Telomeres Flashcards
What are telomeres?
Repetitive DNA structures at the end of eukaryotic linear chromosomes. They protect the ends from DNA repair machinery.
Do prokaryotes have telomeres?
No because their chromosomes are circular.
What is the probable reason that eukaryotes evolved linear chromosomes?
Linear chromosomes can generate great genetic diversity through independent assortment and crossing over in meiosis. This diversity is advantageous.
What would be the outcome of a singular cross over with 2 circular homologous chromosomes, each with 2 sister chromatids?
One large circle, joined at the crossover points of 2 homologous sister chromatids would be generated. This larger circle would still be attached to 2 smaller circular sister chromatids at the 2 centromeres. (Lecture 14, slide 3).
Segregation of this one structure in meiosis would generate 2 random DSBs.
What would be the outcome of an even number of cross overs with 2 circular homologous chromosomes?
The circular chromosomes would not be joined and segregation in meiosis would be successful.
What could evolve to solve the requirement of an even number of crossovers in circular chromosome meiosis?
- Counting of the crossovers. Maybe too complex?
- Linearisation of chromosomes (successful segregation with any number of crossovers). WHAT HAPPENED (eukaryotes).
In a circular chromosome, what does a DNA end mean?
A DNA break (DNA damage).
In a linear chromosome, what does a DNA end mean?
- A DNA break (DNA damage).
- The end of the chromosome (natural end).
How are natural and broken DNA ends distinguished in linear chromosomes?
Presence or absence of telomeres.
Is the DDR activated by telomeres?
No.
What is the DNA damage response (DDR)?
- Phosphorylation cascade
- Triggers cell cycle arrest
- DNA repair enzymes recruited to break site and subsequent NHEJ or HDR (uses sister chromatid)
- OR apoptosis
What would happen if DDR was activated at all DNA ends (including telomeres)?
A DSB could lead to a dicentric chromosome (a telomeric end is used to repair the break, so 2 chromosomes are ligated). This leads to another DSB when these chromosomes are segregated.
What would happen if telomerase was activated at all DNA ends (including DNA breaks)?
There would be terminal deletions of DNA no longer joined to a centrosome. I.e. genetic information is lost after meiosis.
What are the possible DNA sequences of telomeres (non-coding)?
- Long inverted terminal repeats (LITRs)
- Short tandem repeats (STRs)
What are the possible structures of telomeres?
- LITRs with covalently closed single stranded hairpin
- LITRs with terminal protein bound to the 5’ end
- LITRs with ss 3’ overhangs
- STRs with ss 5’ overhangs
How can telomeres vary between organisms?
- Sequence
- Structure (RNA and protein)
- Telomere binding proteins (TBPs)
What are the functions of TBPs?
- Hide the DNA end from DNA repair enzymes, preventing the DDR and cell death.
- Recruit telomerase.
What are the classes of TBPs present in most organisms?
- Recognise dsDNA
- Recognise ssDNA
Which organisms have 3’ overhang telomeres?
- Vertebrates
- Budding yeast
- Fission yeast
What is the name of the TBP complex in humans?
Shelterin
How many proteins does shelterin consist of?
6
How are human telomeric ends protected?
- TBPs (shelterin)
- T loop formation
What is a T loop?
3’ ssDNA base pairs with sequence earlier in the telomere so a loop forms.
Which organism has 5’ overhang telomeres?
C. elegans
Which organisms’ telomeres have blunt ends (no overhang)?
Plants
What happens in NHEJ?
The DNA is ligated back together without monitoring.
What is HDR also known as?
Homologous recombination
How is DDR activated at DNA break?
- Nucleases digest 5’ strands at break, generating ss 3’ overhangs.
- RPA binds ss DNA (non sequence specific). This binding is persistent (not transient).
- This signals DNA damage, so DDR proteins are recruited.
- Several DDR proteins binding in the same place triggers DDR and cell cycle arrest.
What is the RPA protein?
Replication protein A. Binds any ssDNA in a cell (but not RNA).
How is the DDR inhibited at telomeres?
- TBPs outcompete RPA at the 3’ overhang because they have a higher affinity to the ssDNA than RPA. RPA recruitment is inhibited.
- TBPs inhibit 5’ end resection by nucleases. Without this the DNA could be digested past the telomeric sequence and RPA would be recruited (DDR).
- TBPs inhibit NHEJ.
Why do TBPs not bind any ssDNA?
They bind in a sequence specific manner.
How were the functions of TBPs characterised?
Observing ts mutants - cell arrests when a certain temperature is reached.
What happens when TRF2 (shelterin component) in knocked out?
Telomeres ligate to each other through NHEJ. A train of chromosomes is generated (multiple centromeres) which causes mitotic catastrophe.
Why are longer telomeres better at protecting the chromosomes from the DDR than shorter telomeres?
There are more TBPs bound - less likely that there will be an occasion where all TBPs are ‘off’, repair enzymes begin digesting the DNA the DDR is promoted (irreversible).
What is an uncapped telomere?
A telomere that is so short it has a greatly increased probability of end-to-end fusions and activation of the DDR.
What can happen when 2 adjacent sister chromatids have uncapped telomeres?
Breakage fusion bridge cycle:
- They can fuse (end-to-end); an obvious template for repair machinery (looks like a break).
- This creates a dicentric chromosome so in mitosis another random DSB is generated.
- 2 incorrect chromosomes are generated - one has lost material (deletion) and one has some duplicated material (duplication).
- The DSB needs repairing, so the cell uses another uncapped chromosome or another chromosome with a DSB.
- Another dicentric chromosome is created and the cycle repeats.
Which cells often have an ongoing breakage fusion bridge cycle?
Cancer cells
What is an anaphase bridge?
DNA connecting 2 centromeres (in a dicentric chromosome). It links the chromosomes when they are being pulled apart in anaphase.
How is an anaphase bridge broken?
- Mechanical stress
- Cut in cytokinesis
What gross chromosomal rearrangements can the breakage fusion bridge cycle result in?
- Deletions
- Duplications
- Translocations
- Aneuploidy
What was the end replication problem postulated in 1972?
The current model did not explain how the ends of the DNA were replicated. It was unclear how the last part of the lagging strand (Okazaki fragments) was replicated because there was no place for an RNA primer to bind.
What is the transcription mechanism in Tetrahymena?
- DNA is replicated in the micronucleus.
- DNA is transferred to the macronucleus.
- DNA is chopped into fragments.
- DNA is transcribed.
Why is Tetrahymena an ideal organism for studying telomeres?
Because its DNA is fragmented when it is transcribed, a high proportion of Tetrahymena DNA is telomeric (each fragment has telomeres). Lots of telomeres makes it easy to study.
What is a typical characteristic of a telomeric sequence?
T and G on one strand, A and C on the other.
What was the original theory for how cells replicated the telomeres?
HDR (homologous recombination) because all chromosomes had the same telomeres. A long telomere could be used as a template to extend a short telomere.
Are all telomeres the same in one organism?
Yes - just variation between species.
How was telomerase discovered?
Greider and Blackburn took fractions of protein extract from tetrahymena (lots of telomeres = lots of telomerase) and observed which could extend the telomere oligo (known sequence) - length of radioactive oligos observed on a gel after incubation with extract.
What properties of telomerase were discovered from incubating protein extract with oligos?
- It is specific to telomeric DNA sequences.
- It only extends one strand of the DNA (didn’t work with complemetary telomeric sequence).
What type of enzyme is telomerase?
A reverse transcriptase. It is a nucleoprotein because it contains the RNA template for telomere elongation (also complementary to the telomeric 3’ end) and the protein.
How much can telomerase extend the telomere by per cycle?
One telomeric repeat. Then dissociates and anneals further down the DNA.
How long is a telomeric repeat in vertebrates?
6 nucleotides.
What are the functions of telomeres?
- Protect chromosome ends from DNA repair enzymatic activities (nucleases, ligases, recombinases)
- Prevent DNA loss due to incomplete DNA replication by the conventional mechanism. Telomere structure coevolved with telomerase to ensure telomerase-dependent telomere synthesis compensating for the incomplete DNA replication.
Which eukaryotes don’t have telomeres and what do they have instead?
Drosophila - they have repeated transposable elements at the end of their chromosomes. Transposition maintains these repeats.
What is the human telomerase protein name?
hTERT.
What are some structural elements of telomerase’s RNA?
- Hairpins a.k.a. stem loops
- Pseudoknot (for telomerase protein component recognition)
- Template sequence (recognises telomeric DNA)
- Template boundary (tells telomerase to dissociate from DNA).
What is a pseudoknot?
Base pairing of 3 RNA strands.
What is the human telomerase RNA name?
hTR.
What is conserved between different organisms’ telomerase RNA?
Important structural elements (but these are made from different sequences).
In yeast, what are the telomerase subunits called?
Est 1/2/3 - ever shorter telomeres mutant phenotype.
What is the phenotype of telomerase mutants?
Delayed lethality.
Don’t die immediately, but death is guaranteed (≈ 50 generations in yeast).
What bases are telomeres rich in?
G and C.
Why are telomeres hard to replicate?
- They are GC rich so the lagging strand can form G quartets (G4) - non-Watson Crick base pairing structures in a folded DNA strand. There are H bonds between corner (neighbouring) Gs. The replication forks have to pause so specialised helicases can unwind the G4.
- The T loop must also be unwound every cycle by RTEL1 helicase.
Is telomerase abundant in cells?
No - there are relatively few copies. Not every telomere is extended every cell cycle.
Which telomeres is telomerase predominantly recruited to?
The shortest ones.
How does telomerase know which telomeres are the shortest?
It can (somehow - molecular mechanism unknown) count the number of TBPs bound i.e. TBPs inhibit telomerase recruitment.
Works if you artifically tether TBPs to non-telomeric DNA.
What triggers senescence?
Cellular stress causes p53 activation which causes a reversible G1 arrest. The cell can then fix its problems and continue dividing, or not fix its problems and apoptose (rare) or senesce (common).
What is senescence?
After a cell has been in reversible G1 arrest (p53 activated) for too long, the p16 pathway is activated and the cell enters irreversible G1 arrest.
What is the senescence-associated secretory phenotype (SASP)?
Senescent cell releases cytokines, which trigger:
- Neighbouring cells to divide (promotes tissue repair).
- Macrophages to be recruited to the area to eliminate the senescent cell (prevents larger scale problems occurring).
Why is senescence advantageous over apoptosis?
- It allows clearance of all problematic cells in the area as opposed to just the original problem cell.
- Replacement of cells allows embryo development to continue as normal.
How does the presence of senescent cells vary with age?
- Young people rarely have senescent cells - efficiently eliminated.
- In people >40, senescent cells can be detected easily.
- Senescent cells accumulate as we age.
What are the effects of senescent cell accumulation?
SASP continues:
- Tissue dysfunction
- Chronic inflammation
- Tumorigenesis
Why might senescent cells accumulate as we age?
- More senescent cells are produced.
- Immune clearance becomes less efficient.
- Both of the above.
What happens when mice are engineered to trigger apoptosis instead of senescence?
- The mice appear healthier.
- The mice live longer.
Why is telomere length generally thought to be an indicator of lifespan?
Telomere erosion triggers senescence, which is an indicator of lifespan.
However many other factors also trigger senescence, so this is not a direct correlation.
What happens to telomeres as we age?
They get shorter / erode.
What telomerase activity do germline and ESCs have?
Constitutively high activity. Telomere length is maintained with divisions.
What telomerase activity do ASCs have?
Regulated low telomerase activity. Telomere length decreases slowly with divisions.
What telomerase activity do somatic cells have?
No telomerase activity. Telomere length decreases quickly with divisions. Senescence is triggered.
How is telomerase activity regulated?
Transcriptional inactivation of the catalytic subunit.
What is the usual result of mutations in p53 or p16 pathways in cells?
These senescent cells can continue to divide without having their problems fixed. Telomeres shorten even further. Essential DNA is lost and the cells die.
How can senescent cells become cancer cells?
There are mutations in the p53 or p16 pathways so the cells continue to divide, however their telomeres are maintained either by:
- Reactivation of telomerase (90%)
- Alternative lengthening of telomeres (ALT) (recombination dependent telomere maintenance)
and the cells survive (10%).
What is telomerase haploinsufficiency?
Only one telomerase gene is functional. The shortening of telomeres is faster than normal. Dividing tissues become non-functional (bone marrow, skin, blood). Ultimately results in early mortality.
What condition do mutations in the Dyskerin component of telomerase result in?
Dyskeratosis congenita.
- X-linked (more common in males).
- Manifests as a skin disorder.
- Associated with early onset of many other conditions associated with ageing / reducing telomeres.
What is the impact of having double the usual telomerase?
Early onset of cancer (early 20s) - familial mutation. Cancer is promoted because the cancer cells take longer to reach crisis point (telomeres shorten slower) (don’t senesce due to mutations in p53), and are rescued from death as they never become uncapped.
What is the impact of downregulating telomerase expression in somatic cells?
Cancer is suppressed; cancer cells reach crisis point (skip senescence due to p53 mutation) and die.
Why is there only downregulation of telomerase transcription in embryonic development in larger animals?
Larger animals are more likely to die from cancer because:
- They have more cells.
- They live longer.
What is ALT?
Telomeres use each other to recombine and lengthen because they are all homologous.
They can use:
- Upstream telomeric sequence
- Sister chromatid’s telomeric sequence
- Different chromosome’s telomeric sequence
- Circular telomeric DNA
What is commonly found in cancer cells using ALT?
Circular telomeric DNA.
What do yeast cells use for telomere maintenance?
HDR (homologous recombination) via circular telomeric DNA.
How do we immortalise human cells in culture?
Transform them by expressing the telomerase catalytic subunit.
Why does cancer develop when a mutation inactivates p53?
- Senescence is overridden
- Cells continue to divide and telomeres shorten
- The shortest telomeres become dysfunctional and the cell reaches a crisis point
- CANCER CELLS ARE RESCUED BY TELOMERASE REACTIVATION OR ALT. Telomeres are stabilised.
How is telomerase reactivated in cancer cells?
There is a mutation (SNP) in the promoter of the telomerase catalytic subunit gene. Expression of this gene is reactivated, only in the allele with this mutation. One allele is still inactivated.
Which cells gain an immediate advantage when telomerase is reactivated?
Cells with short telomeres that are senescent. They are able to divide again.
Which cells does reactivation of telomerase make little difference to?
Cells with long telomeres. Their division is not arrested anyway.
How does telomerase expression correlate with survival in cancer patients?
Higher telomerase expression correlates with poorer survival.
Why is telomerase a good anti-cancer drug target?
90% of cancers are telomerase + and this decreases survival of patients. Inhibition of telomerase could inhibit cancer cell survival.
What is the mechanism of the anti-cancer drug being developed by Geron?
It is an oligo that binds the template RNA in telomerase, therefore blocking telomerase from binding telomeric ends.
What is the issue with developing telomerase targeting drugs?
Hard to target delivery - don’t want to inhibit telomerase function in all cells!
What are the 5 clinical approaches to telomerase inhibition?
- Telomerase (active site) inhibitors.
- Immunotherapy - eliminate cells with telomerase activity so cancers can’t progress. However may also target ASCs and germline cells.
- Disrupting agents that remove shelterin from cancer cell telomeres so the DDR is triggered.
- Engineering so telomerase promoter activity promotes apoptosis.
- Interfering with a step in telomerase biogenesis (complex process).
