DNA

1. DNA • The Universal Code of Life

2. W O R K T O G E T H E R • To which class of biological molecules does DNA belong? • What are the monomers of DNA?

3. DNA is a: • Carbohydrate • Protein • Lipid • Nucleic Acid • Depends on the organism

4. The monomers of DNA are: • Sugars • Amino acids • Fatty acids • Nucleotides • Depends on the organism

5. Early History • 1869: Friederich Mieschner isolates “nuclein” from nuclei of cells. His student Richard Altman later renames the substance “nucleic acid.” • Mid 1800s: Biochemists identify two distinct nucleic acids. • 1929: Phoebus Levine identifies four distinct bases in DNA.

6. Heredity as a Science • Genetics arose as a new science in the late 19th and early 20th centuries, spurred by questions raised by Darwin’s On the Origin of Species: • Are there patterns to inheritance? • Are traits handed on intact (particle theory) or blended together in each generation (blending theory)?

7. Mendel’s Answers • Gregor Mendel’s work was rediscovered in 1900, answering both questions: • Inheritance of many traits follows predictable patterns. • Traits are handed on intact via some kind of particle: “elementen.”

8. Hereditary Molecule? • Question in the 20th century: What is the hereditary molecule? • Cell nucleus associated with inheritance. • Both proteins and nucleic acids are in the nucleus. Which contains information coding for traits?

9. Protein or DNA? • Linus Pauling favored protein: DNA has only four bases, protein has over 20 amino acids. Seemed like protein could store more information. • Others favored DNA, which is found only in the nucleus.

10. Frederick Griffith • In 1928, Frederick Griffith carried out experiments on pneumonia bacteria, trying to create a vaccine against pneumonia. Among his findings were early clues about hereditary factors.

11. Griffith’s Experiment Bacterial strain(s) injected into mouse Results Conclusions Mouse remains healthy. R-strain does not not cause pneumonia. Living R-strain Mouse contracts pneumonia, dies. S-strain causes pneumonia. Living S-strain Mouse remains healthy. Heat-killed S- strain does not cause pneumonia. Heat-killed S-strain A substance from heat-killed S-strain can transform the harmless R-strain into a deadly S-strain. Mouse contracts pneumonia, dies. Living R strain, heat-killed S-strain

12. W O R K T O G E T H E R Why were living S-strain bacteria recovered from dead mice injected with dead S-strain and live R-strain bacteria?

13. Oswald Avery • Avery learned of Griffith’s experiment and thought it might hold a clue to the identity of the hereditary molecule. • Avery isolated carbohydrates, proteins, lipids, and nucleic acids from the bacteria to discover which, if any, would transform the non-virulent R-strain bacteria.

14. Of the substances isolated and tested, only DNA from killed S-strain bacteria transformed R-strain bacteria.

15. Hershey & Chase • Early 1950’s: Alfred Hershey and Martha Chase used the bacteriophage virus in another series of experiments to identify the hereditary material. • Bacteriophages, like other viruses, contain both protein and DNA, but are non-living.

16. DNA Bacteriophage head Protein coat tail 1 Phage attaches to bacterium. 6 Bacterial wall destroyed; phage released. 2 Phage injects its DNA into bacterium. 5 Complete phages assembled. 3 Phage DNA is replicated. 4 Phage parts synthesized, using bacterial metabolism.

17. Radio-tagged DNA Radio-tagged Protein Radioactive phosphorus (P32) Radioactive sulfur (S35) Radioactive DNA (blue) Radioactive protein (yellow) 1 Label phages with P32 or S35. 2 Infect bacteria with labeled phages; phages inject genetic material into bacteria. 3 Whirl in blender to break off phage coats from bacteria. 4 Centrifuge to separate phage coats (low density: stay in liquid) from bacteria (high density: sink to bottom as a “pellet”) 5 Measure radioactivity of phage coats and bacteria. Results: Bacteria are radioactive; phage coats are not. Results: Phage coats are radioactive; bacteria are not. Conclusion: Infected bacteria are labeled with radioactive phosphorus but not with radioactive sulfur, supporting the hypothesis that the genetic material of bacteriophages is DNA, not protein.

18. These early experiments showed that DNA is the hereditary molecule because: • Only DNA could break down proteins. • Only DNA caused changes in hereditary traits. • Only bacteria and viruses have DNA.

19. DNA Structure? • While many research teams were trying to discover the hereditary molecule, other researchers were working to discover the nature of DNA.

20. Erwin Chargaff • Chargaff took apart DNA into its component nucleotides and studied the proportions. • Found consistent ratios between certain nucleotides.

21. In DNA, Chargaff consistently found equal amounts of adenine compared with thymine, and equal amounts of cytosine compared with guanine. Did that mean the bases were always paired?

22. If a strand of DNA is 30% adenine, how much thymine does it have? • 15% • 20% • 30% • Impossible to predict

23. If a strand of DNA is 30% adenine, how much cytosine does it have? • 15% • 20% • 30% • Impossible to predict

24. Franklin and Wilkins • Rosalind Franklin worked in Maurice Wilkins’ lab in the late 1940s, using X-ray crystalography to find clues about the structure of DNA. • Franklin’s images were the first to suggest a helical structure.

25. The X-shape on the radiograph was characteristic of helical molecules. Franklin also measured distances between bases and other dimensions using her images.

26. Watson and Crick • James Watson and Francis Crick worked at the same time as Franklin and Wilkins. • Applying Chargaff’s rule, they concluded that A pairs with T, C with G. • Used their knowledge of molecular geometry to try to discover the structure of DNA.

27. Wilkins consulted with Watson and Crick. Without Franklin’s knowledge, he handed them several of Franklin’s X-ray images. • Watson immediately recognized their significance, though he’d criticized Franklin’s work earlier.

28. By adding Franklin’s data to their own (without her permission!), Watson and Crick assembled the first plausible model of DNA and published an article on the structure of DNA in 1953.

29. DNA Structure DNA contains four bases. RNA also has four bases, but has uracil instead of thymine.

30. How many rings? How many rings? How many H-bonds? How many H-bonds? As Chargaff’s work suggested, Adenine always pairs across the DNA ladder with Thymine, while Cytosine always pairs with Guanine.

31. 5’ end 5 4 6 3 5’ 1 2 1’ 4’ 2’ 3’ 3’ end Nucleotides are 3-dimensional, with an orientation that affects the shape of the entire nucleic acid.

32. 5’ end The 3’ end of one nucleotide binds with the 5’ end of the next nucleotide in the chain. 5 4 6 3 5’ 1 2 1’ 4’ 3’ 2’ 7 8 5’ 9 5 4 1’ 6 3 4’ 1 2 3’ 2’ 3’ end

33. 5’end 3’ end Two chains of DNA nucleotides are held together by hydrogen bonds between the bases of each strand. Notice that the strands run in opposite directions. They are antiparallel. 3’end 5’end

34. free phosphate free sugar The 3-dimensional shape of the nucleotides creates the helical structure of DNA.

35. The sugar in the backbone of DNA is: • Glucose • Ribose • Deoxyribose • Lactose

36. In the DNA double helix, adenine always matches thymine because: • Adenine is polar and thymine is nonpolar. • Both can form two hydrogen bonds with each other. • Both are single-ring bases. • Wrong! Adenine always matches adenine.

37. W O R K T O G E T H E R • Suppose that one side of a DNA double-helix reads:A T A A C A G T T A G C A G G According to the base-pairing rule, what is the sequence of bases on the other side of the DNA double-helix?

38. Label the four bases in this diagram. (Look back several slides for a hint.) T A A T Circle one complete nucleotide on each side. (Hint: look back several slides to see which carbon on the sugar attaches to the phosphate.) G C C G

39. DNA Replication • When cells divide, the two resulting daughter cells must have exactly the same DNA as the original cell. • Therefore, before cell division happens, the cell must replicate (copy) its DNA.

40. replication bubbles DNA DNA helicase replication forks The enzyme DNA helicase “unzips” DNA by breaking hydrogen bonds holding the two strands together. “Unzipping” occurs at multiple points on the DNA strand.

41. DNA helicase replication forks DNA polymerase #1 3′ continuous synthesis 5′ 3′ discontinuous synthesis 5′ DNA polymerase #2 Within each replication bubble, the enzyme DNA polymerase builds a new strand of DNA, using the original strands as templates.

42. DNA polymerase #1 3′ 5′ continuous synthesis 3′ discontinuous synthesis 5′ DNA polymerase #2 DNA polymerase #1 continues along parental DNA strand 3′ DNA polymerase #2 leaves 5′ 5′ continuous synthesis 3′ discontinuous synthesis 3′ 5′ DNA polymerase #3 Because DNA polymerase always travels from the 3’ to the 5’ end of DNA, one polymerase is always moving away from the replication fork

43. DNA polymerase #1 continues along parental DNA strand 3′ DNA polymerase #2 leaves 5′ 5′ continuous synthesis 3′ discontinuous synthesis 3′ 5′ DNA polymerase #3 3′ 5′ 3′ 5′ DNA polymerase #3 leaves 3′ 5′ 3′ DNA polymerase #4 5′ DNA ligase joins daughter DNA strands together. Multiple DNA polymerase molecules are required for the strand where discontinuous replication is happening.

44. How does DNA polymerase “know” which bases to use when replicating? Remember Chargaff’s rule: A and T always match, C and G always match. Practice DNA base-pair matching: http://learn.genetics.utah.edu/content/begin/dna/builddna

45. W O R K T O G E T H E R • Suppose a segment of a DNA double-helix reads:A G T C A A T G CT C A G T T A C GAfter replication, what will the two resulting DNA double-helices read?

46. The enzyme that “unzips” DNA is: • DNA polymerase • Helicase • Ligase

47. The enzyme that “pastes” in new bases during replication is: • DNA polymerase • Helicase • Ligase

48. The enzyme that mends gaps in the sugar-phosphate backbone is: • DNA polymerase • Helicase • Ligase

49. W O R K T O G E T H E R • Helicase, DNA polymerase, and ligase are enzymes. To which class of biological molecules do enzymes belong? • Where are the instructions for making DNA polymerase found?

50. Mutations • Though many enzymes patrol your DNA, looking for replication errors, some errors do creep in. • Most cells with a DNA error will die. A few may turn cancerous. • If mutated cells are sex cells, the mutation can be passed on and will affect all cells in the offspring.