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CH7 key concepts genetic material: DNA DNA structure DNA replication

LECTURE 12: DNA: STRUCTURE & REPLICATION I. CH7 key concepts genetic material: DNA DNA structure DNA replication. CHAPTER 7: KEY CONCEPTS. transforming principle in bacteria is DNA experiments with labeled T2 phage establish that DNA is the hereditary material

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CH7 key concepts genetic material: DNA DNA structure DNA replication

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  1. LECTURE 12: DNA: STRUCTURE & REPLICATION I • CH7 key concepts • genetic material: DNA • DNA structure • DNA replication

  2. CHAPTER 7: KEY CONCEPTS • transforming principle in bacteria is DNA • experiments with labeled T2 phage establish that DNA is the hereditary material • Watson & Crick DNA model: anti-parallel double helix = nucleotides linked by phosphodiester bonds; helices are held together by hydrogen bonds between pairs of nitrogenous bases • DNA structure ensures fidelity of replication; base pairing specified by hydrogen bonding • DNA replication is semiconservative; daughter duplex has 1 parental & 1 new strand • DNA replication depends on many enzymes

  3. DNA = GENETIC MATERIAL • Griffith (1928), Streptococcus pneumoniae • transforming principle in bacteria is DNA

  4. DNA = GENETIC MATERIAL • Avery et al. (1944), Streptococcus pneumoniae • more proof ... transforming principle is DNA

  5. DNA = GENETIC MATERIAL • Hershey & Chase (1952), T2 bacteriophage • DNA (not protein) is genetic material

  6. DNA = GENETIC MATERIAL • why was the scientific community reluctant to accept DNA (rather than protein) as hereditary material?... • DNA is a simple molecule... • how is complexity of life encoded by such simplicity? • how is information accurately transmitted from one generation to the next?

  7. DNA STRUCTURE • chemical structure of the 4 nucleotides in DNA

  8. DNA STRUCTURE • hydrogen bonded nucleotides on opposite helices • DNA helices are antiparallel • carbons on sugar define ends... 5' and 3' • pyrimidines bond with purines • T  A • C  G

  9. DNA STRUCTURE • bonding is specific: pyrimidines bond with purines • implication: mechanism for accurate replication

  10. DNA STRUCTURE • helical structure of DNA • major & minor groves • 10Å radius & 20Å diameter • 3.4Å between nucleotide base pairs • 34Å / 360° turn • 10 nucleotide base pairs / 360° turn

  11. DNA STRUCTURE • clues for Watson & Crick (1953) ... X-ray data • & ratios ( A + G ) = ( T + C ) ... Chargaff

  12. DNA STRUCTURE • DNA double helix (space filling model) • several forms... A, B & Z DNA

  13. DNA REPLICATION • model of replication proposed by Watson & Crick (1953) • parental strand = template • semiconservative model (new double helix has 1 template + 1 new daughter strand) • 3 possible models: • conservative • semiconservative • dispersive

  14. DNA REPLICATION • 3 possible models of DNA replication:

  15. DNA REPLICATION • Meselson & Stahl (1958), tested the models

  16. DNA REPLICATION • what would have happened if...

  17. DNA REPLICATION • plant root cells incubated with [3H] thymidine • colchicine to inhibit spindles (chromosomes do not separate) • autoradiography

  18. DNA REPLICATION • explanation of [3H] thymidine incubation / colchicine treatment / autoradiography at DNA level

  19. DNA REPLICATION • “harlequin chromosomes”, incubated with bromodeoxyuridine (BUdR)

  20. DNA REPLICATION • harlequin chromosomes of Chinese hamster ovary cells, incubated with bromodeoxyuridine (BUdR) chromatid exchange

  21. DNA REPLICATION • semiconservative replication • linear gene arrangement • ... chromatid = single DNA molecule + protein matrix chromatid exchange

  22. DNA REPLICATION • model of replication proposed by Watson & Crick (1953) • parental strand = template • semiconservative model (new double helix has 1 template + 1 new daughter strand)

  23. DNA REPLICATION • model of replication proposed by Watson & Crick (1953) • parental strand = template • semiconservative model (new double helix has 1 template + 1 new daughter strand) • replication fork

  24. DNA REPLICATION • replication fork ? • bacteria incubated with [3H] thymidine... • daughter molecules  1 “hot” + 1 “cold” strand • autoradiograph...

  25. DNA REPLICATION • replication... theta () structures • replication fork

  26. DNA REPLICATION • some molecules use rolling-circle replication

  27. MECHANISM OF DNA REPLICATION • Kornberg (late ‘50s) • enzyme that catalyzes DNA replication ... DNA polymerase I • several others

  28. MECHANISM OF DNA REPLICATION • DNA replication requirements... 1. H bonds between bases must be broken 2. chain separation/unwinding 3. available pools of 4 dNTPs: A = T, C  G 4. enzymes • E. coli... • ~4.7 × 107 bp (47,000,000) • molecule has ~ 4.7 × 106 turns (10 bp/turn) • 1500 bp/s, ~ 20 min to divide with 1 origin

  29. MECHANISM OF DNA REPLICATION • prokaryotes have a single origin of replication • bidirectional replication with 2 forks  single terminus • ~ 20 minutes (*... book says 40)

  30. MECHANISM OF DNA REPLICATION • E. coli origin of replication • tandem repeat consensus sequence • protein binding sites

  31. MECHANISM OF DNA REPLICATION • replication to completion...

  32. MECHANISM OF DNA REPLICATION • eukaryotes have multiple origins of replication • bidirectional replication with 2 forks • 1.4 h (yeast) to 24 h (cultured animal cells)... or more • issues... • linear vs circular • coordination of multiple chromosomes • pulse/chase and autoradiography ...

  33. MECHANISM OF DNA REPLICATION • eukaryotes have multiple origins of replication • bidirectional replication with 2 forks • 1.4 h (yeast) to 24 h (cultured animal cells)... or more • issues... • linear vs circular • coordination of multiple chromosomes • pulse/chase and autoradiography ... Drosophila

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