1. DNA REPLICATION WC: DNA replication is semi-conservative strands melt: form templates for copy Copy is reverse & complement of template Copy of other strand

2. Meselson & Stahl proved DNA replication is semi-conservative 1)grew E. coli on 15 N to tell new DNA from old make dense DNA

3. Meselson & Stahl 1) grew E. coli on 15 N to tell new DNA from old make dense DNA 2) Xfer to 14 N

4. Meselson & Stahl 1) grew E. coli on 15 N to tell new DNA from old make dense DNA 2) Xfer to 14 N new DNA is light

5. Meselson & Stahl 1) grew E.coli on 15 N to tell new DNA from old make dense DNA 2) Xfer to 14 N new DNA is light 3) measure  of F1 & F2 DNA by centrifuging in CsCl

6. Meselson & Stahl measure  of F1 & F2 DNA by centrifuging in CsCl forms  gradients when spun @500,000 x g

7. Meselson & Stahl measure  of F1 & F2 DNA by centrifuging in CsCl forms  gradients when spun @500,000 x g DNA bands where  is same as CsCl

8. Meselson & Stahl Results F0 DNA forms HH band control Parental

9. Meselson & Stahl F0 DNA forms HH band F1 DNA forms one higher band Control Parental F1

10. Meselson & Stahl F0 DNA forms HH band F1 DNA forms one higher band: HL Control Parental F1

11. Meselson & Stahl F0 DNA forms HH band F1 DNA forms one higher band: HL F2 DNA forms 2 bands: #1 same as F1 #2 same as 14 N DNA Control Parental F1 F2

12. Meselson & Stahl F2 DNA forms 2 bands: # 1 same as F1 #2 same as 14 N DNA # 1 = HL # 2 = LL : DNA replication is semiconservative

13. DNA replication 1) Replication begins at origins of replication

14. DNA replication 1) Replication begins at origins of replication Polymerases are dumb!

15. DNA replication 1) Replication begins at origins of replication DNA polymerases are dumb! other proteins tell where to start

16. DNA replication 1) where to begin? 2) “melting” DNA

17. DNA replication 1) where to begin? 2) “melting” DNA must melt DNA @ physiological T

18. DNA replication must melt DNA @ physiological T Helicase melts DNA

19. DNA replication must melt DNA @ physiological T Helicase melts DNA Forms “replication bubble”

20. DNA replication helicase melts DNA Forms “replication bubble” SSB proteins separate strands until they are copied

21. DNA replication helicase melts DNA unwinding DNA increases supercoiling elsewhere

22. DNA replication helicase melts DNA unwinding DNA increases supercoiling elsewhere DNA gyrase relieves supercoiling DNA gyrase

23. DNA replication 1) where to begin? 2) “melting” 3) “priming” DNA polymerase can only add

24. DNA replication “priming” DNA polymerase can only add primase makes short RNA primers

25. DNA replication “priming” primase makes short RNA primers DNA polymerase adds to primer

26. DNA replication “priming” primase makes short RNA primers DNA polymerase adds to primer later replace primers with DNA

27. DNA replication 1) where to begin? 2) “melting” 3) “priming” 4) DNA replication

28. DNA replication 4) add bases bonding 5’ P to 3’ OH @ growing end

29. DNA replication 4) add bases bonding 5’ P to 3’ OH @ growing end Template holds next base until make bond

30. DNA replication Template holds next base until make bond - only correct base fits

31. DNA replication Template holds next base until make bond - only correct base fits - energy comes from 2 PO 4

32. DNA replication energy comes from 2 PO 4 "Sliding clamp" keeps polymerase from falling off

33. DNA replication energy comes from 2 PO 4 "Sliding clamp" keeps polymerase from falling off Proof-reading: only correct DNA can exit

34. DNA replication Proof-reading: only correct DNA can exit Remove bad bases & try again

35. DNA replication Only make DNA 5’ -> 3’

36. Leading and Lagging Strands Only make DNA 5’ -> 3’ strands go both ways!

37. Leading and Lagging Strands Only make DNA 5’ -> 3’ strands go both ways! Make leading strand continuously

38. Leading and Lagging Strands Make leading strand continuously Make lagging strand opposite way

39. Leading and Lagging Strands Make leading strand continuously Make lagging strand opposite way wait for DNA to melt, then make Okazaki fragments

40. Leading and Lagging Strands Make lagging strand opposite way wait for DNA to melt, then make Okazaki fragments each Okazaki fragment has its own primer made discontinuously

41. Leading and Lagging Strands each Okazaki fragment has its own primer made discontinuously DNA replication is semidiscontinuous

42. Leading and Lagging Strands each Okazaki fragment has its own primer made discontinuously DNA replication is semidiscontinuous Okazaki fragments grow until hit one in front

43. RNAse H removes primer & gap is filled

44. Okazaki fragments grow until hit one in front RNAse H removes primer & gap is filled DNA ligase joins fragments

45. Okazaki fragments grow until hit one in front RNAse H removes primer & gap is filled DNA ligase joins fragments Energy comes from ATP-> AMP

46. DNA replication Real process is far more complicated! Proteins replicating both strands are in replisome

47. DNA replication Real process is far more complicated! Proteins replicating both strands are in replisome: feed DNA through it

48. DNA replication Proteins replicating both strands are in replisome: feed DNA through it lagging strand loops out so make both strands in same direction

49. DNA pol detaches when hits previous primer, reattaches at next primer

50. Bacterial DNA has one origin Euk have ARS ~ every 100,000 bp - speed DNA replication

51. Euk have ARS ~ every 100,000 bp - speed DNA replication ORC (Origin Recognition Complex) binds ARS A B1B2B3

52. ORC binds ARS licensing factors ensure each ARS is only replicated once/S fall off when ARS is replicated, don't reattach until G1

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

54. 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

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

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

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

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

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

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

61. Telomerase reverse transcriptase with attached RNA template 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

62. Telomeres Aging theory:” mature” cells lose telomerase

63. Telomeres Aging theory:” mature” cells lose telomerase lose DNA each cell cycle

64. Telomeres Aging theory:” mature” cells lose telomerase lose DNA each cell cycle Die when lose too much Telomeres Aging theory:” mature” cells lose telomerase lose DNA each cell cycle

65. Telomeres Aging theory:” mature” cells lose telomerase lose DNA each cell cycle Die when lose too much Cancer cells reactivate telomerase

66. Telomeres Aging theory:” mature” cells lose telomerase lose DNA each cell cycle Die when lose too much Cancer cells reactivate telomerase Can use serum telomerase to diagnose cancer

67. Telomeres Aging theory:” mature” cells lose telomerase lose DNA each cell cycle Die when lose too much Cancer cells reactivate telomerase Can use serum telomerase to diagnose cancer Can kill cultured cancer cells by inhibiting telomerase

68. Telomeres Aging theory:” mature” cells lose telomerase lose DNA each cell cycle Die when lose too much Cancer cells reactivate telomerase Can use serum telomerase to diagnose cancer Can kill cultured cancer cells by inhibiting telomerase Telomerase is symptom cf cause of cancer

69. DNA repair DNA is constantly damaged

70. DNA repair DNA is constantly damaged UV makes pyrimidine dimers

71. DNA repair DNA is constantly damaged UV makes pyrimidine dimers Photolyase uses light energy to cleave dimers

72. DNA repair DNA is constantly damaged UV makes pyrimidine dimers Photolyase uses light energy to cleave dimers Also fixed by Nucleotide Excision Repair

73. DNA repair UV makes pyrimidine dimers Photolyase uses light energy to cleave dimers Are also fixed by Nucleotide Excision Repair NER also fixes chemical damage

74. Nucleotide excision repair 1)XPC finds damage 2) Cut bad strand on each side of lesion, then remove it

75. Nucleotide excision repair 1)XPC finds damage 2) Cut bad strand on each side of lesion, then remove it 3) DNA polymerase fills gap

76. Nucleotide excision repair 1)XPC finds damage 2) Cut bad strand on each side of lesion, then remove it 3) DNA polymerase fills gap 4) Ligase seals it

77. DNA repair base excision repair fixes altered and missing bases

78. DNA repair base excision repair fixes altered and missing bases 1) Enzymes find & remove bad bases

79. DNA repair base excision repair fixes altered and missing bases 1) Enzymes find & remove bad bases 2) DNA pol replaces missing base

80. DNA repair base excision repair fixes altered and missing bases 1) Enzymes find & remove bad bases 2) DNA pol replaces missing base 3) Ligase seals gap

81. DNA repair mismatch repair : fix new DNA to match old DNA

82. DNA repair mismatch repair : fix new DNA to match old DNA DNA is modified t/o cell cycle

83. mismatch repair 1) enzymes find mismatched bases 2) replace base on new strand using old strand

84. DNA repair Repairing 2 -strand breaks 1)MRN finds broken DNA

85. DNA repair Repairing 2-strand breaks 1)MRN finds broken DNA 2)Activates enzymes that ligate broken ends

86. DNA Repair Clinical significance Important for preventing cancers Many genetic disorders are due to bad DNA repair bad BRCA1 or BRCA2 predispose to cancer

87. DNA Repair Many human genetic disorders are due to DNA repair bad BRCA1 or BRCA2 predispose to cancer NER defects cause Xeroderma Pigmentosum 11 genes have "same" phenotype

88. DNA Repair Many human genetic disorders are due to DNA repair bad BRCA1 or BRCA2 predispose to cancer NER defects cause Xeroderma Pigmentosum 11 genes have "same" phenotype bad mismatch repair causes hereditary nonpolyposis colon cancer (HNPCC)