Enabling Replicative Immortality Sept 19, 2017

Hallmarks of Cancer, 2011

Telomeres 2 Issues End of Replication problem DNA Repair problem

DNA replication Problem END 3’ 5’ 5’ 3’

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

DNA replication Problem 3’ 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

DNA replication Problem 3’ 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?

DNA replication Problem 3’ END? 5’ 5’ 3’ 5’ 3’ 3’ 5’ RNA primers are removed!

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

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.

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

2009 Nobel Prize

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!

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

Enzyme Function???

Telomere Video https://www.youtube.com/watch?v=AJNoTmWsE0s http://www.scientificamerican.com/article/a-life-in-science-elizabeth-blackburn/

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?

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

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

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 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548426/figure/F2/ Can immortality be achieved by activating telomerase activity?

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

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)

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)

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)

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!!!

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

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

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