1. Enabling Replicative Immortality
Sept 19, 2017

2. Hallmarks of Cancer, 2011

3. Telomeres
2 Issues
End of Replication problem
DNA Repair problem

4. DNA replication Problem
END 3’ 5’ 5’ 3’

5. DNA replication Problem3’
Leading strand 5’ 3’
END 5’ 5’ 3’ 3’
Lagging strand
RNA primer 5’
Newly synthesized DNA

6. DNA replication Problem3’ 5’
END
1) Extend DNA replication to the end on the leading strand
2) Extend DNA replication to the end on the lagging strand
3) How do they differ at the ends?
Leading strand
Lagging strand

7. DNA replication Problem3’
Leading strand
END? 5’ 5’ 3’ 5’ 3’ 3’
Lagging strand 5’
1) Extend DNA replication to the end on the leading strand
2) Extend DNA replication to the end on the lagging strand
3) How do they differ at the ends?

8. DNA replication Problem3’
END? 5’ 5’ 3’ 5’ 3’ 3’ 5’
RNA primers are removed!

9. Telomeres were discovered in Tetrahymena
Tetrahymena contains many (~300) very short linear chromosomes
Elizabeth Blackburn discovered that the chromosomal ends had repetitive sequences
Did this result suggest that the repetitive sequence protected the ends of chromosomes?
“Pond Scum”: Tetrahymena
A ciliated protozoa

10. Telomeres Protect Chromosome ends
Normal yeast chromosomes
Degraded artificial minichromosome
Jack Szostak
Can you think of an experiment that would test if the repetitive
sequence (telomeres) identified tetrahymena could protect the artificial
minichromsomes in yeast.

11. Telomeres Protect Chromosome ends
Tetrahymena telomeres
Artificial minichromosomes
Did not degrade!

12. 2009 Nobel Prize

13. Discovery of telomerase
Elizabeth Blackburn and her graduate student, Carol Greider tested “cell extracts” from tetrahymena and discovered a fraction that had the ability to maintain/elongate telomeres!
extract x: telomeres were lost
extract y: telomeres were lost
extract z has ability to maintain telomeres!

14. DNA replication Problem Solved
-Telomeres are repetitive sequences: In humans the repeat unit is GGGTTA.
-A telomere can be long—measured telomeres in humans are 10,000-15,000 nucleotides in length.
-A telomere does NOT contain genes 3’
Telomere 5’
Chromosomal DNA
During DNA replication, some telomeric DNA is lost

16. Enzyme Function???

17. Telomere Video https://www.youtube.com/watch?v=AJNoTmWsE0s

19. DNA repair problem
Appreciated by Hermann Muller and Barbara McClintock in the 1930’s: termed the word “telomere”
X-rays
Creation of the new ends led to chromosomal arrangements (chromosomes fusing to each other)
Part of DNA repair pathways because of double-stranded breaks.
Chromosomal arrangements led to aneuploidy
Telo: end
Mere: part
Natural ends of chromosomes are protected from chromosomal rearrangements
Why did natural ends not look like double-stranded breaks?

20. DNA repair problem Solution is a T-Loop
The 3’protruding end has shown to loop back and “tuck” into the telomeric sequence of DNA duplex.
Protects it from degradative enzymes and distinguishes them from broken DNA

21. Telomeres tackle two problems
1. DNA repair problem
2. End-Replication problem
Telomeres helps shield the ends of chromosomes so that chromosomes to not fused (stick) together
X-rays
Telo: end
Mere: part
Telomeres helps ensure the genomic DNA is replicated 3’
Telomere 5’
Chromosomal DNA

23. Somatic cell telomeres shorten over time
A schematic drawing that illustrates the typical range of telomere lengths by age in, for example, peripheral blood lymphocytes. At every age, telomere length displays a normal distribution that is defined by the percentile lines labelled on the right. Telomere length in individuals with four different clinical presentations across the age range is indicated. The dashed lines represent a typical age range in which these disorders may first manifest
Can immortality be achieved by activating telomerase activity?

24. Cells have different levels of telomerase
Telomerase low levels/absent
Telomerase present
iPS: addition of a component of TERT increases the induction of stem cells from older samples
Sirtuin 6, or SIRT6, has recently been identified as a critical regulator of transcription, genome stability, telomere integrity, DNA repair, and metabolic homeostasis. A knockout mouse model of SIRT6 has displayed dramatic phenotypes of accelerated aging. In keeping with its role in aging, we demonstrated that human dermal fibroblasts (HDFs) from older human subjects were more resistant to reprogramming by classic Yamanaka factors than those from younger human subjects, but the addition of SIRT6 during reprogramming improved such efficiency in older HDFs substantially
Most somatic (adult) cells
Fetal cells
Stem cells
Cell senescence: adult cells have limited cell divisions and will stop dividing.
Cancer cells

25. Telomere Shortening in Aging cells
Tumor suppressors (Rb/p53)
Senescence (cell arrest) **
telomere is critically short
1. Why would it be advantageous to activate tumor suppressor pathways that promote cell senescence when telomeres get critically short?
Long (12 kb)
Telomere length
Short
(4 kb)
young
70+
Age (years)

26. Telomere Shortening in Aging cells
Tumor suppressors (Rb/p53)
Senescence (cell arrest) **
telomere is critically short
2. Can you think of mutations that might result in cells becoming cancerous with critically short telomeres?
Long (12 kb)
Telomere length
Short
(4 kb)
young
70+
Age (years)

27. X Telomeres and Cancer * ***
Tumor suppressors (Rb/p53)
Chromosome
instability/aneuploidy
continued
proliferation with loss of telomeres
Long (12 kb)
cancer
Telomere length
3. What would happen if cancer cells reactivate telomerase?
Some investigations have demonstrated that shorter telomere length in PBLs is associated with increased risk of several cancers including lung, bladder, gastric, esophageal, ovarian, head and neck and renal cancer[10], [19]–[25]. In contrast, some reports have suggested that longer telomere length may be associated with increased risk of melanoma and breast cancer, non-Hodgkin lymphoma and soft tissue sarcoma[26]–[29].
Short
(4 kb)
young
70+
Age (years)

28. Process to Immortalization
Telomeres shorten
Telomeres lose their protective function
Creation of end-to-end fusions
Entrance into crisis
Few cells develop unlimited replication
Immortalization!!!

29. Telomerase Levels
Very low in Nonimmortalized cells, resulting in senescence or crisis followed by apoptosis
Significant levels in spontaneously immortalized cells resulting in replicative immortality

30. Process to Immortalization
Telomere Shortening
Senescence
Chromosomal Fusions, Crisis
Apoptosis
Immortalization

31. Cancerous Cells
Maintenance of Telomere Length resulting in endless replication regardless of Chromosomal Issues