lecture 3 - telomeres and telomerase

replicative sensecence

end of lifespan - unable to divide any further but remain alive for years. change of phenotype. increase with age, cause aging

telomeres basic function

mechanism in each cell that counts division - replication counter

structure-function relationships of telomeres

stop ends of chromosomes fusing with each other

stop ends of chromosomes activating genome damage checkpoints

prevent loss of sequence from exonuclease attack

prevents the “end replication problem”

substrate for telomerase

TTAGGG

Current definition of telomeres

structure present at the natural end of a linear chromosome that enables it to behave differently from a simple double-stranded break in the genome

structure of a telomere

TTAGGG at either end of a double stranded region of telomeric DNA (4-10kbp)

followed by a single stranded 3’ overhang of G-rich telomeric DNA (100s bases long)

how does the end of a chromosome structure itself

3’ overhang folds back on itself anneals to earlier part of telomere. sequesters end of chromosome away

theory about why 3’ end of chromosome is sequestered away

to hide from enzymes that can detect DNA damage (such as ATM/ATR) that may mistake the end as a DNA break and initiate apoptosis

2 main protein complexes that associate with telomeres

TRF1 and TRF2 complexes.

TRF1 counts telomere repeats to determine length

TRF2 helps the T-loop form (kinks DNA back on itself) and inhibits DNA repair enzymes.

end-replication problem

DNA replication of lagging strand - could lose a section of DNA potentially up to the size of an Okazaki fragment (~200 nucleotides). means that every time a cell divides, a bit of DNA is lost from the end of chromosomes.

role of telomerase

telomeres shorten with each cell division - telomerase is a reverse transcriptase (RNA to DNA) that adds TTAGGG onto chromosome ends to compensate for the erosion

hTERT

catalytic subunit of telomerase

hTERC

RNA molecule - provides template for the synthesis of TTAGGG

how does telomerase do its thing

binds to end of chromosome. fill-in synthesis of the nucleotides. translocates along by 1 unit and repeats again and again. etc.

effects of telomere erosion

limits replicative lifespan, prevents unlimited cell growth, provides a strict tumour suppressive mechanism

which cells express telomerase

male/female germline cells, immortal cell lines, 85% of malignancies, stem cells.

effect of adding telomerase into cells

replicative immortality. cells live forever.

what happens to telomeres with age

they shorten, ~31bp per year. females have longer telomeres than males ~240 bps (7-8 years)

what is the rate of telomere erosion dependent on

cell turnover - rates differ between tissues: liver 60bp per year, spleen 29 bp per year.

what disease/lifestyle is associated with telomere shortening

obesity, cigarette smoking, atherosclerosis. but is not a good biomarker of biological age in humas.

higher risk of disease in shorter telomere lengths but cancer in higher lengths.

zebrafinches

telomere length in early life predicts their lifespan (model organism). shorter telomers → shorter lives

telomere syndromes and examples

mutations in components of the telomerase complex: start life with very short telomeres.

bone marrow failure (no immune system), pulmonary fibrosis, liver disease, GI disease, skin/hair/mucosal abnormalities

ALT: alternate lengthening of telomeres

lengthening telomeres not using telomerase. involves recombination of telomeric DNA from 2 different chromosomes.

hallmark of cancer when looking at genome

chromosomes look completely wrong, bits missing, additional bits, wrong shapes, etc.

role of telomeres in tumour suppression

double stranded dna break → p53 dependednt dna-damage checkpoint → G1-S arrest and replicative sensecence.

role of telomeres in tumour promotion

telomere fusion, anaphase bridging, breakage and fusion cycles → non reciprocal translocations

what can telomere erosion and unprotected chromatid ends lead to

end-to-end fusion of sister chromatids, during mitosis can lead to non-homologous chromosomes and new breakages → non reciprocal translocations

removal of trf2 protein effects on genome (in vitro)

chromosomes joined end-to-end throughout genome

telomere fusion at molecular level

dna sequence goes from one chromosome into a TTAGGG rich domain (telomere) straight into the next chromosome. +telomere sandwiches

effects of telomere fusion on cancers

sporadic compared to in vitro

drives genetic diversity and loss of chromosomes of cancer cells

very mutagenic

colorectal polyps

small lesions within the colon that can evolve into cancer. large chunks of dna are being lost and this correlates with the sizes of the telomeres.

how long are telomeres in cancers/early stage lesions

usually very short.

clinical consequences of short telomeres in cancers

correlates with substantial worsening of prognosis and higher likelihood of death.

how do cancers overcome the short telomeres

telomerase is overexpressed in 85% of cancers - therapeutic target to remove telomerase in cancers

imetelstat

telomerase inhibitor. modified oligonucleotide. binds TERC template and induces telomere erosion.

but efficacy endpoint not met.

G-quadruplex stabilising ligands

interfere with telomere structure itself. planar molecules.

locks telomeres into g-quadruplex state (hoogsteen g-g bonds) preventing lengthening and inducing uncapping.

e.g. telomestatin.

hoogsteen g-g bonds

the G rich domains in telomeres can anneal to each other

OBP-301

oncolytic virus that replicates inside tumours with telomerase.

suicide gene therapy

TERT promotor. converts pro drugs. induces cell death.