Telomeres

Question 1

What are telomeres and their functions?

Answer 1

Physical ends of eukaryotic chromosomes consisting of specific DNA sequences and proteins
Functions:
- Allow replication of extreme ends of linear chromosomes
- Protect ends of DNA molecules, preventing end-end fusion

Question 2

Describe the End Replication Problem

Answer 2

Watson (1971) and Olovnikov (1973)
Leading strand is synthesised normally
When primers on the lagging strand are removed, gaps are created
A gap in the middle of the sequence can be filled but gaps at the ends can’t because there’s no primer for DNA Polymerase to add to
Shortens around 50-100nt after each round of cell division
Raised the question of how do eukaryotic cells replicate the ends of their linear chromosomes?

Question 3

Why are Ciliates useful for studying telomeres?

Answer 3

They have two nuclei:
- Small Micronucleus: typical eukaryotic C, many genes, linear, very long
- Big Macronucleus: Unusual C, one gene, linear and very short, each mini C is amplified

Because they have so many genes, they have loads of telomeres - some have 25x10^6

Question 4

What is the structure of a telomere?

Answer 4

Many repeats of a short unit
TG rich on DNA strand running 5’ to 3’ towards chromosome end
Humans (TTAGGG)n length = 1-20kb
Yeast (TGGG)1-3 length = 700bp
Tetrahymena (TTGGGG) length = 200bp

Question 5

What happens to Tetrahymena telomeres in yeast?

Answer 5

Blackburn and Szostak made linear plasmid with Tetrahymena telomeres and put it into yeast
DNA at end was no longer Tetrahymena telomere but yeast telomeric DNA (TG1-3)n
Something in yeast was capable of recognising Tetrahymena telomeres and able to add on their own
No DNA template so must be RNA (RTase)

Question 6

What is the telomeric RNA component (TERC or hTR)?

Answer 6

The Template complementary to ~1.5 copies of telomeric repeat
Information from the RNA template is copied into DNA
Tetrahymena: 159 nt
Yeast: 1150 nt
Humans (hTR): 451 nt

Question 7

Describe the process of telomerase extending telomeres.

Answer 7

Telomerase binds to the 3’ overhang of the telomere and the internal RNA component aligns with the existing telomere repeats.
Telomerase synthesises new repeats using its own RNA component as a template.
Telomerase repositions itself on the chromosome and the RNA template hybridises with the DNA once more.
In this cycle of synthesis, disassociation and reassociation to the 3’ end of the G rich strand, we can synthesise additional copies of the G rich unit repeat.
When the overhang is long enough, DNA Polymerase then extends an RNA primer to synthesise a complementary strand.
This primer may not be positioned right at the chromosome end and cannot be replaced with DNA so an overhang will still be there but the whole length of the telomere will be longer.

Question 8

What is the catalytic component of telomerase?

Answer 8

Protein component - Telomerase reverse transcriptase (TERT)
In vitro: Only TER and TERT (EST2p) required for activity
In yeast in vivo: EST1p and EST3p also required
In humans: NOP10, NHP2, dyskerin, GAR1, TCAB1, H2A, H2B

Question 9

Describe the evidence that protein TERT works in vivo.

Answer 9

Yeast mutants (RT domain) which have extra short telomeres (EST) die because they can’t maintain linear integrity
Found EST lined up with TERT sequence so knew EST2 encoded yeast paralogue of Euplotes TERT

Question 10

Which other proteins are involved in the telomerase finding the telomere?

Answer 10

hTERT synthesised in cytoplasm and associates with chaperones HSP90 and p23
hTR cotranscriptionally binds with dyskerin, NOP10, NHP2 and NAF1 (subsequently replaced by GAr1)
Telomerase TRT associated with telomeres through specific protein interactions with TPP1

Question 11

How do drosophila maintain telomeres?

Answer 11

Lack TG rich telomeric DNA so have specific retrotransposons at the ends
HeT-A 6kb element with 2 overlapping ORFs
(utilises cellular enzyme, encodes no own RT)
TART 10kb element with 2 sequential ORFs (encodes own RT)
Synthesis of retro-element telomeres requires RTase and may resemble mechanism from which telomeres evolved

Question 12

Describe Alternative Lengthening of Telomeres pathway (ALT).

Answer 12

Absence of telomerase activity
Promyelocytic leukaemia protein (PML)
Extra-chromosomal circular telomeric repeats

Break induced replication
One of the strands is cut away, resulting in a G rich single strand of DNA that bind onto a matching template, forming a displacement loop (d-loop)
DNA Polymerase extends single strand further than initial breakpoint, causing initiation of lagging strand synthesis.
Two strands produced containing entirely new DNA (non-conservative)

Question 13

What are the 6 proteins involved in the shelterin complex and their functions?

Answer 13

TRF1 and TRF2 bind to ds region of telomere and regulate length by inhibiting or facilitating telomerase activity
POT1 binds to ss overhang and prevents it from being degraded or recombined
RAP1 interacts TRF1 and stabilises T-loop
TIN2 connects TRF1and2 with TPP1andPOT1 and mediates recruitment of other factors to the telomere
TPP1 forms heterodimer with POT1 and enhances binding to the overhang; also serves as docking site for telomerase and other proteins involved in telomerase maintenance

Question 14

What are the 3 key roles of shelterin?

Answer 14

1. T loop formation
2. Regulate telomerase (POT1)
3. Inhibits DNA damage response (POT1 and TRF2)

Question 15

Describe T-loop formation

Answer 15

The ss end loops back and pairs with complementary sequences in ds DNA region.
In mammals, T loop requires TRF2

Question 16

What evidence supports the idea that TRF2 inhibits the damage response?

Answer 16

Experiment deleting TRF2 gene
Activation of ATM (Ataxia Telangiectasia Mutated) kinase
Cell cycle arrest at G1/S
Cell sees chromosome ends as ds breaks so tries to fix it by fusing chromosomes together in a chain

Question 17

What evidence supports the idea that POT1 also regulates telomerase?

Answer 17

Experiment where we conditionally knockout POT1 in chicken cells resulting in unregulated telomeres
Experiment knocking down in human cells resulted in very long telomeres

Question 18

Describe what is meant by the term Hayflick limit.

Answer 18

The Hayflick limit refers to the limited doubling potential of normal mammalian cells when cultured.
In human fibroblasts, this is 50-80 doublings and leads to replicative senescense
Fibroblasts from newborn will proliferate for much longer than the fibroblasts from an adult

Question 19

Describe what is meant by the Telomere Clock Hypothesis.

Answer 19

Theory that proposes that the length of telomeres determines the biological age and lifespan of an organism. According to this theory, each time a cell divides, the telomeres shorten and eventually reach a critical length that triggers cellular senescence or death. Therefore, telomere length can be seen as a molecular clock that measures the number of cell divisions and the cumulative damage from oxidative stress and inflammation

Question 20

How do short telomeres trigger senescence?

Answer 20

Short telomeres activate p53
p53 activates p21
p21 inhibits Cdk2/Cyclin E promoting Rb repression of E2F dependent transcription

If p53 mutated in cancer, response in inhibited, won’t be able to inhibit kinases, Rb won’t be phosphorylated, continue to cycle, telomeres continue to shorten, telomere-telomere fusion, chromosome rearrangements/damage

Question 21

Describe the evidence for the link between telomerase and cell senescense using telomerase negative cell lines.

Answer 21

Telomerase negative cell lines e.g. retinal pigment epithelial cells and foreskin fibroblasts
Express hTERT to reactivate telomerase (RNA component present in all cells)
Controls lacking TERT - telomere shortening and senescence
Cells expressing TERT - longer telomeres and continued division

Question 22

Describe the evidence for the link between telomerase and cell senescense using the telomerase KO mouse

Answer 22

Mice viable and fertile, no obvious effects through 4 generations
Couldn’t be bred past G6 - heart failure, immuno- and haemato-senescence
Homozygous mouse lacking telomerase RNA gene, no telomerase activity in any tissue
Short telomeres promote upregulation of p53 and function as tumour suppressors

Question 23

How do cancer cells develop?

Answer 23

Multiple genetic changes
Loss of checkpoint responses allows cells to continue dividing with short telomeres leading to genetic instability
In the absence of tumour suppressors such as p53 and RB cell growth isn’t checked
Often associated with aneuploidy and can give rise to chromothripsis and kataegis

Telomerase can be reactivated in cancer cells after crisis - telomeres stabilised at much shorter length than comparable normal cells

Question 24

How can telomere shortening lead to chromosome fusion, chromothripsis and kataegis?

Answer 24

Took cells which had no telomerase and shortened telomeres
Telomere-telomere fusions as expected
As cells divide, chromosomes segregated to opposite ends with chromatin bridge forming
New membrane and nuclear envelope around long chromatin bridge - envelope stretched and breaks down
Enzyme Trex1 given access to chromatin bridge
Degrade 1 strand of chromatin to ssDNA
ssDNA bound by RPA (replicating protein A1)
ssDNA became target for cytosine deaminase

Question 25

What are the problems with telomerase as a target for anti-cancer drugs?

Answer 25

1. Would take time to act while (TTAGGG)n is eroded.
2. Would damage proliferating stem cells i.e. not specific
3. Emergence of resistant cells using ELT pathway
4. Possibility of increased chromosome instability amongst survivors

Question 26

What are Telomere Biology Disorders (TBDs)/Telomere Syndromes?

Answer 26

Diverse inherited disorders that share short telomeres - defect acquired with age
Most prevalent monogenic premature aging disorders
Unique inheritance patterns that distinguish them from other Mendelian disorders
Affect many organ systems

Question 27

Describe the evidence behind the idea that telomerase shows haploinsufficiency.

Answer 27

Greider (2005) mouse model
Suggest telomere length also sensitive to volume not just presence/absence of TERT
Hemi for telomerase, hetero for KO (1 allele telomerase, 1 with KO)
HG1 = hetero KO of Tr 1st gen; signal free ends, no telomeres
Breed hetero together for successive gen
By 5th gen, telomeres in hetero stock shorter than 1st gen hetero stock (these sick mice)
Haven’t got enough telomerase so telomeres still shorten
Cells sensitive to length of shortest telomere and not average telomere length

Question 28

Describe how telomeres can shorten due to the collapse of replication fork.

Answer 28

Telomeric strand is G rich, leaving it susceptible to 2 types of damage:
1. Oxidation of guanine residues to 8-oxo-Guanine
2. G quadruplexes
Impediments to progress of replication fork and can’t get replicated from other direction leading to sudden shortening of telomeric DNA

Question 29

Describe Dyskeratosis congenita (DKC) as an example of a telomerase syndrome.

Answer 29

First disorder linked to mutant telomere gene-dyskerin DKC1 gene on X chromosome
Cancer occurs mainly in tissues with high turnover

Question 30

How can genes mutated in TBD sometimes be rescued by somatic mutations?

Answer 30

1. Back mutations restore wild-type sequences - substituting mutated section back to original bases (can happen spontaneously or as a result of mutagens)
2. Second-site (or reversion) mutations result in compensatory changes - Different site within gene to germline mutation - do not revert to original sequence but could restore reading frame, correct splicing defect or modify protein structure - associated with milder phenotypes