BIOLOGY OF TELOMERES

1. BIOLOGY OF TELOMERES Prof. H. A. Ranganath National Assessment and Accreditation Council Bangalore, India Univ. of Mysore; 20.9.2010

2. GREGOR JOHANN MENDEL (1822-1884) ORIGIN OF GENE CONCEPT • Gregor Mendel- Pea plant- 1865 • Rediscovery of Mendelism-1900 • Carl Correns, Hugo de Vries, Erich von Tschermak • Period of Classical Genetics: 1900-1940

3. WHERE ARE THESE GENES? Cytologists and Geneticists began to see parallels between the behaviour of chromosomes during cell division and the expected behaviour of Mendel’s Factors. Around 1902, Walter S. Sutton,Theodor Boveri and others independently noted these parallels andCHROMOSOMAL THEORY OF INHERITANCE began to take form, which meant Mendelian genes are located on chromosomes.

4. Early 1930s H J Muller B. McClintock described the Telomere (Telo=end, Meros=part) as a protective devise at the terminal end of the chromosome. When this structure was absent, end-to-end fusion of the chromosomes may occur with ensuing cell death. The importance of telomeres was appreciated. However, they had no tools to understand the molecular nature of these chromosome ends.

5. CHROMOSOME ANALYSIS

6. CHROMOSOME ANALYSIS

7. What are these Genes? How do they control different characters?

8. 1940: NEO-CLASSICAL PERIOD Birth of Genetic Chemistry 1944: DNA: the Hereditary material Gene is a chemical substance called Deoxyribo Nucleic acid (DNA) Structures of the complementary base pairs found in DNA

9. DNA Double Helix

10. CHROMATIN – CHROMOSOME ORGANISATION

11. 1955: onwards-Prokaryotes-E. coli & Viruses Nucleic Acid Transactions Replication Transcription Translation Recombination Mutation Repair Regulation etc…

12. 1960s L. Hayflick’s hypothesis • “Hayflick limit” • Human diploid cells proliferate a limited number of times in a cell culture. When cells reach this limit, they undergo morphologic and biochemical changes that eventually lead to arrest of cell proliferation, a process called “cell senescence”. • Why? Reasons were not known.

13. 1970 Onwards : Modern Period in Genetics Structure and functions of eukaryotic genes • Lessons learnt from Prokaryotes (E. coli) particularly with reference to DNA transactions. • How far those mechanisms were applicable to eukaryotes was the focus of study. • Jacques Monad’s dictum “What is true for E. coli is true for an elephant” • How far is it true?

14. DNA Replication DNA Polymerase Template dependent Primer dependent Polymerization direction is from 5`to 3`

15. DNA Replication • Semi discontinuous • Bidirectional • Semi-conservative

16. DNA from Bacterial chromosome: CIRCULAR DNA from Eukaryotic chromosome: LINEAR

17. Replication Model: Circular Chromosome RNA primer Okazaki fragmnet Gap after the removal of final RNA primer Gap is filled up by DNA polymerase by using tip of the chromosome as the primer Final RNA primer No end replication problem

18. End Replication Problem of Linear eukaryotic chromosomes J D Watson 1970

19. CONSEQUENCES OF END-REPLICATION PROBLEM

20. “End Replication Problem” • of Linear eukaryotic • Chromosomal DNA. • DNA polymerase does not completely replicate the extreme 5` terminal end of the linear chromosome - leaving a small region of the telomere uncopied. • A compensatory mechanism was needed to fill this terminal gap in chromosome, if not the chromosome was shortened with each successive cell division. • The mechanism was not known. J D Watson 1970

21. END PROTECTION PROBLEM OF EUKARYOTIC CHROMOSOMES The DNA of EUKARYOTIC CHROMOSOMES is linear Therefore it has “free ends” at both the tips. These free ends are likely to be mistaken for damaged or broken DNA If this happens, “Cellular DNA damage response pathways” will be activated to “repair” the chromosomal ends. The natural free ends of chromosomal DNA have to be protected for this repair mechanism. How ?

22. End PROTECTION Problem

23. What next? 1975-2010 • The chromosomes are the vehicles of inheritance. Their integrity has to be maintained. In this direction several questions on TELOMERES need to be addressed. For instance • Sequence organization • Non stickiness • End replication • End protection and • Possible impact and implications of these findings

26. TELOMERES GO MOLECULAR: Mysterious DNA termini • Blackburn – Doctoral student at Sanger’s Lab in Cambridge, • England (Sanger’s technique for DNA sequencing) • Blackburn 1975 post doc with Prof. Joseph Gall at Yale • University , USA • Tetrahymena, a ciliate, contains a multitude of short linear DNA • molecules called “Minichromosomes” • Blackburn analysed the ends of these Minichromosomes • The result was that at the tip of each chromosome, a short DNA • sequence, CCCCAA was repeated twenty to seventy times that • is, Tandem repeats

27. TELOMERES GO MOLECULAR: Mysterious DNA termini • Sequence repeats varied from one chromosome to the other. • Function of this tandem repeat sequence was obscure • Relationship with end replication problem and this repeat sequence, if any, how?

28. TELOMERES GO MOLECULAR:

29. Molecular STRUCTURE of TELOMERE • Telomeric DNA is made up of Tandem repeats • It is not a coding sequence • In some cases the DNA double strand is incomplete. • i.e. 3` end of the strand is longer with more number of nucleotides than that of the complimentary 5` strand • The extra length at 3` end overhangs

30. TELOMERE STRUCTURE • The overhanging portion loops back to have a secondary pairing

31. Elizabeth H. Blackburn and Jack Szostak 1980 • Blackburn presented her findings about Tetrahymena at • a scientific conference where Jack Szostakwas also • present. • Szostak had a scientific problem: artificial linear DNA • sequences inserted into yeast cells quickly broke down • Reasons were not known • Discussion between Blackburn and Szostak – • could the telomeric sequences from Tetrahymena • protect artificial chromosomes from being broken • down in yeast?

32. 1982 : Telomeric DNA of Tetrahymenaprotected the artificial chromosomes in yeast from degradation

33. MESSAGE The telomeric DNA from one organismcould protect DNA in a completely different organism-indicated that this was a fundamental mechanism common for several widely different species

34. Discovery of Telomerase The Telomere ends change length ! • Carl Greider– doctoral student of E. Blackburn • Experiment: (a) Extract of Tetrahymena cells, • (b) a mixture of short telomeric sequences and • (c) radio actively tagged nucleotides • After incubation - the telomeric DNA fragments which were identical when they were mixed into the extract, were now of different lengths - it means they have grown. • This growth cannot be contributed by DNA polymerase Could there be a unknown enzyme that constructs DNA in this way?

35. Discovery of Telomerase The Telomere ends change length ! • This growth, that is, addition of new nucleotides • was due to a “new enzyme”, a component of the • extract from Tetrahymena cells = Telomerase. Questions: 1. How did the enzyme in the cell extract promoted the “growth” of the DNA ? 2. How did it “know” the order in which the nucleotides to be added?

36. Telomerase Complex • Telomerase is a Ribonucleo-Protein complex = RNA + Protein = RNP • The protein domain is a Telomeric reverse transcriptase (TERT) • THE RNA COMPONENT OF THIS RNP HAS THE SAME SEQUENCE AS ONE OF THE DNA STRANDS OF THE TELOMERE • The telomere RNA acts as a template. • On this template, the reverse transcriptase (TERT) synthesise DNA whose sequence is identical to one of the strands of telomeric DNA • Dyskerin (DKC1) a key auxiliary protein CAJAL BODIES Telomerase complex + TCAB1 + Reptin + ATPases (RUVBL1)

38. Examples to illustrate the complimentarity between Telomeric repeat DNA sequence and Telomerase RNA template. Hence when this template directs the addition of new nucleotides to the tip of the telomeric DNA, the same telomeric sequence is repeated and results in the growth of the telomere.

39. Telomerase as Reverse Transcriptase and Telomeric RNA as template facilitates the growth of Telomeric DNA during replication • The end replication problem is shifted from original tip of the telomere to the new tip. By this, the original size of the chromosome is protected during replication

40. Answers to the earlier questions How did the enzyme in the cell extract promoted the “growth” of the DNA ? Ans. The protein domain of the telomerase is a reverse transcript enzyme 2. How did it “know” the order in which the nucleotides to be added? Ans. The nucleotide sequence of the telomeric RNA template determines the sequence to be added to the growing tip of the telomere

41. Drosophila Telomeres • These telomeres are not made up of short repeated sequences. • It consists of tandem arrays of much longer repeats, 6 to 10 kb in length. • These repeats are full length copies of of two DrosophilaRetrotransposons, called Het-A and TART. • Mechanism of maintenance?

42. END PROTECTION OF EUKARYOTIC CHROMOSOMES • Mammalian telomeres have solved in this problem through the agency of a SIX PROTEIN complex called “SHELTERIN”. • TRF1 and TRF2 bind to the TTAGGG sequences in the double strand telomeric DNA; • POT1 binds to the sequences in single strand form • TIN2 and TPP1 proteins keep TRF1, TRF2 and POP1 together. • This six protein complex, i.e SHELTERIN prevents the activation of the DNA damage response. • SHELTERIN is required for the recruitment of telomerase

43. AN OVERVIEW OF THE CHEMISTRY OF THE TELOMERE

44. TELOMERE CAP CHEMISTRY contd….. The tip of the chromosome, that is telomere is CAPPED by aggregation of telomeresequence specific proteins. This CAP protects the integrity of the chromosome. Thus, the telomeres effectively “Cap” the end of the chromosome in a manner similar to the way the plastic on the ends of our shoelaces “Caps” and protects the shoelaces from unraveling.

45. TELOMERE AND HUMAN BIOLOGY • Ageing: • 1970: Prof. A.M. Olovnikov’s theory of “Marginotomy” • Relates cell scenescence with end replication problem. • The telomeric shortening was proposed as an intrinsic clockwise mechanism of aging that tracks the number of cell divisions before the arrest of cell growth or replicative scenescence sets in. (Hayflick limit) • 1988: Greider and her colleagues • Corrobrated this theory when they observed a progressive loss of Telomere length in dividing cells cultured in vitro • Correlation between Telomere shortening and a reduction in the replicative life span of human cultured cells (Cell scenescence;).

46. TELOMERE AND HUMAN BIOLOGY • Ageing: • Telomerase activity is almost absent or undetectable in most differentiated cells. • Telomerase activity is highest in germ cells and in totipotent stem cells. • Telomerase deficient mice: organismal ageing is related to telomerase activity and telomere length

47. TELOMERE AND HUMAN BIOLOGY Maintenance of telomere length and protection are important factors in the control of cellular life span, but organismal ageing is a highly complex process influenced by many factors.

48. TELOMERE AND HUMAN BIOLOGY Contd… Hereditary Disease Syndrome: Mutations in Genes encoding TERT (Protein), Telomeric RNA and Telomeric Binding Proteins lead to Telomerase dysfunction Ex. Congenital aplasticanemias DyskeratosisCongenita (DKC) Idiopathic Pulmonary Fibrosis Cancer: 90% of tumor lines (Cancer) possess Telomerase activity. This has led to the development of “anti-telomerase” therapeutic strategies against cancer.

49. R. J. O’Sullivan and J. Karlseder (2010 Nature reviews) • Biochemical Purification of the Telomeric Proteome produced a list of 210 PROTEINS that interacted with and might influence Telomeric Structure. • In addition there is a growing number of proteins that localize to telomeres that are involved in the Assembly and Regulation of Telomerase in cells while it is expressed.

50. Recent Developments contd….(2010) • For many years, telomeres were viewed as Transcriptionally inert. However, transcription of the C-strand of telomeres by RNA polymerase II produces a long UUAGGG-containing transcripts. These are called Telomeric Repeat-containing RNA (TERRA). • TERRAs have a strong inverse correlation with Telomere activity. • TERRAs, non-coding structure RNAs contribute to higher order telomeric structure.