1. Transformation-Griffith’s Expt 1928

2. DNA Mediates Transformation Convert IIR to IIIS By DNA?

3. Avery MacLeod and McCarty ExperimentCirca 1943

4. Transforming Principle

6. + means that activity is present DNAse activity All RNA gets degraded during enzyme preparation

8. A-DNA, B-DNA and Z-DNA The Z-DNA helix is left-handed and has a structure that repeats every 2 base pairs. The major and minor grooves, unlike A- and B-DNA, show little difference in width

9. Non-B DNA in disease

12. Chapter 10 Replication of DNA and Chromosomes

13. DNA Replication is Semiconservative  Each strand serves as a template  Complementary base pairing determines the sequence of the new strand  Each strand of the parental helix is conserved

14. Possible Modes of DNA Replication

15. The Meselson-Stahl Experiment: DNA Replication in E. coli is Semiconservative

16. Visualization of Replication in E. coli

17. Replication in E. coli

18. The Origin of Replication in E. coli Note: OriC is 245bp The Core Origin of Replication in SV 40

19. Prepriming at oriC in E. coli

20. DNA Polymerases and DNA Synthesis In Vitro

21. Requirements of DNA Polymerases  Primer DNA with free 3'-OH  Template DNA to specify the sequence of the new strand  Substrates: dNTPs  Mg 2+ ( where?) Nucleophilic attack of alpha phosphate which releases pyrophosphate

22. 

23.  Mg 2+ ( where?)

24.   

25. DNA Polymerase I: 5'  3' Polymerase Activity Often called: Kornberg Polymerase

26. DNA Polymerase I: 5'  3' Exonuclease Activity Cleaves ahead of itself

27. DNA Polymerase I: 3'  5' Exonuclease Activity Proofreading

28. Klenow fragment…..is?

30. DNA Polymerases  Polymerases in E. coli –DNA Replication: DNA Polymerases III and I –DNA Repair: DNA Polymerases II, IV, and V  Polymerases in Eukaryotes –Replication of Nuclear DNA: Polymerase  and/or  –Replication of Mitochondrial DNA: Polymerase  –DNA Repair: Polymerases  and   All of these enzymes synthesize DNA 5' to 3' and require a free 3'-OH at the end of a primer

31. DNA Polymerase III is the True DNA Replicase of E. coli

32. DNA replication is a complex process, requiring the concerted action of a large number of proteins.

33. E. coli DNA Polymerase III Holoenzyme

34. Replication in E. coli

35. The Origin of Replication in E. coli Note: OriC is 245bp

36. Prepriming at oriC in E. coli

38. DNA Replication  Synthesis of the leading strand is continuous.  Synthesis of the lagging strand is discontinuous. The new DNA is synthesized in short segments (Okazaki fragment) that are later joined together.

39. What’s wrong with this picture?

40. RNA Primers are Used to Initiate DNA Synthesis

42. DNA Helicase Unwinds the Parental Double Helix

43. DNA Ligase Covalently Closes Nicks in DNA

44. DNA ligase forms a high energy intermediate that

45. Calf Intestinal Phosphotase? GAATTC CTTAAG G -OH p- AATTC CTTAA -p HO- G Cut with EcoR1 Aside:

46. Calf Intestinal Phosphotase? G -OH p- AATTC CTTAA -p HO- G Cut with EcoR1 G -OH HO- AATTC CTTAA -OH HO- G

47. Calf Intestinal Phosphotase? p- AATTCgatacagagagactcatgacgG -OH HO- GctatgtctctctgagtactgcCTTAA -p Cut with EcoR1 G -OH HO- AATTC CTTAA -OH HO- G Vector won’t religate, But will take in insert

48. Single-Strand DNA Binding (SSB) Protein

49. Supercoiling of Unwound DNA

50. DNA Topoisomerase I Produces Single- Strand Breaks in DNA

51. DNA Topoisomerase II Produces Double-Strand Breaks in DNA

52. The Replication Apparatus in E. coli

53. The E. coli Replisome

54. DNA Replication in Eukaryotes  Shorter RNA primers and Okazaki fragments  DNA replication only during S phase  Multiple origins of replication  Telomeres

55. Bidirectional Replication from Multiple Origins in Eukaryotes

57. The Eukaryotic Replisome

58. Eukaryotic Replication Proteins  DNA polymerase  -DNA primase—initiation; priming of Okazaki fragments  DNA polymerase  — processive DNA synthesis  DNA polymerase  — DNA replication and repair in vivo  PCNA (proliferating cell nuclear antigen)—sliding clamp  Replication factor-C Rf- C)—loading of PCNA  Ribonuclease H1 and Ribonuclease FEN-1— removal of RNA primers

59. The E. coli Replisome

60. The Telomere Problem

61. Telomerase

62. Telomere Length and Aging  Most human somatic cells lack telomerase activity.  Shorter telomeres are associated with cellular senescence and death.  Diseases causing premature aging are associated with short telomeres.

63. BACs

64. Geometric Doubling Progression 1 2 4 8 16 32 64 128 256 512 1024=10 3 =2 10 ….10 more doublings is another 2 10 So 20 doublings is 2 20 =10 3+3 =10 6 So 30 doublings is 2 30 =10 3+3+3 =10 9 So 40 doublings is 2 40 =10 3+3+3+3 =10 12

65. Molecular Weight of Nucleosides s Base plus ribose Single phosphate 330 Da= 330g/mol/nt (nucleotide) 660 Da= 660g/mol/bp (base pair)

66. Molecular Weight of Plasmid DNA 330 Da= 330g/mol/nt (nucleotide) 660 Da= 660g/mol/bp (base pair) For 3000bp of DNA (a starting plasmid vector) 3000 bp x 660 g/mol/bp= 1000 x 3 x 660 = 1x 10 3 x 2 x 10 3 = 2 x 10 6 g/mol for a 3kb plasmid 2 x 10 6 g/mol is how many grams per molecule 6 x 10 23 molecules/mol Thus 2 x 10 6 / 6 x 10 23 = g/molecules 1g/ 3 x 10 17 molecules for a given 3kb plasmid

67. 2 x 10 6 g/mol is how many grams per molecule 6 x 10 23 molecules/mol Thus 2 x 10 6 / 6 x 10 23 = g/molecules 1g/ 3 x 10 17 molecules for a given 3kb plasmid 1g/ 3 x 10 17 molecules is the same as 1mg/ 3 x 10 14 molecules 1ug/ 3 x 10 11 molecules 1ng/ 3 x 10 8 molecules 1pg/ 3x 10 5 molecules 1fg/ 3 x 10 2 (300) molecules If each bacterium can hold 3000 molecules, then each Bacterium makes 10fg of plasmid DNA If one makes 1mg of plasmid DNA, then this is 10 12 fg as well

68. If each bacterium can hold 3000 molecules, then each Bacterium makes 10fg of plasmid DNA If one makes 1mg of plasmid DNA, then this is 10 12 fg as well Since each bacterium has 10fg DNA, then only 10 11 Are needed to produce 1mg DNA. …..So 40 doublings is 2 40 =10 3+3+3+3 =10 12 3 cell divisions per hour or about 12 hours 2 36 =~10 11 36 doublings for 10 11 bacteria What are the factors that affect DNA replication?

69. Geometric Doubling Progression 1 2 4 8 16 32 64 128 256 512 1024=10 3 =2 10 ….10 more doublings is another 2 10 So 20 doublings is 2 20 =10 3+3 =10 6 So 30 doublings is 2 30 =10 3+3+3 =10 9 So 40 doublings is 2 40 =10 3+3+3+3 =10 12