeast Rap1 Rap1 Rap1 Cdc13 3’ Sir Sir Rif1 Rif1 swi6 Rif2 Rif2 Fission yeast Taz1 Taz1 Taz1 3’ Pot1 Pot1 Rap1 Rap1 Rap1 Rif1 Rif1 Rif1 HP1 HP1 HP1 TRF2 RAP1 TRF1 RAP1 3’ Mammals TRF1 TRF2 Pot1 TPP1 Pot1 TPP1 Pot1 TPP1 Pot1 Tin2 Tin2 TPP1 E. Gilson and V. Géli, 2007.

7 Titia de Lange, 2004, 2005

8 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 guanine-quadraduplex

9 Dysfunctional telomeres
Genomic instability

10 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?

11 Eukaryotes — TELOMERES and TELOMERASE
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 TTGGGG Humans TTAGGG 5-15 kbp

12 Unprotected telomere Apoptosis Senescence
Telomeric DNA is synthesized by the semi-conservative replication machinery 3’ 3’ 3’ Gradual telomere shortening Unprotected telomere Apoptosis Senescence

13 La télomérase est régulée au cours du développement
Cellules germinales Taille des télomères Télomérase Cellules somatiques Taille des télomères Télomérase Naissance Mort

14 Structure des télomères temps

15

16

17 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 remains viable for extended periods of time changes in morphology, nuclear structure gene expression, metabolism Diapositive 23 Diapositive 23

18 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)

19

20 Sénescence (M1) Crise (M2) Immortalité
hTERT +++ P53 RB Sénescence (M1) Crise (M2) Immortalité

21 can initiate genome instability
Telomere dysfunction can initiate genome instability End-to-end fusion Random DNA break Anaphase Repeated generation of DNA breaks through breakage-fusion-bridge cycles

22 Telomere length dynamics during malignant transformation
Telomerase activity Abnormal proliferation Extensive proliferation

23 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

24

25 Most telomeric DNA is synthesized by the semi-conservative replication machinery
3’ Telomerase 3’ 3’ Gradual telomere shortening (only 7% of cell cycles)

26 The telomerase complex

27 5'TTGGGGTTGGGGTTGGGGTTG 3'AACCCC 5'
These repeat sequences are found as 3' overhangs: (TTGGGG)n 5'TTGGGGTTGGGGTTGGGG 3' 3'AACCCC 5' AACCCCAAC 3' 5' RNA TELOMERASE T 3' T G 5'TTGGGGTTGGGGTTGGGG 3'AACCCC 5' AACCCCAAC 3' 5' RNA GGGTTG 5'TTGGGGTTGGGGTTGGGGTTG 3'AACCCC 5' Translocation

28 How is telomerase recruited to the telomere?
How is telomerase action coupled to DNA replication? What tell us the yeast system model

29 Budding yeast 3’ E. Gilson and V. Géli, 2007. Ten1 Stn1 Rap1 Rap1 Rap1
Cdc13 Sir Sir Rif1 Rif1 Rif2 Rif2 E. Gilson and V. Géli, 2007.

30 Late S-phase 5’ 3’ Est 2 3’ 5’ 5’ 3’ FEN-1 DNA ligase 1 Rnase H1
Okasaki fragments Primase ADN Pol a Helicase TLC1 Est 2 3’ Rap1 Rap1 Ku 5’ RFC ADN Pol d e Cdc13-Stn1-Ten1 RPA PCNA 5’ 3’

31 N. Hug and J. Lingner Chromosoma, june 2006

32 Est 2 Est 3 Est1 Ku 5’ 3’ Cdc13 3’ Ku 5’ wt 60 50 Fold enrichment
(Tel/Background) 40 30 20 10 15 30 45 60 75 Time (min) Est1-Myc Est2-Myc Cdc13-Myc yKu80-Myc

33 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)

34 Late S Early S G1 M Est 2 Lagging strand Ku Rap1 Rap1 Leading strand
Stn1 Lagging strand Ten1 Ku Cdc13 3’ Rap1 Rap1 Leading strand Rap1 Late S Early S Ten1 Est 2 TLC1 Ku Rap1 Cdc13 Stn1 Ten1 3’ Rif1 Rif2 Sir G1 M

35 Rap1 Est 2 Stn1 Lagging strand Ten1 Rap1 Rap1 Ku Cdc13 3’ Leading strand Rap1 Late S Early S Lagging telomerase Leading telomerase Ku ? TLC1 Ku ? TLC1 RPA Est2 Cdc13 Est2 Rap1 Cdc13 Rap1 Est3 Est1 MRX Exo? Est3 ? Est1 Cdk1 Tel1 ? Cdk1 G1 S/G2 M Telomerase activation is linked to the passage of the replication fork

36 Late S Early S G1 S/G2 M Lagging telomerase Leading telomerase ? ?
Ku ? TLC1 Ku ? TLC1 RPA Est2 Cdc13 Est2 Rap1 Cdc13 Rap1 Est3 ? Est1 MRX Exo? Est3 Est1 Cdk1 RPA Tel1 ? RPA Cdk1 G1 S/G2 TLC1 Rif1 Rif2 Est2 Pif1 M Cdc13 Cdc13 Rap1 Rap1 Rap1 Rap1 Rap1 Pol/Pol12 Primase Est1 Est3 RPA

37 Late S Early S G1 S/G2 M Ten1 Est 2 TLC1 Ku Rap1 3’ TLC1 Ten1 Rap1
Cdc13 Stn1 Ten1 3’ Rif1 Rif2 Sir G1 S/G2 Stn1 TLC1 Ten1 Sir Rif1 Rif2 Rif1 Rif2 Rif1 Rif2 Ten1 Rap1 Rap1 Rap1 Ku Cdc13 3’ Rap1 Rif1 Rif2 Est2 Pif1 M Cdc13 Cdc13 Rap1 Rap1 Rap1 Rap1 Rap1 Pol/Pol12 Primase Est1 Est3 RPA

38 The telomerase cycle Late S Early S G1 S/G2 M
Rap1 Est 2 Stn1 Lagging strand Ten1 3’ Rap1 Rap1 Ku Cdc13 Leading strand Rap1 Late S Early S Lagging telomerase Leading telomerase Ten1 Est 2 TLC1 Ku Rap1 Cdc13 Stn1 Ten1 3’ Rif1 Rif2 Sir Ku ? TLC1 Ku ? TLC1 RPA Est2 Cdc13 Est2 Rap1 Cdc13 Rap1 Est3 Est3 ? Est1 MRX Exo? Est1 Cdk1 Tel1 ? Cdk1 G1 S/G2 Stn1 TLC1 Sir Rif1 Ten1 Rif2 Rif1 Rif2 Rif1 Rif2 Ten1 Ku Cdc13 3’ Rap1 Rap1 Rap1 Rap1 Rif1 Rif2 Est2 Pif1 M Cdc13 Cdc13 Rap1 Rap1 Rap1 Rap1 Rap1 Pol/Pol12 Primase Est1 Est3 RPA E. Gilson and V. Géli, 2007

39 Late S Early S G1 S/G2 M E. Gilson and V. Géli, 2007
Rap1 Est 2 Stn1 Lagging strand Ten1 Ku Cdc13 3’ Rap1 Rap1 Leading strand Rap1 Late S Early S Lagging telomerase Leading telomerase Ten1 Est 2 TLC1 Ku Rap1 Cdc13 Stn1 Ten1 3’ Rif1 Rif2 Sir Ku ? TLC1 Ku ? TLC1 RPA Est2 Cdc13 Est2 Rap1 Cdc13 Rap1 Est3 ? Est1 MRX Exo? Est3 Est1 Cdk1 RPA Tel1 ? RPA Cdk1 G1 S/G2 Stn1 TLC1 Sir Rif1 Ten1 Rif2 Rif1 Rif2 Rif1 Rif2 Ten1 Ku Cdc13 3’ Rap1 Rap1 Rap1 Rap1 Rif1 Rif2 Est2 Pif1 M Cdc13 Cdc13 Rap1 Rap1 Rap1 Rap1 Rap1 Pol/Pol12 Primase Est1 Est3 RPA E. Gilson and V. Géli, 2007


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