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Chapter 16

Chapter 16. Chapter 16 Molecular Basis of Inheritance (DNA structure and Replication). Helicase Enzyme. What is the genetic material? DNA or protein?. The Amazing Race. 1928 Griffith – transformation of pneumonia bacterium.

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Chapter 16

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  1. Chapter 16 Chapter 16 Molecular Basis of Inheritance (DNA structure and Replication) Helicase Enzyme

  2. What is the genetic material? DNA or protein? The Amazing Race

  3. 1928 Griffith – transformation of pneumonia bacterium

  4. 1944 Avery – further studied transformation by destroying lipids, CHO, and proteins

  5. 1947 Chargaff – • Quantified purines and pyrimidines • Suggested base pairing rules (A=T, C=G)

  6. 1950 Wilkins and Franklin – DNA X-rays (a) Rosalind Franklin (b) Franklin’s X-ray diffraction photograph of DNA

  7. 1952 Hershey and Chase – bacteriophages – incorporation of radioactive viral DNA in new phages

  8. EXPERIMENT Radioactive protein Phage Bacterial cell DNA Batch 1: radioactive sulfur (35S) Radioactive DNA Batch 2: radioactive phosphorus (32P)

  9. EXPERIMENT Empty protein shell Radioactive protein Phage Bacterial cell DNA Batch 1: radioactive sulfur (35S) Phage DNA Radioactive DNA Batch 2: radioactive phosphorus (32P)

  10. EXPERIMENT Empty protein shell Radioactivity (phage protein) in liquid Radioactive protein Phage Bacterial cell DNA Batch 1: radioactive sulfur (35S) Phage DNA Centrifuge Pellet (bacterial cells and contents) Radioactive DNA Batch 2: radioactive phosphorus (32P) Centrifuge Radioactivity (phage DNA) in pellet Pellet

  11. 1953 Watson and Crick – DNA Model

  12. 1962 Nobel Prize awarded to Watson and Crick and Wilkins** Conclusion: DNA = Genetic Material, not Protein

  13. Models of DNA Replication

  14. Semi-Conservative Model(1950s - Meselson and Stahl)

  15. Fun DNA Replication Facts • 6 billion bases in human cell = 2 hours of replication time • 500 nucleotides added per second • Accurate (errors only 1 in 10,000 base pairs)

  16. Anti-Parallel Structure of DNA

  17. Mechanism of Replication Step 1 • Origins of Replication= Special site(s) on DNA w/Specific sequence of nucleotides where replication begins • Prokaryotic Cells = 1 site (circular DNA) • Eukaryotic Cells = several sites (strands)

  18. Steps 2 - 5 • Helicase: (enzyme) unwinds DNA helix forming a “Y” shaped replication fork on DNA • Replication occurs in two directions, forming a replication bubble • To keep strands separate, DNA binding proteins attach to each strand of DNA • Topoisomerases: enzymes that work w/helicase to prevent “knots” during unwinding.

  19. Step 6 - Priming • Priming = due to physical limitation of DNA Polymerase, which can only add DNA nucleotides to an existing chain • RNA primase – initiates DNA replication at Origin of Replication by adding short segments of RNA nucleotides. • Later these RNA segments are replaced by DNA nucleotides by DNA Pol.

  20. Step 7 • DNA Pol. = enzyme that elongates new DNA strand by adding proper nucleotides that base-pair with parental DNA template • DNA Pol. can only add nucleotides to the 3’ end of new DNA, so replication occurs from a 5’ to 3’ direction • Leading vs. Lagging Strand results

  21. Leading vs. Lagging Strand • Leading Strand: strand that can elongate continuously as the replication for progresses • Lagging Strand: strand that cannot elongate continuously and moves away from replication fork. • Short Okazaki fragments are added from a 5’ to 3’ direction, as replication fork progresses.

  22. 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’

  23. Step 8 • DNA Ligase = enzyme that “ligates” or covalently bonds the Sugar-Phosphate backbone of the short Okazaki fragments together • Primers are required prior to EACH Okazaki fragment

  24. DNA i Flash Overview

  25. Step 10: Fixing Errors • DNA Pol. Proofreads as it elongates • Special enzymes fix a mismatch nucleotide pairs • Excision Repair: • Nuclease: Enzyme that cuts damaged segment • DNA Pol. Fills in gap with new nucleotide

  26. Mutations • Thymine Dimers (covalent bonding btwn Thymine bases) –often caused by over-exposure to UV rays  DNA buckeling  skin cancer results, unless corrected by excision repair • Substitutions: incorrect pairing of nucleotides • Insertions and Deletions: an extra or missing nucleotide  causes “frameshift” mutations (when nucleotides are displaced one position)

  27. Problems with Replication • Since DNA Polymerase can only add to a 3’ end of a growing chain, the gap from the initial 5’ end can not be filled • Therefore DNA gets shorter and shorter after each round of replication

  28. Solution? • Bacteria have circular DNA (not a problem) • Ends of some eukaryotic chromosomes have telomeres at the ends (repeating nucleotide sequence that do not code for any genes) • Telomeres can get shorter w/o compromising genes • Telomerase = enzyme that elongates telomeres since telomeres will shorten

  29. Telomerases are not in most organisms • Most multicellular organisms do not have telomerases that elongate telomeres (humans don’t have them) • So, telomeres = limiting factor in life span of certain tissues • Older individuals typically have shorter telomeres

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