Telomeres Flashcards
How is chromosomal stability achieved?
Telomeres protect chromosomal stability.
Outline telomeres.
A telomere is a region of repetitive hexanucleotide sequences (TTAGGG)n at each end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. In humans telomeres are 10-15 kb long, and significantly longer in mice at ~50 kb.
Telomeres have a T-loop structure at their ends which is maintained by shelterin complexes. This stops recognition by DDR proteins and subsequent chromosome end-to-end fusions.
Telomeres are maintained by telomerase in germ and stem cells.
What is the end replication problem? How does telomerase solve it?
The end replication problem was first realised in 1969: the conventional replicating enzymes cannot replicate at the end of chromosomes. The replication fork contains a leading and a lagging strand, which creates a problem at telomeres: the lagging strand is replicated in segments and requires a primer to start synthesis, so at the very end there is a gap where DNA cannot be synthesised, and when the last primer is removed at the very end of the telomere a small fragment of DNA is lost each time the cell replicates. RNA primers are ~10 nt. Each end should have an overhang: there is a post replication mechanism that uses an exonuclease to degrade the strands and create an overhang, which removes ~150 nt each cell division
How are the very ends of chromosomes protected?
The formation of a T-loop structure by shelterin proteins.
What are the components of shelterin?
- TRF1: DNA remodelling activity, dsDNA binding
- TRF2: DNA remodelling activity, dsDNA binding
- RAP1
- TIN2: acts as a bridge between all of the shelterin proteins, binds TRF1, TRF2, TPP1/POT1
- POT1: ssDNA binding, binds the 3’ G overhang, 5’ nuclease regulation
- TPP1
What is the function of shelterin?
Shelterin determines the structure of the telomere terminus and is implicated in the generation of t-loops.
Collectively the subunits:
- Inhibit the chromosome ends from eliciting a DDR
- Inhibit the chromosome ends from being subjected to unregulated end processing, NHEJ, or HR
- Regulate the activity and access of telomerase both positively and negatively
- Promote replication duplex of telomeric DNA
- Promote proper sister chromatid adhesion
How does shelterin control the action of telomerase?
What are the consequences of dysfunctional shelterin?
Dysfunctional shelterin means that telomere end structures cannot be formed correctly, which leads to activation of the DDR pathway. This leads to either apoptosis or senescence.
- Loss of TRF2 activates the ATM kinase pathway, leading to p53 upregulation and p21-mediated G1/S arrest.
- When TRF2 is inhibited or chromosomes become critically short, 53BP, gamma-H2AX, the MRN complex, and phosphorylated ATM (S1981) accumulates at chromosome ends. These form structures called telomere dysfunction induced foci (TIFs).
- TIFs are also formed when other components of shelterin are inhibited.
What are the different components of telomerase?
- TERT: catalytic reverse transcriptase unit
- hTR (TERC): RNA template unit
- Nop10
- NHP2
- GAR1
- Dyskerin: facilitates telomerase assembly and TERC stability
What diseases arise from dysfunctional components of telomerase?
- Mutations in the genes TERT or TERC (encoding hTR) are associated with autosomal dominant pulmonary fibrosis, bone marrow failure syndromes, and dyskeratosis congenita.
- Mutations in DKC1, encoding dyskerin, cause X-linked recessive dyskeratosis congenita.
- Autosomal recessive DKC has been described in a family with a homozygous mutation in NOLA3, encoding NOP1.
Outline dyskeratosis congenita (DKC)
DKC is a rare inherited multisystem disorder characterised by the triad of:
- reticulated skin pigmentation
- nail dystrophy
- leukoplakia (white patches) in the mouth
The prevalence is around 1 in a million individuals and the median age of death is 16. Individuals appear normal at birth, later developing bone marrow, pulmonary, GI, skeletal, immunologic and neurologic abnormalities. Bone marrow failure is the leading cause of death, followed by pulmonary disease and cancer.
Mutations in genes involved with telomere maintenance have been identified in ~40% of clinically diagnosed DKC cases.
There are three forms of the disease:
- X-linked autosomal recessive
- Autosomal recessive
- Autosomal dominant
What is the consequence of a mutated DKC1 gene?
DKC1 encodes the protein dyskerin, which is a nucleolar protein that associates with hTR (TERC) of telomerase. Mutations in DKC1 cause X-linked recessive dyskeratosis congenita (DKC).
- Cell lines have reduced hTR which limits telomere length.
- Most mutations affect the N-terminal or PUA domain, which are needed for hTR interaction.
What is the consequence of a mutated TERC gene?
Mutations in the TERC gene, encoding hTR, causes the rare autosomal dominant form of dyskeratosis congenita (DKC).
- Many mutation affect hTR accumulation and/or catalytic activity, and all demonstrate telomere shortening.
- Autosomal dominance arises from the interaction and inhibition of the WT gene product by the mutant gene product in a multiprotein complex. In most cases it is due to haploinsufficiency leading to a reduced amount of telomerase.
NOP10
What is the consequence of mutated NOLA3 gene?
Mutations in NOLA3, encoding NOP10, can cause autosomal recessive dyskeratosis congenita (DKC).
- Saudi Arabian family: thought to be a substitution (R34W) in a high conserved region in NOP10. None of the other 15 AR families showed linkage to this region, and none of the probands of 171 uncharacterised DKC cases, underscoring the mutation rarity.
What is a common observation in all cases of DKC?
Shortened telomeres
SSqQF
What are methods available for analysing telomere length? [5]
- Terminal restriction fragment (TRF) analysis: this the original technique, considered a ‘gold standard’. DNA is digested using a cocktail of restriction enzymes that do not recognise telomeric regions. The intact telomeres are resolved based on size using agarose gel electrophoresis. The fragments are visualised by Southern blotting or in-gel hybridisation. This can be seen as a ‘smear’ of varying lengths of all the telomeres. This method is highly susceptible to accidental DNA shortening in experimental procedure, so extra care is needed.
- qPCR analysis: this method overcomes the need for large quantities of DNA for telomere length evaluation. The amount of DNA sequence of interest is quantified through the use of a fluorophore that intercalates with dsDNA (SYBR green). After each cycle fluorescence is measured and quantified. This calculates the average telomere length. This method produces variable results due to compromised precision of the assay.
- Single telomere length analysis (STELA): this qPCR-based method was designed based on the need for analysis of individual telomere lengths for a subset of chromosomes. Subtelomeric sequences of specific chromosomes are targeted for amplification through use of specific primers. Only a small subset of chromosomes can be analysed this way due to the lack of specific subtelomeric primers (Xp, Yp, 2p, 11q, 12q, 17p). This methods allows recognition of short telomeres on single chromosomes from specimes of small amounts of DNA.
- Quantitative fluorescence in situ hybridisation (Q-FISH): metaphase chromosomes or interphase nuclei are assessed following hybridisation/labeling with a fluorescent (CCCTAA)3 probe. The assessed cells can be fresh, frozen, fixed, embedded, or permeabilised. The probe used is typically a synthetic peptide nucleic acid (PNA) probe. Can measure individual telomeres and is the only method allowing recognition of telomere-free ends.
- Flow-FISH: an adaptation of Q-FISH combining flow cytometry with FISH, allowing for hybridisation to cells in suspension. Seperate populations can be distinguished. The same PNA probe as in Q-FISH is used. Average cell length for different cell populations is calculated. Can be used for categorising cells.
