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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

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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

Telomeres & cancer

Conclusion

References


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.


Overview of Eukaryotic DNA replication


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

  • 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.


  • Telomere length are the measuring stick that determines the no. of cell divisions .

  • Hayflick limit

  • Mortality stage 1 ( M1)

  • Mortality stage 2 ( M2)


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 :

  • 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

  • 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 :

  • 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.


  • 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


hTR


  • 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

  • Initially purified from Euplotes aediculatus as p123 containing RT’ase activity


Telomere replication


Telomerase regulation

  • hTR associated proteins.

  • hTERT 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.


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?


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 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.


  • 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

  • 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


  • Human papilloma virus 16E6

  • in primary human keratinocytes & mammary epithelial cells.

  • Steroid hormones > estrogen and androgen.


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.


Are Telomeres the Key to Aging and 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.


  • Importantly, telomerase can cooperate

  • with oncogenes or with inactivation of tumor suppressor genes to induce tumorigenic conversion of normal human epithelial cells and fibroblasts.


  • 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?

  • 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

  • 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


  • 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


  • 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

  • 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


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


  • 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)


Extension of Life-Span byIntroduction of Telomerase intoNormal Human Cells


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


  • 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


THANK YOU


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