Telomeres, Senescence, and Crisis

Define telomeres.

Telomeres are terminal structures that protect chromosome ends from NHEJ and exonucleolytic damage. They consist of repeated 5’-TTAGGG-3’ sequences (G-rich) in vertebrates. In humans, telomeres are between 5-10k bps. In mice, they vary between 20-160k bps, which has implications for how mouse models are being used to study human disease.

What is the end replication problem?

During DNA replication, DNA polymerase primes internal to the 3’ template. When primers are removed, a 3’ overhang of the template strand is left over the 5’ end of the newly synthesized strand. Thus, if a cell divides a lot, it will eventually run out of DNA at the ends.

What is telomerase? Describe the steps used by telomerase to extend the telomeres.

Telomerase is an enzyme that adds new 3’ telomere sequences onto the template strand, allowing extension of the newly synthesized lagging strand. Notably, telomerase expression is absent in most normal somatic cells but active in a majority of human cancers to maintain endless replicative potential.

Structure: TERC (RNA template) + TERT (reverse transcriptase catalytic subunit). The TERC RNA sequence is a C-rich strand that primes new DNA synthesis of the G-rich strand. TERC tends to be expressed in all cells, but TERT is expressed only in germ cells, stem cells, and rapidly dividing lymphocytes.

1. Telomerase with bound TERC sequence binds to the 3’ end of the parental strand (G-rich strand), acting as a primer.

2. TERT subunit facilitates reverse transcription of DNA from RNA to elongate the parental strand

3. The lagging strand is filled in by DNA pol, increasing the length of the telomere

What protects ends of chromosomes?

1. Terminal loop (T-loop) acts as a protective cap on the end of the chromosome. Within the telomere, there’s a C-rich strand (the lagging strand) and a G-rich strand (overhanging template strand). The G-rich strand has a 3’ overhanging end that can anneal to an upstream portion of the C-rich strand, displacing a portion of the C-rich strand that was originally there to create the displacement loop.

2. Shelterin Complex (TRF1, TRF2, POT1, TPP1, RAP1, TIN2) binds telomere in either the T-loop or linear structure, preventing the ends of chromosomes from being recognized as DSBs. Losing any component of the Shelterin complex can result in chromosomal fusions.

3. Proteins associated with DSB repair also bind to telomeres, notably NHEJ proteins (Ku70::Ku80, DNA-PKcs, MRN sensor complex)

What is replicative senescence? How can it be bypassed?

Replicative senescence is permanent (theoretical) exit from the cell cycle associated with morphological changes and expression of senescence associated beta-galactosidase, P15INK4b, P16INK4a, etc. – similar to oncogene induced senescence.

Human cells undergoing replicative senescence display increased p53, p19ARF, P16INK4a, SA-BGal, and unphosphorylated Rb, indicating that loss of telomeric length can activate cell cycle checkpoints and a subsequent senescent phenotype.

Ex. Cultured cells that express the dominant negative form of Trf2 (Shelterin complex protein) look like cells that have entered replicative senescence despite having long telomeres. This indicates that p53 and Rb related checkpoints are active, since loss of Trf2 creates what looks like a DSB, which prompts multiple responses (replicative senescence, apoptosis, and worse case – NHEJ = chromosomal fusion from BFB cycles).

Replicative senescence can be bypassed by expressing the TERT subunit in human fibroblasts, leading to stabilized telomeres and subsequent immortalization.

What is crisis?

Crisis is massive cell death caused by cells continuing to divide without telomeres (due to unprotected ends and/or loss of cell cycle checkpoints).

Early Crisis: Caused by telomere attrition and inactivation of cell cycle checkpoints → DNA damage signals have been activated.

Late Crisis: Due to inactive p53 → severe telomere dysfunction and rampant genetic instability from breakage-fusion-bridge (BFB) cycles

• BFB cycles: During M phase, chromosomes without functional telomeres break → replicate → sister chromatids fuse together or with other chromatids to form a bridge → resulting fused chromosome lacking telomeres breaks → cycle repeats

How can a cell be saved from crisis?

1. TERT expression to generate new telomeres

2. Alternative lengthening of telomeres (ALT): Similar to HR

   1. Exonuclease resects 5’ end of the C-rich strand to create 3’ overhang in the G-rich strand

   2. 3’ overhang can invade into the telomeric sequences on a chromosome with a long telomere – they can anneal because all telomeres consist of the 5’-TTAGGG-3’ sequence

   3. Invading strand is extended with DNA pol using the other chromosome’s telomere seq as a template

How is telomere dysfunction involved in contradictory processes?

a) Tumor suppression by activating senescence

b) Tumor promotion when replicative senescence is bypassed due to chromosomal rearrangements increasing mutagenic load. Ultimately, cancer cells will need to generate new telomeres (TERT expression, ALT) to restore genomic stability after crisis.