1. Final Journal Club Monday April 27 & Wed April 29 1.New techniques for genome editing & other purposes CRISPR TALEN Zn Finger Cre-Lox 2.New techniques for DNA sequencing Illumina Ion Torrent Nano-Pore Pac-Bio Mass-Spec Other? 3.Molecular biology for fun and profit

2. Telomeres sequences at chromosome ends humans have 250-7,000 repeats of TTAGGG special proteins bind them

3. Telomeres sequences at chromosome ends humans have 250-7,000 repeats of CCCTAA special proteins bind them can't replicate 5' end of lagging strand since remove primer

4. Telomeres can't replicate 5' end of lagging strand since remove primer telomeres lose ~ 200 bp each S

5. Telomeres can't replicate 5' end of lagging strand since remove primer telomeres lose ~ 200 bp each S telomerase replaces missing bases

6. Telomerase telomeres lose ~ 200 bp each S telomerase replaces missing bases reverse transcriptase with attached RNA template

7. Telomerase telomeres lose ~ 200 bp each S telomerase replaces missing bases reverse transcriptase with attached RNA template 1) RNA bonds leading strand

8. Telomerase reverse transcriptase with attached RNA template 1)RNA bonds leading strand 2) Forms template to extend leading strand

9. Telomerase reverse transcriptase with attached RNA template 1)RNA bonds leading strand 2) Forms template to extend leading strand 3) Translocates 6 bases & repeats

10. Telomerase 1)RNA bonds leading strand 2) Forms template to extend leading strand 3) Translocates 6 bases & repeats 4) Extend lagging strand with primer & DNA pol

11. Telomere structure Bound by complex called shelterin or the telosome

12. Telomere structure Bound by complex called shelterin or the telosome Protects DNA, prevents recombination

13. Telomere structure Bound by Shelterin/telosome complex Protects DNA, prevents recombination DNA proposed to form T-loop structure IF long enough

14. Telomere structure Bound by Shelterin/telosome complex Protects DNA, prevents recombination DNA proposed to form T-loop structure IF long enough G-rich SS-DNA invades and displaces C-rich strand

15. Telomere structure Bound by Shelterin/telosome complex Protects DNA, prevents recombination DNA proposed to form T-loop structure IF long enough G-rich SS-DNA invades and displaces C-rich strand POT1/TPP1 binds & protects D-loop

16. Telomere structure Bound by Shelterin/telosome complex Protects DNA, prevents recombination DNA proposed to form T-loop structure IF long enough G-rich SS-DNA invades and displaces C-rich strand POT1/TPP1 binds & protects D-loop If can’t form T-loop trigger apoptosis

17. Telomere Length Maintenance Bound by shelterin/telosome complex Proposed to form T-loop structure IF long enough If can’t form T-loop trigger apoptosis Telomere length proposed to be regulated by TRF1 & PINX1

18. Telomere Length Maintenance Telomere length proposed to be regulated by TRF1 & PINX1 If too short, not enough TRF1 bind so telomerase is active

19. Telomere Length Maintenance Telomere length proposed to be regulated by TRF1 & PINX1 If too short, not enough TRF1 bind so telomerase is active Once long enough TRF1 binds telomere

20. Telomere Length Maintenance Telomere length proposed to be regulated by TRF1 & PINX1 If too short, not enough TRF1 bind so telomerase is active Once long enough TRF1 binds telomere PINX1 then binds TRF1 & stops telomerase

21. Telomeres Aging theory:” mature” cells lose telomerase lose DNA each S

22. Telomeres Aging theory:” mature” cells lose telomerase lose DNA each S Die when lose too much: Hayflick limit ~50 divisions for 1˚ cultures

23. Telomeres Aging theory:” mature” cells lose telomerase lose DNA each S Die when lose too much: Hayflick limit ~50 divisions for 1˚ cultures Ramunas et al (2015) DOI: 10.1096/fj.14-259531 found that transient expression of TERT in cultured cells added 1000 bp to telomeres and allowed 28 x more divisions in fibroblasts10.1096/fj.14-259531

24. Telomeres Aging theory:” mature” cells lose telomerase lose DNA each S Die when lose too much: Hayflick limit ~50 divisions for 1˚ cultures Ramunas et al (2015) DOI: 10.1096/fj.14-259531 found that transient expression of TERT in cultured cells added 1000 bp to telomeres and allowed 28 x more divisions in fibroblasts10.1096/fj.14-259531 Cancer cells must maintain telomere length 90% reactivate telomerase

25. Telomeres Cancer cells must maintain telomere length 90% reactivate telomerase [serum telomerase] can diagnose cancer

26. Telomeres Cancer cells must maintain telomere length 90% reactivate telomerase [serum telomerase] can diagnose cancer Inhibiting telomerase kills most cancer cells

27. Telomeres Cancer cells must maintain telomere length 90% reactivate telomerase [serum telomerase] can diagnose cancer Inhibiting telomerase kills most cancer cells Telomerase is symptom cf cause of cancer not viewed as good target for cancer therapy

28. Telomeres 90% of cancer cells reactivate telomerase [serum telomerase] can diagnose cancer Inhibiting telomerase kills most cancer cells Telomerase is symptom cf cause of cancer 10 % of cancer cells lengthen telomeres by alternative mechanism involving homologous recombination

29. Telomeres 10 % of cancer cells lengthen telomeres by alternative mechanism involving homologous recombination normally inactive: therefore good target for cancer therapy

30. Telomeres 10 % of cancer cells lengthen telomeres by alternative mechanism involving homologous recombination normally inactive: therefore good target for cancer therapy See high frequency of SCE at telomeres in ALT cells

31. Telomeres 10 % of cancer cells lengthen telomeres by alternative mechanism involving homologous recombination normally inactive: therefore good target for cancer therapy See high frequency of SCE at telomeres in ALT cells Also see high frequency of telomere fragments Substrates for HR and telomere elongation

32. Telomeres 10 % of cancer cells lengthen telomeres by alternative mechanism involving homologous recombination normally inactive: therefore good target for cancer therapy See high frequency of SCE at telomeres in ALT cells Also see high frequency of telomere fragments Substrates for HR and telomere elongation Key common feature = chromatin remodeling allowing HR Perhaps due to depletion of shelterin, especially TRF2

33. DNA damage DNA gets damaged a lot!

34. DNA damage DNA gets damaged a lot!

35. DNA damage DNA gets damaged a lot! >200,000 events/human cell/day

36. DNA damage Occurs 2 ways 1) spontaneously

37. DNA damage Occurs 2 ways 1) spontaneously 2) mutagens : damage DNA

38. Spontaneous mutations 1) In S a) mispairs C tautomers bind A T tautomers bind G

39. Spontaneous mutations 1) In S a) mispairs C tautomers bind A T tautomers bind G 1/10 6

40. Spontaneous mutations 1) In S a) mispairs C tautomers bind A T tautomers bind G 1/10 6 6000/S

41. Spontaneous mutations Wobbling: no numbers!

42. Spontaneous mutations 1) In S a) mispairs b) misaligned bases

43. Spontaneous mutations 1) In S a) mispairs b) misaligned bases -> indels

44. Spontaneous mutations 1) In S a) mispairs b) misaligned bases -> indels 2) Hydrolysis a) C deaminates to U

45. Spontaneous mutations 2) Hydrolysis a) C deaminates to U 100-500/cell/day

46. Spontaneous mutations 2) Hydrolysis a)C deaminates to U b)5-meC deaminates to T!

47. Spontaneous mutations 2) Hydrolysis a)C deaminates to U b)5-meC deaminates to T! c)Adenine deaminates to hypoxanthine: bonds C!

48. Spontaneous mutations 2) Hydrolysis a) C deaminates to U 100-500/cell/day b) Depurination (lose A or G) (also depyrimidation, but 20 x less)

49. Spontaneous mutations 2) decomposition a) C deaminates to U 100-500/cell/day b) Depurination (lose A or G) (also depyrimidation, but 20 x less) Replace missing base randomly

50. decomposition a) C deaminates to U 100-500/cell/day b) Depurination (lose A or G) (also depyrimidation, but 20 x less) Replace missing base randomly 10,000 – 50,000 depurinations /cell / day 1,000 – 3,000 depyrimidations /cell / day

51. Spontaneous mutations 2) decomposition 3) Oxidation: > 20 base lesions due to oxidation have been identified

52. Spontaneous mutations 2) decomposition 3) Oxidation: > 20 base lesions due to oxidation have been identified G is most reactive: most frequent lesion is 8-hydroxy G

53. Spontaneous mutations 2) decomposition 3) Oxidation: > 20 base lesions due to oxidation have been identified G is most reactive: most frequent lesion is 8-hydroxy G 8oxoG (syn) bonds A well enough to evade proofreading!

54. Spontaneous mutations 3) Oxidation: > 20 base lesions due to oxidation have been identified G is most reactive: most frequent lesion is 8-hydroxy G 8oxoG (syn) bonds A well enough to evade proofreading! (8oxoG (anti) bonded to C gets removed!)