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TELOMERES & TELOMERASE. Prasamit Saurav Baruah MODERATOR Dr. Nalini K. Objectives :. Introduction Overview of DNA replication End replication problem Telomeres – structure & functions Telomerase – hTR & hTERT Telomere replication Regulation of Telomerase TRAP assay _ overview

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telomeres telomerase

TELOMERES &TELOMERASE

Prasamit Saurav Baruah

MODERATOR

Dr. Nalini K.

objectives
Objectives :

Introduction

Overview of DNA replication

End replication problem

Telomeres – structure & functions

Telomerase – hTR & hTERT

Telomere replication

Regulation of Telomerase

TRAP assay _ overview

Telomeres & cancer

Conclusion

References

introduction
Introduction
  • Telomeres , the DNA structures at the termini of linear chromosomes have been implicated in regulating the lifespan of normal human cells.
  • Replication of chromosome ends pose a special problem for cells .
  • Telomeres cant be replicated each time a cell divides and they shorten until reaching a critical length that signals replicative senescence.
telomere biology
Telomere biology
  • Observation by HENRY MULLER , while studying Drosophila chromosomes & of BARBARA McCLINTOCK studying maize chromosomes suggested end capping of chromosomes.
  • MULLER coined telomere .
  • It’s a simple sequence of 5’-TTAGGG-3’ in humans .
telomeres structure
Telomeres : structure
  • Majority of them are double helical with GC rich sequence.
  • The 3’ end of the DNA protrudes as a single strand overhang
  • Telomeres shrink at a rate of approx. 100bps per cell division from their original size of about 15 kbp.
slide9
Telomere length are the measuring stick that determines the no. of cell divisions .
  • Hayflick limit
  • Mortality stage 1 ( M1)
  • Mortality stage 2 ( M2)
slide10
TE L

O

M

E

R

E

L

E

N

G

T

H

Germ line cell

hTERT transduced cell

Stem cell

Telomerase stabilization

Abrogation of p53

M1

Telomerase activation

M2

Crisis

Senescence

CELL DIVISIONS

functions of telomeres
Functions of telomeres :
  • Protects the chromosome from end to end fusion .
  • Provides a means for complete replication of chromosomes.
  • Contributes to the functional organization of chromosomes within the nucleus.
  • Participates in the regulation of gene expression.
  • Serves as a molecular clock that controls the replicative capacity of human cells and their entry into senescence.
telomerase
Telomerase
  • Its an RNA dependent DNA polymerase that synthesizes telomeric DNA sequences.
  • First discovered in Tetrahymena thermophila .
  • Telomerase activity was found to be almost absent in normal human somatic cells but present in over 90% of cancerous and in vitro immortalized cells.
structure
Structure :
  • Consists of two essential components –
  • hTR ( HUMAN TELOMERASE)

– RNA component serving as a template for telomeric synthesis.

  • h TERT( HUMAN TELOMERASE REVERSE TRANSCRIPTASE)

- catalytic protein with Reverse Transcriptase activity.

slide14
it provides a scaffold for binding of RNP proteins
  • telomerase RNA feature a pseudoknot structure located close to the template sequence
  • 3'-half of telomerase RNA carries a box H/ACA small nucleolar RNA-like (snoRNA-like) domain
slide16
four conserved secondary structure elements, namely the 5'-terminal pseudoknot domain, the CR4–CR5 domain, the box H/ACA snoRNA-like domain and the CR7 domain .
  • play important roles in the function, stability, processing and intracellular trafficking of telomerase RNAs.
  • the box H/ACA snoRNA-like domain direct the 3'-endprocessing of precursor telomerase RNA, provide metabolic stability for the mature RNA
htert
hTERT
  • Initially purified from Euplotes aediculatus as p123 containing RT’ase activity
telomerase regulation
Telomerase regulation
  • hTR associated proteins.
  • hTERT associated proteins.
telomere associated proteins
Telomere associated proteins
  • Telomere repeat binding factor- TRF1 & TRF2.
  • TRF1 interacts with Tankyrase &TRF 1interacting protein 2 ( TIN2).
  • TRF2 interacts with hRap 1 and Mre11/ Rad 50/ Nbs 1 DNA repair complex.
  • Ku 70/80 heterodimer interacts with TRF2.
slide24
Protein Interacting region Function

hTERT associated

TEP1 aa 1–350, 601–927 Unknown

P23/p90 aa 1–195 Assembly/conformation

14-3-3 aa 1004–1132 Nuclear localization

hTR associated

TEP1 nt 1–871 Unknown

hGAR1 hTR H/ACA domain Stability, maturation, localization

Dyskerin/NAP57 hTR H/ACA domain Stability, maturation, localization

hNOP10 hTR H/ACA domain Unknown

hNHP2 hTR H/ACA domain Stability, maturation, localization

C1/C2 nt 33–147 Stability, maturation, localization

La nt 1–205, 250–451 Accessibility to telomeres?

A1/UP1 nt 1–208 Unknown

hStau nt 64–222 Accessibility to telomeres?

L22 nt 64–222 hTR processing, localization?

structure of telomeres
Telomeres are tucked into double strand repeat forming a loop , which may serve to protect the 3’ overhang.

Displacement of TG rich strand internally stabilized by TRF2.

Rest duplex is bound by TRF1.

Structure of telomeres
telomerase regulation contd
Telomerase regulationcontd….
  • In normal human cells telomerase activity appears to be strictly regulated during development.
  • Remains active in some tissues – male germ cells, activated lymphocytes & certain types of stem cell populations.
slide28
The regulation of telomerase activity occurs at –
  • Transcription
  • mRNA splicing
  • Maturation & modification of hTR and hTERT
  • Transport and subcellular localization of each component.
  • Assembly of telomerase.
activation of htert transcription
Activation of hTERT transcription
  • C-Myc
  • oncogene induces hTERT expression and telomerase activity in normal human mammary epithelial cell and primary fibroblasts.
  • Sp1
  • hTERT core promoter contains five GC-boxes that are binding sites for the transcription factor Sp1. Sp1 is found to co operate with c-Myc
slide31
Human papilloma virus 16E6
  • in primary human keratinocytes & mammary epithelial cells.
  • Steroid hormones > estrogen and androgen.
slide32
NEGATIVE REGULATORS:
  • Mad1 > Mad / Max heterodimers inhibit hTERT promoter.
  • p53 > inhibits tumor formation & hTERT promoter along with E2F, p16,p21,p15.
  • IFN–α inhibits hTERT promoter.
  • Progesterone.
cancer
Cancer
  • Most human cancers ‘ve short telomeres and express high levels of telomerase .
  • Reactivation and upregulation of telomerase activity and its hTR and hTERT are associated with all cancers with few exceptions.
  • Studies have found shortened telomeres in many cancers, including pancreatic, bone, prostate, bladder, lung, kidney, and head and neck.
slide36
Importantly, telomerase can cooperate
  • with oncogenes or with inactivation of tumor suppressor genes to induce tumorigenic conversion of normal human epithelial cells and fibroblasts.
slide37
Measuring telomerase may be a new way to detect cancer.
  • If scientists can learn how to stop telomerase, they might be able to fight cancer by making cancer cells age and die.
  • Blocking telomerase could impair fertility, wound healing, and production of blood cells and immune system cells
do telomeres play a role in other diseases
Do telomeres play a role in other diseases?
  • Dyskeratosis congenita
  • mis-formed nails, altered skin pigmentation and hair loss.
  • spots on the skin, intestinal disorders, softening of the bones and learning disabilities
telomeres and transplanted cells
Telomeres and transplanted cells
  • In SCID,
  • One approach to gene therapy is to
  • remove cells from the patient,
  • transform them with the gene for the product that the patient has been unable to synthesize,
  • return them to the patient
slide40
this procedure used with children suffering from severe combined immunodeficiency (SCID)
  • The cells described by Bodnar et. al.
  • these cells continue to divide indefinitely as cancer
slide41
show contact inhibition as normal cells do when grown in culture.
  • do not grow into tumors when injected into immunodeficient mice (as cancer cells do).
  • still fussy about their diet — unable to grow on the simple media that supports cancer cells in culture.
inhibition of telomerase
Inhibition of telomerase
  • One of the strategies for the development of anti-cancer therapies is to inhibit telomerase activity in cancer cells.
  • Inhibiting telomerase activity should result in telomere shortening and therefore cause aging and death of cancer cells.
  • oligonucleotides, that target the template region, or active site, of telomerase.
  • GRN163 and GRN163L
slide43
GRN163L is a 13-mer oligonucleotide N3’ --> P5’ thio-phosphoramidate (NPS oligonucleotide)

directly bind to the RNA component of telomerase at the active site of the enzyme

slide44
Telomerase inhibition with an oligonucleotide telomerase template antagonist
  • 3'-azido-2',3'-dideoxy-2-aminoadenosine (AZddAA)
  • 3'-azido-2',3'-dideoxyadenosine (AZddA),
  • 9-(3-azido-2,3-dideoxy-ß-D-ribofuranosyl)-2-aminopurine (AZddAP),
  • 3'-azido-2-chloro-2',3'-dideoxyadenosine (AZddClA)
references
References
  • Telomerase and cancer –jerry w. shay,Woodring W. Right et . al.- Human Molecular genetics – 2001,vol 10 , no. 7.
  • Telomere , telomerase and cancer – Scientific American 2/96
  • Telomeres and telomerase – Genes and development ,1999, 13 :2353-2359
  • Telomere and telomerase – Maria A.Blaso,Susan M. Gasser, - meeting review-genesdev.org
  • Telomere dynamics in cancer – Nature medicinés, 6,849-851:2000
  • Telomere shorten during aging – nature, 345,458-460:1990
slide47
The multistep nature of cancer-Trends inGenetics,9,138-144:1993
  • Harrington et.al.- A Mammalian Telomere Associated Protein – Science-275, 973-7
  • Alberts et,al.- Molecular Biology Of TheCell-2002
  • Watson et.al. – Molecular Biology Of the Gene – 5th edition
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