Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. CHAPTER 14 LECTURE SLIDES To run the animations you must be

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Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. CHAPTER 14 LECTURE SLIDES To run the animations you must be in Slideshow View. Use the buttons on the animation to play, pause, and turn audio/text on or off. Please note: once you have used any of the animation functions (such as Play or Pause), you must first click in the white background before you advance the next slide. Slide 2 DNA: The Genetic Material Chapter 14 Slide 3 Frederick Griffith 1928 Studied Streptococcus pneumoniae, a pathogenic bacterium causing pneumonia 2 strains of Streptococcus S strain is virulent R strain is nonvirulent Griffith infected mice with these strains hoping to understand the difference between the strains 3 Slide 4 4 Griffiths results Live S strain cells killed the mice Live R strain cells did not kill the mice Heat-killed S strain cells did not kill the mice Heat-killed S strain + live R strain cells killed the mice Slide 5 5 Slide 6 6 Slide 7 7 Transformation Information specifying virulence passed from the dead S strain cells into the live R strain cells Our modern interpretation is that genetic material was actually transferred between the cells Slide 8 8 Avery, MacLeod, & McCarty 1944 Repeated Griffiths experiment using purified cell extracts Removal of all protein from the transforming material did not destroy its ability to transform R strain cells DNA-digesting enzymes destroyed all transforming ability Supported DNA as the genetic material Slide 9 9 Hershey & Chase 1952 Investigated bacteriophages Viruses that infect bacteria Bacteriophage was composed of only DNA and protein Wanted to determine which of these molecules is the genetic material that is injected into the bacteria Slide 10 10 Bacteriophage DNA was labeled with radioactive phosphorus ( 32 P) Bacteriophage protein was labeled with radioactive sulfur ( 35 S) Radioactive molecules were tracked Only the bacteriophage DNA (as indicated by the 32 P) entered the bacteria and was used to produce more bacteriophage Conclusion: DNA is the genetic material Slide 11 11 Slide 12 12 DNA Structure DNA is a nucleic acid Composed of nucleotides 5-carbon sugar called deoxyribose Phosphate group (PO 4 ) Attached to 5 carbon of sugar Nitrogenous base Adenine, thymine, cytosine, guanine Free hydroxyl group (OH) Attached at the 3 carbon of sugar Slide 13 13 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Purines Pyrimidines Adenine Guanine NH 2 C C N N N C H N C CH O H H OC NC H N C H C H O O C N C H N C H3CH3C C H H O O C N C H N C H C H C C N N N C H N C CH H Nitrogenous Base 44 55 11 3322 2 8 76 39 4 5 1 O P OO OO Phosphate group Sugar Nitrogenous base O CH 2 N N O N NH 2 OH in RNA Cytosine (both DNA and RNA) Thymine (DNA only) Uracil (RNA only) OH H in DNA Slide 14 Phosphodiester bond Bond between adjacent nucleotides Formed between the phosphate group of one nucleotide and the 3 OH of the next nucleotide The chain of nucleotides has a 5-to-3 orientation 14 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Base CH 2 O 55 33 O P O OH CH 2 OOO C Base O PO 4 Phosphodiester bond Slide 15 Chargaffs Rules Erwin Chargaff determined that Amount of adenine = amount of thymine Amount of cytosine = amount of guanine Always an equal proportion of purines (A and G) and pyrimidines (C and T) 15 Slide 16 16 Rosalind Franklin Performed X-ray diffraction studies to identify the 3-D structure Discovered that DNA is helical Using Maurice Wilkins DNA fibers, discovered that the molecule has a diameter of 2 nm and makes a complete turn of the helix every 3.4 nm Slide 17 17 James Watson and Francis Crick 1953 Deduced the structure of DNA using evidence from Chargaff, Franklin, and others Did not perform a single experiment themselves related to DNA Proposed a double helix structure Slide 18 Double helix 2 strands are polymers of nucleotides Phosphodiester backbone repeating sugar and phosphate units joined by phosphodiester bonds Wrap around 1 axis Antiparallel 18 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 55 33 P P P P OH 5-carbon sugar Nitrogenous base Phosphate group Phosphodiester bond O O O O 44 55 11 33 22 44 55 11 33 22 44 55 11 33 22 44 55 11 33 22 Slide 19 19 Slide 20 Complementarity of bases A forms 2 hydrogen bonds with T G forms 3 hydrogen bonds with C Gives consistent diameter 20 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. A H Sugar T G C N H N O H CH 3 H H N N N H N N N H H H N O H H H N NH N N H N N Hydrogen bond Hydrogen bond Slide 21 21 DNA Replication 3 possible models 1.Conservative model 2.Semiconservative model 3.Dispersive model Slide 22 22 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Conservative Slide 23 23 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. ConservativeSemiconservative Slide 24 24 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. ConservativeSemiconservativeDispersive Slide 25 25 Meselson and Stahl 1958 Bacterial cells were grown in a heavy isotope of nitrogen, 15 N All the DNA incorporated 15 N Cells were switched to media containing lighter 14 N DNA was extracted from the cells at various time intervals Slide 26 26 Conservative model = rejected 2 densities were not observed after round 1 Semiconservative model = supported Consistent with all observations 1 band after round 1 2 bands after round 2 Dispersive model = rejected 1 st round results consistent 2 nd round did not observe 1 band Slide 27 27 Slide 28 28 DNA Replication Requires 3 things Something to copy Parental DNA molecule Something to do the copying Enzymes Building blocks to make copy Nucleotide triphosphates Slide 29 29 DNA replication includes Initiation replication begins Elongation new strands of DNA are synthesized by DNA polymerase Termination replication is terminated Slide 30 30 Slide 31 DNA polymerase Matches existing DNA bases with complementary nucleotides and links them All have several common features Add new bases to 3 end of existing strands Synthesize in 5-to-3 direction Require a primer of RNA 31 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 55 33 55 55 55 33 33 RNA polymerase makes primerDNA polymerase extends primer Slide 32 Prokaryotic Replication E. coli model Single circular molecule of DNA Replication begins at one origin of replication Proceeds in both directions around the chromosome Replicon DNA controlled by an origin 32 Slide 33 33 Slide 34 34 E. coli has 3 DNA polymerases DNA polymerase I (pol I) Acts on lagging strand to remove primers and replace them with DNA DNA polymerase II (pol II) Involved in DNA repair processes DNA polymerase III (pol III) Main replication enzyme All 3 have 3-to-5 exonuclease activity proofreading DNA pol I has 5-to-3 exonuclase activity Slide 35 Unwinding DNA causes torsional strain Helicases use energy from ATP to unwind DNA Single-strand-binding proteins (SSBs) coat strands to keep them apart Topoisomerase prevent supercoiling DNA gyrase is used in replication 35 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Supercoiling Replisomes No Supercoiling Replisomes DNA gyrase Slide 36 Semidiscontinous DNA polymerase can synthesize only in 1 direction Leading strand synthesized continuously from an initial primer Lagging strand synthesized discontinuously with multiple priming events Okazaki fragments 36 Slide 37 37 Slide 38 38 Partial opening of helix forms replication fork DNA primase RNA polymerase that makes RNA primer RNA will be removed and replaced with DNA Slide 39 Leading-strand synthesis Single priming event Strand extended by DNA pol III Processivity subunit forms sliding clamp to keep it attached 39 Slide 40 Lagging-strand synthesis Discontinuous synthesis DNA pol III RNA primer made by primase for each Okazaki fragment All RNA primers removed and replaced by DNA DNA pol I Backbone sealed DNA ligase Termination occurs at specific site DNA gyrase unlinks 2 copies 40 Slide 41 41 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5 3 Primase RNA primer Okazaki fragment made by DNA polymerase III Leading strand (continuous) DNA polymerase I Lagging strand (discontinuous) DNA ligase Slide 42 Replisome Enzymes involved in DNA replication form a macromolecular assembly 2 main components Primosome Primase, helicase, accessory proteins Complex of 2 DNA pol III One for each strand 42 Slide 43 43 Leading strand Lagging strand Primase Clamp loader Helicase DNA polymerase III DNA gyrase RNA primer Single-strand binding proteins (SSB) RNA primer clamp 1. A DNA polymerase III enzyme is active on each strand. Primase synthesizes new primers for the lagging strand. 5 3 5 3 5 3 RNA primer Loop grows Second Okazaki fragment nears completion First Okazaki fragment 2. The loop in the lagging-strand template allows replication to occur 5-to- 3 on both strands, with the complex moving to the left. 5 3 5 3 5 3 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5 3 3. When the polymerase III on the lagging strand hits the previously synthesized fragment, it releases the clamp and the template strand. DNA polymerase I attaches to remove the primer. clamp releases Lagging strand releases DNA polymerase III DNA polymerase I 5 3 5 3 Slide 44 44 Clamp loader 4. The clamp loader attaches the clamp and transfers this to polymerase III, creating a new loop in the lagging-strand template. DNA ligase joins the fragments after DNA polymerase I removes the primers. DNA ligase patches nick DNA polymerase I detaches after removing RNA primer 5 3 5 3

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