Lecture 13: Telomerase and Step Cells Flashcards
What is the Hayflick limit?
Limits the ability of cells to multiply in culture
What is senescence?
Normal somatic cells: finite replicative capacity
Tumour cells: infinite in vitro and vivo
Replicative senescence is a tumour suppressor mechanism:
1. most cancer cells have bypassed the replicative limit
2. p53 and Rb are essential for establishing and/or maintaining
senescence growth arrest
3.cells from individuals with germline p53 mutation are more likely to spontaneously immportalise
What is the problem with replicating ends?
DNA polymerase only works 5’-3’
It cannot reconstruct the ends of chromosome strands - 50-200bp remain replicated
ALSO, exonucleases chew at the ends of telomeric DNA
What is the role of telomerase in senescence?
Progressive decline of proliferative potential demonstrated by ageing cells is directly correlated with progressive shortening of telomerases
What affects telomerase length
Type of tissue
Persons age
What are telomerases?
Nucleoprotein structures at ends of linear chromosomes
Essential for proper maintenance of chromosomes:
a. maintain integrity and stability
b. stop chromosome fusion
c. anchor chr to nuclear matrix
Chromosome ends hidden from DNA repair systems by telomer binding proteins
No protein coding genes
Shorten each round of DNA replication
At a particular short length, senescence triggered or crisis
Loss or shortening of telomeres linked to genome instability
a. degradation
b. fusion
c. recombination
What is the repeat found at telomeres
TTAGGG repeated 100s - 1000s of times
Same in mammals, birds, reptiles, amphibians, bony fish, plant species’.
Double stranded with short single-sided 3’ overhang
What is the T-loop
Structure at telomeres
What happens to cells lacking Trf2?
Telomeres lose protective function, resulting in massive end-to-end fusion of chromosomes
What is telomerase?
- Holoenzyme
- Two main components: Protein (TERT) and RNA (TR)
- rNA contains CCCUAA repeats (complementary to telomere)
- RNA seq. acts as template for adding new TTAGGG to end
- Plus several other proteins: hsp90, p23, dyskerin, L22, hStain, and
Est1A/B, all associated with telomerase activity in cell extrac - Reverse transcriptase
What does the telomerase do?
Maintains telomere length, ensuring chromosome integrity and avoidance of crisis and death. In normal development, telomerase expressed early in embryo, counterbalancing proliferation
What happens when cancer induced by telomere dysfunction
- mechanisms leading to de-repression of hTERT complex and still obscure (potential involvement of myc transcription factor)
- Telomerase activity does not induce transformation, but is req for immortalisation
How is telomerase regulated?
Multifactorial:
1. gene regulation
2. post-translation protein-protein interactions
3. phosphorylation
4. differences at telomere
HPV E6 & c-myc can increase expression
How can telomerase activity be detected?
Telomeric repeat amplification protocol (TRAP)
Terminal restriction fragment length - measure size of telomere
How can telomeres be maintained without telomerase?
10-15% cancers
Alternative Lengthening of Telomeres (ALT)
ALT-associated PML bodies (APBs) in nucleus (contain telomeric DNA, TF1, TRF2, DNA recombination enzymes -RAD50,51,52-)
