TELOMERES What are they? Why are they important? Telomere shortening and the end-replication problem Telomerase Telomere hypothesis of aging

Telomeres Ends of linear chromosomes Centromere Telomere Repetitive DNA sequence (TTAGGG in vertebrates) Specialized proteins Form a 'capped' end structure

Telomeres 'cap' chromosome ends

TELOMERE STRUCTURE 5’3’ 5' 3' Telomeric t loop Telomeric proteins: TRF1 TRF2 TIN2 RAP1 TANKS 1,2 POT1 etc NUCLEAR MATRIX

Why are telomeres important? Telomeres allow cells to distinguish chromosomes ends from broken DNA Stop cell cycle! Repair or die!! Homologous recombination (error free, but need nearby homologue) Non-homologous end joining (any time, but error-prone)

Why are telomeres important? Prevent chromosome fusions by NHEJ (non-homologous end joining) NHEJ Mitosis FUSION BRIDGE BREAKAGE Fusion-bridge-breakage cycles Genomic instability Cell death OR neoplastic transformation

Telomere also provide a means for "counting" cell division Proliferative capacity Number of cell divisions Finite Replicative Life Span "Mortal" Infinite Replicative Life Span "Immortal" How do cells "know" how many divisions they have completed??

The End Replication Problem: Telomeres shorten with each S phase Ori DNA replication is bidirectional Polymerases move 5' to 3' Requires a labile primer 3' 5' 3' 5' 3' 5' Each round of DNA replication leaves bp DNA unreplicated at the 3' end

Telomere Length (humans) Number of Doublings Cellular (Replicative) Senescence Normal Somatic Cells (Telomerase Negative) Telomere also provide a means for "counting" cell division: telomeres shorten with each cycle Telomeres shorten from kb (germ line) to 3-5 kb after doublings (average lengths of TRFs) Cellular senescence is triggered when cells acquire one or a few critically short telomeres.

How do replicatively immortal cells avoid complete loss of telomeres (how do they solve the end-replication problem)?

TELOMERASE: Key to replicative immortality Enzyme (reverse transcriptase) with RNA and protein components Adds telomeric repeat DNA directly to 3' overhang (uses its own RNA as a template) Vertebrate repeat DNA on 3' end: TTAGGG Telomerase RNA template: AAUCCC

TELOMERASE: Key to replicative immortality + TELOMERASE Overcomes telomere shortening and the end- replication problem Expressed by germ cells, early embryonic cells Not expressed by most somatic cells (human) May be expressed by some stem cells, but highly controlled Expressed by 80-90% of cancer cells Remaining still need to overcome the end replication problem; do so by recombinational mechanisms -- ALT (alternative lengthening of telomeres) mechanisms

Telomere Length (humans) Number of Doublings Cellular (Replicative) Senescence Normal Somatic Cells (Telomerase Negative) Germ Cells (Telomerase Positive) + Telomerase Telomere Length and Cell Division Potential

HOWEVER, CELLS THAT EXPRESS TELOMERASE STILL UNDERGO SENESCENCE (E.G., IN RESPONSE TO DNA DAMAGE, ONCOGENES, ETC.) Inducers of cellular senescence Cell proliferation (short telomeres) DNA damage Oncogenes Strong mitogens/ stress Potential Cancer Causing Events

Telomerase: Biomedical uses Expand cells for replacement therapies (burns, joint replacements, etc) Telomerase inhibitors to selectively kill cancer cells

The telomere hypothesis of aging Telomeres shorten with each cell division and therefore with age TRUE Short telomeres cause cell senescence and senescent cells may contribute to aging TRUE HYPOTHESIS: Telomere shortening causes aging and telomerase will prevent aging TRUE OR FALSE?

The telomere hypothesis of aging Telomere length is not related to life span (mice vs human; M musculus vs M spretus) Telomeres contribute to aging ONLY if senescent cells contribute to aging Telomerase protects against replicative senescence but not senescence induce by other causes

SUMMARY Telomeres are essential for chromosome stability Telomere shortening occurs owing to the biochemistry of DNA replication Short telomeres cause replicative senescence (other senescence causes are telomere-independent) Telomerase prevents telomere shortening and replicative senescence The telomere hypothesis of aging depends on the cellular senescence hypothesis of aging