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Transformation-Griffith’s Expt

Transformation-Griffith’s Expt. 1928. DNA Mediates Transformation. Convert IIR to IIIS By DNA?. Avery MacLeod and McCarty Experiment. Circa 1943. Transforming Principle. DNAse activity. + means that activity is present. All RNA gets degraded during enzyme preparation.

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Transformation-Griffith’s Expt

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  1. Transformation-Griffith’s Expt 1928

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

  3. Avery MacLeod and McCarty Experiment Circa 1943

  4. Transforming Principle

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

  6. 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

  7. Non-B DNA in disease

  8. Chapter 10Replication of DNA and Chromosomes

  9. 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

  10. Possible Modes ofDNA Replication

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

  12. Visualization of Replication in E. coli

  13. Replication in E. coli

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

  15. Prepriming at oriC in E. coli

  16. DNA Polymerases and DNA Synthesis In Vitro

  17. Requirements of DNA Polymerases • Primer DNA with free 3'-OH • Template DNA to specify the sequence of the new strand • Substrates: dNTPs • Mg2+ (where?) Nucleophilic attack of alpha phosphate which releases pyrophosphate

  18.  

  19. Mg2+ (where?)

  20. b g

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

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

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

  24. Klenow fragment…..is?

  25. 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

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

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

  28. E. coli DNA Polymerase III Holoenzyme

  29. Replication in E. coli

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

  31. Prepriming at oriC in E. coli

  32. 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.

  33. What’s wrong with this picture?

  34. RNA Primers are Used to Initiate DNA Synthesis

  35. DNA Helicase Unwinds the Parental Double Helix

  36. DNA Ligase Covalently Closes Nicks in DNA

  37. DNA ligase forms a high energy intermediate that

  38. Aside: Calf Intestinal Phosphotase? Cut with EcoR1 GAATTC CTTAAG G-OHp-AATTC CTTAA-pHO-G

  39. Calf Intestinal Phosphotase? Cut with EcoR1 G-OHp-AATTC CTTAA-pHO-G G-OHHO-AATTC CTTAA-OHHO-G

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

  41. Single-Strand DNA Binding (SSB) Protein

  42. Supercoiling of Unwound DNA

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

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