1. DNA: The Genetic Material Chapter 14

2. 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 2

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5. 5 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

6. 6 Avery, MacLeod, & McCarty – 1944 Repeated Griffith’s 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

7. 7 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

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9. 9 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

10. 10 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 4´4´ 5´5´ 1´1´ 3´3´2´2´ 2 8 76 39 4 5 1 O P O–O– –O–O 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

11. 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 11 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Base CH 2 O 5´5´ 3´3´ O P O OH CH 2 –O–OO C Base O PO 4 Phosphodiester bond

12. Chargaff’s 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) 12

13. 13 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

14. 14 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

15. Double helix 2 strands are polymers of nucleotides Phosphodiester backbone – repeating sugar and phosphate units joined by phosphodiester bonds Wrap around 1 axis Antiparallel 15 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´5´ 3´3´ P P P P OH 5-carbon sugar Nitrogenous base Phosphate group Phosphodiester bond O O O O 4´4´ 5´5´ 1´1´ 3´3´ 2´2´ 4´4´ 5´5´ 1´1´ 3´3´ 2´2´ 4´4´ 5´5´ 1´1´ 3´3´ 2´2´ 4´4´ 5´5´ 1´1´ 3´3´ 2´2´

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17. Complementarity of bases A forms 2 hydrogen bonds with T G forms 3 hydrogen bonds with C Gives consistent diameter 17 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

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19. Fig. 16-10 Parent cell First replication Second replication (a) Conservative model (b) Semiconserva- tive model (c) Dispersive model DNA Replication Meselson and Stahl – 1958

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22. 22 DNA Replication Requires 3 things –to copy Parental DNA molecule –to do the copying Enzymes –Building blocks to make copy Nucleotide triphosphates DNA replication includes –Initiation – replication begins –Elongation – new strands of DNA are synthesized by DNA polymerase –Termination – replication is terminated

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24. DNA polymerase –All have several common features Add new bases to 3’ end of existing strands(3’- free) Synthesize in (5’  3’) direction Require a primer of RNA 24 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´5´ 3´3´ 5´5´ 5´5´ 5´5´ 3´3´ 3´3´ RNA polymerase makes primerDNA polymerase extends primer

25. 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 25

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27. 27 E. coli has 3 DNA polymerases -- Polymerase activity (5’  3’ polermerization and need primer ) 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 –Nuclease activity (exo-, endo-) All 3 have (3’  5’) exonuclease activity – proofreading DNA pol I has (5’  3’) exonuclase activity

28. 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 28 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Supercoiling Replisomes No Supercoiling Replisomes DNA gyrase DNA gyrase can relieve supercoiling)

29. 29 Semidiscontinous Okazaki fragments

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31. 31 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 Discontinuous synthesis DNA pol III All RNA primers removed and replaced by DNA Backbone sealed –DNA gyrase unlinks 2 copies

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33. 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 33

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36. 36 Eukaryotic Replication Complicated by –Larger amount of DNA in multiple chromosomes – Linear structure Basic enzymology is similar –Requires new enzymatic activity for dealing with ends only

37. Fig. 16-12 Origin of replication Parental (template) strand Daughter (new) strand Replication fork Replication bubble Two daughter DNA molecules (a) Origins of replication in E. coli Origin of replicationDouble-stranded DNA molecule Parental (template) strand Daughter (new) strand Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes 0.5 µm 0.25 µm Double- stranded DNA molecule Multiple replicons Initiation phase of replication requires more factors

39. Telomeres Specialized structures found on the ends of eukaryotic chromosomes Protect ends of chromosomes from nucleases and maintain the integrity of linear chromosomes Gradual shortening of chromosomes with each round of cell division –Unable to replicate last section of lagging strand 39

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41. 41 Telomerase enzyme makes telomere of lagging strand using and internal RNA template (not the DNA itself) –Leading strand can be replicated to the end Telomerase developmentally regulated –Relationship between senescence and telomere length Cancer cells generally show activation of telomerase

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43. 43 DNA Repair Errors due to replication –DNA polymerases have proofreading ability Mutagens – any agent that increases the number of mutations above background level –Radiation and chemicals Importance of DNA repair is indicated by the multiplicity of repair systems that have been discovered

44. 44 DNA Repair Falls into 2 general categories 1.Specific repair –Targets a single kind of lesion in DNA and repairs only that damage 2.Nonspecific –Use a single mechanism to repair multiple kinds of lesions in DNA

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