Lecture 9 - Telomeres and telomerase

Question 1

What is the end replication problem?

Answer 1

DNA polymerases can only extend an already existing piece of DNA, and only if there is an available 3’ end to be extended also requires a primer.
DNA polymerase leaves a 3’ overhang after synthesis, as there is no reforming with complementary base pairs as no primer can join to.

Question 2

What are three ways you can solve the end replication problem?

Answer 2

• use circular chromosome
• use special mechanism (viral) e.g. like a protein binding to a nucleotide
• use repetitive sequences at the end of chromosomes (telomeres) (allow primers to change position)

Question 3

What is the bacterial mechanism to solve end replication problem?

Answer 3

Use bidirectional replication of circular bacterial chromosome initiated at a single origin
1-Parental DNA is circular complex
2-Bidirectional relication imitates at the origin (no loose ends)
3-Replication forks move around the chromosomes
5-replica chromosome may be catenated at completion
5-replica chromosomes are decatenated and can segregate to form daughter cells

Question 4

Problem with circulatar chromosome replication?

Answer 4

If replication fork gets stuck the mechanism to repair it involves BIR, induces a HJ behind newly establish RF, HJ resolved
Problem: 50% may be cut so that there is crossing over leading to a dimeric genome
-use XerCD system to fix to monomers

Question 5

How do virus’ get around the end replication problem?

Answer 5

1- protein linked to cysteine nucleotide binds to DNA polymerase
2- allows binding of complementary cytodine to guanine at the end of the chromosome
3-this can be extended and at the end of replication protein remains covelently bound end

Question 6

What mechanism is there of replication in retrovirus?

Answer 6

1) TRNA can be used as a primer as has a region of complimentaryity to viral DNA
2) DNA synthesis can be primed by reverse transcriptase to produce DNA at one end
3) As each end is a repeat structure newly synthesised DNA can switch template as it complimentary to other end of DNA
4) synthesise rest of the DNA
5) RNA degraded

Question 7

What are telomeres?

Answer 7

Repeat stucture at the end of chromosomes

GT rich region found at 3’ overhang of DNA generated when RNA primer removed

Question 8

What is telomerase?

Answer 8

A reverse transcriptase enzyme

Highly associated to RNA molecule bound to it which is complementary to 3’ overhang at end of chromosome

Question 9

How does telomerase extend telomeres?

Answer 9

1) telomerase associated to an RNA molecule that is complimetary to 3’ overhang at the end of chromosomes generated through replication binds to the telomere
2) extend the incomplete parental strand 3’ end by reverse transcriptase activity, long enough for lagging strand to be completed by DNA polymerase

Question 10

What is the purpose of telomerase/telomere activity?

Answer 10

counteract the degredation of chromosomes

Question 11

In what states can plasmids be maintained in yeast?

Answer 11

In circular form
In chromosome
NOT linear form

Question 12

How can plasmids be maintained in linear form in yeast?

Answer 12

the addition of telomere sequence allows plasmids to replicate in linear form e.g. YAC (yeast artificial chromosome)

Question 13

When is telomerase expressed?

Answer 13

expressed in actively dividing cells, not in quiesent cells or normal human cells

Question 14

What does loss of telomerase result in?

Answer 14

senescence (biological aging)

Question 15

How can cells escape from senescence? Why is this dangerous?

Answer 15

Through homologous recombination to restore telomeres and if telomerase is acitvated, this is dangerous as have no control and e.g. cancer can mjultiply forever

Question 16

What is the Hayflick limit?

Answer 16

Limit on telomere length before senescence e.g. cultured human cells divide 50 times before senescence

Question 17

What does mutations in telomerase result in?

Answer 17

Causes telomeres to shorten in each division

Question 18

How can senescence be prevented in somatic cells, where telomerase is not normally expressed?

Answer 18

Artificial expression of telomerase

Question 19

What is telomere length homeostasis?

Answer 19

The regulation of the length of telomeres

Homeostatsis different in every organism

Question 20

How is telomere length homeostasis shown?

Answer 20

Insert artificially longened/shortened telomeres into an organism
after divisions telomere length will be regulated to normal length

Question 21

How is telomere length measured?

Answer 21

Rap 1 binds to telomere repeat structure and regulates length, deciding whether telomerase binds and elongates telomere

Question 22

How was it shown that Rap1 regulates telomere length?

Answer 22

1)artificial binding sites for Rap1 created by binding protein to Rap1 which is able to bind to pieces of DNA different to telomere sequences
2)Total number of Rap1 binding sites increase because of the addition of artifical binding sites
3)leads to shortening of telomeric repeats
Number of Rap1 proteins counting mechanism for size of telomere

Question 23

What are the three roles of telomeres?

Answer 23

• prevent chromosome shortening
• protect ends from being used in repair process as this would lead to chromosome fusions and genome instability
• act as damage sensor, if damaged trigger senescence stopping becoming caner cells

Question 24

What protein are telomere ends associated with?

Answer 24

Telomere ends are associated with large protein complex shelterin

Question 25

What occurs in the shelterin complex?

Answer 25

Within loop, 3’ overhanging end invades pieces of DNA upstream upstream in D loop
Forms T loop

Question 26

What happens if proteins in the shelterin complex mutate?

Answer 26

lead to genetic disease e.g. WRN protein loss leads to Werner syndrome

Question 27

What occurs if the chromosome ends are not protected?

Answer 27

1) DNA repair system fuses ends of different chromsomes together leading to dicentric chromosome
2) during cell division if centromeres migrate to different poles leads to breakage of fused chromoseome (anaphase bridge)
3) ends are then also not protected leading to further breakage

Question 28

When does nonhomologous end joining occur?

Answer 28

before chromosomes have replicated when no sister chromatid available for HR

Question 29

How is nonhomologous end joining occur?

Answer 29

1) binding of protein complex to loose ends
2) Loose ends are trimmed resulting in the loss of a few bp
3) 5’ to 3’ exonuclease prevented from producing long 3’ overhangs (would be a substrate for HR)
4) Ends joined

Question 30

What does Cdk1 regulate

Answer 30

NHEJ
-if low Cdk1 = NHEJ,
- if high = HR acts on 5’ ends processed to 3’ overhangs
telomere extension
-3’ ends must be extended by exonucleases but at low Cdk1 telomerase cannot access telomeres

Question 31

How does the DNA damage response different at non-telomeric DNA and telomeres?

Answer 31

At non telomeric DNA
-DSB initiates DNA damage response (complex signalling process) leads to Transient proliferation arrest and Repair
At telomeric DNA
-DSB leads to permenent DNA damage response leads to Permenant proliferation arrest (senescence)
HOW? protein complex (shelterin) inhibits repairs
WHY? repair of telomeres could lead to chromosome fusions or ectopic recombination and loss of stability

Question 32

What happens with ectopic localisation of TRF2 (shelterin protein) next to a DSB?

Answer 32

Impedes repair and leads to prolonged DDR activation

Question 33

How was ectopic localisation of TRF2 near DSB shown experimentally?

Answer 33

1) Introduced an artifical TRF binding site into chromosome not at telomeric end by fusing TRF2 to lacI which can bind to lac operator in chromosome
2) I-Scel cut site and fluoresent protein binding site introduced nearby
3) I-scel site cut by nuclease I-scel, break next to TRF2
4) These sites were not repaired

Question 34

What key regulator triggers cell senescece when telomeres reach critical length?

Answer 34

P53

Question 35

What happens if P53 is mutated?

Answer 35

aging does not occur cells escapse senescene leading to cancer

Question 36

How are telomeres naturally shortened?

Answer 36

-environmental stresses and ROI affect
-genetic defects
ROI affects telomeres in G rich regions

Question 37

What is the purpose of telomerase gene therapy?

Answer 37

delays aging and increases logevity without increasing cancer

Question 38

How is telomerase gene therapy shown in mice?

Answer 38

• Treat 1-2 year old mice with adenovirus expressing TERT (telomerase reverse transcriptase)
• increased telomerase expression, reducing aging associated telomerase erosion, leading to extension of short telomeres
• led to inncreased health in mice

Question 39

What are the effects of Telomerase reverse transcription ectopic expression late in life?

Answer 39

• decreases the incidence of age related osteoporosis and glucose intolerance
• significantly extends lifespan without increasing cancer
• improves scores in neuromuscular and object recognition tests