1. Chapter 16: Molecular Basis of Inheritance
The Race for the Double Helix .

2. The Search for the Genetic Material
Friedrich Miescher (1868; Swiss)
nuclein
Robert Feulgen (1914; German)
DNA staining
C. Frederick Griffith (1928; English)

3. Streptococcus pneumoniae
Injected mice with a virulent strain (smooth because of a coat)

4. Streptococcus pneumoniae
Injected mice with a nonvirulent strain (rough)
It lacks a capsule; is harmless

5. Streptococcus pneumoniae
Boiled the virulent strain.
Heat-killed S cells are harmless

6. Streptococcus pneumoniae
Boiled the virulent S strain.
Combined it with non-virulent R strain
What do you predict happened to the mice?

7. Figure 16.1 Transformation of bacteria

8. We know this process today as bacterial transformation.
But what is the transforming factor?
Is it DNA or Protein?

9. THE SEARCH FOR THE TRANSFORMING FACTOR D. Avery, Macleod and McCarty
S strain fractionated
RNA
Protein
Lipid
Carbohydrate
DNA

10. HERSHEY AND CHASE bacteriophages

11. Figure 16.2ax Phages

12. Figure 18.4 The lytic cycle of phage T4

13. Figure 16.2b The Hershey-Chase experiment
The experiment showed that T2 proteins remained outside the host cell during infection, while T2 DNA enters the cell

14. F. Hershey-Chase Experiment
Results published in 1952
1969 Nobel Prize
Delbruck, Luria and Hershey

15. F. Other Evidence Amount of DNA doubles prior to mitosis
Diploid chromosomes have twice as much DNA as haploid sets found in the gametes of the same organism.

16. G. Erwin Chargaff 1947 Studied the DNA of various species
While the % of A,T,C,G varied between species, the amount of A = T, and C = G
Chargaff’s Rules

17. Figure 16.3 The structure of a DNA stand

18. Figure 5.1 Building models to study the structure and function of macromolecules

19. Figure 16.4 Rosalind Franklin and her X-ray diffraction photo of DNA

20. Deductions about DNA made from Franklin’s Photo
1. DNA is a helix with a uniform width of 2 nm.
2. Purine and pyrimidine bases are stacked .34 nm apart
3. The helix makes one full turn every 3.4 nm along its length.
4. There are ten layers of nucleotide pairs in each turn of the helix.

21. Model postulates:
1.The sugar-phosphates alternated on the exterior of the molecule
2.The nitrogen bases were in the interior, each one bonded to a sugar.

22. Model postulates:
3. The two sugar-phosphate backbones of the helix are antiparallel.
4. A purine (adenine or guanine) must pair with a pyrimidine (cytosine or thymine).
5. This base pairing dictates which pairs of bases can hydrogen bond: A-T, C-G
6. Base pairing rule explains Chargaff’s rules

23. Figure 16.5 The double helix

27. Unnumbered Figure (page 292) Purine and pyridimine

28. Figure 16.6 Base pairing in DNA

29. Figure 16.8 Three alternative models of DNA replication

32. Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 2)

33. ONE Layers was obtained. What does this show?
Figure The Meselson-Stahl experiment tested three models of DNA replication (Layer 3)
ONE Layers was obtained.
What does this show?

34. After 2nd replication TWO bands appear. What does this show?
Figure The Meselson-Stahl experiment tested three models of DNA replication (Layer 4)
After 2nd replication
TWO bands appear.
What does this show?

35. B. Replication Background
Just how big is your genome?
Your genome is 6B bp (3B X 2 chromosomes)
If printed out the size of your textbook font, this would fill
1200 Campbell Biology Texts!

36. B. Replication Background
5M bp with E. coli; 3B bp in humans!
Complex
Rapid (up to 500 nucleotides/second in bacteria, 50/sec in human cells)
Accurate ( errors only 1/10B)
Enzymes: Requires the cooperation of over a dozen

37. C. Process of Replication
Origins of Replication: Bacteria have only 1
Replication forks

38. Eukaryotes may have hundreds or thousands of origins of replication

39. Incorporation of a nucleotide into a DNA strand
The nucleotides added are actually triphosphates; the
hydrolysis of the pyrophosphate is the exergonic reax
that drives polymerization.

40. Figure 16.12 The two strands of DNA are antiparallel
New nucleotides can only be added at the 3’ end. This presents a problem!

41. Figure 16.14 Priming DNA synthesis with RNA
But first . . .
Primers: short segments of RNA polymerized by RNA primase

42. Figure 16.14 Priming DNA synthesis with RNA
Then . . .
DNA polymerase III
13 known active sites
Self-correction unit (limits errors to 1/107 bp)
Can only add new bp to 3’ end

43. DNA Synthesis All synthesis is done from 3’ to 5’ end.
Proceeds smoothly in one direction = LEADING STRAND
Discontinuous in the other direction =LAGGING STRANDS
OKAZAKI FRAGMENTS

50. Synthesis of leading and lagging strands during DNA replication
LIGASE
Is the “glue” required to connect the fragments

52. A summary of DNA replication

53. Enzymes of Synthesis Helicase Untwists Topoisomerase Nicks SSB
Holds apart

54. Enzymes of Synthesis Primase Makes RNA Primer Ligase
Connects Okazaki fragments
DNA Polymerase III
Adds new nucleotides 5’ to 3’
DNA Polymerase I
Removes RNA primer; replaces it with DNA

55. A--CATALYZES 5’-3’ ADDITON OF NUCLEOTIDES
E- PROOFREADS 3’-5’
B2 DIMER CLAMPS AROUND DOUBLE HELIX (yellow and blue)

56. Figure 16.15 The main proteins of DNA replication and their functions

59. Nobel Prize 1959
Kornberg for DNA Polymerase
Nobel Prize 1962
Watson, Crick and Wilkins

60. PROOFREADING AND REPAIR
Amnesty Order of Caesar:
EXECUTE NOT, LIBERATE!
However, slightly altered:
EXECUTE, NOT LIBERATE!

61. PROOFREADING AND REPAIR
DNA is only macromolecule to be repaired
a. Correct nucleotide has higher affinity for moving polymerase
b. DNA polymerase is a “self-correcting” enzyme; has a 3’ to 5’ proofreading exonuclease

62. PROOFREADING AND REPAIR
Errors in completed molecule: 1/1 billion
Mismatch repair enzymes; mutation in one of these assoc. with colon cancer.
Reactive chemicals and UV can contribute to DNA alterations.
There are over repair enzymes in humans!

63. A. Causes of DNA Damage Environmental Agents a. UV Light
1. Thymine or cytosine dimers
2. Distortion interferes with replication and protein synthesis

64. Figure 16.17 Nucleotide excision repair of DNA damage

65. A. Causes of DNA Damage Environmental Agents
b. Ionizing Radiation: X rays, Atom bomb, 1986 Chernobyl
1. Radiation reacts w/ DNA or water molecules

66. A. Causes of DNA Damage Environmental Agents
c. Chemical Agents (Carcinogens)
1. Benzopyrene
2. Cigarette smoke, auto exhaust
3. Dioxin

67. PROOFREADING AND REPAIR
Nucleotide Excision Repair:
DNA Polymerase I (a nuclease) snips out wrong base, puts in correct nucleotide
DNA ligase seals it back
Thymine dimers are common with UV damage; Xeroderma pigmentosum

68. B. DNA Repair: Enzymatic Processes
1. Selection of Correct Nucleotide
Correct base is most energetically favorable
Error occurs about every 1/100,000 bases

69. B. DNA Repair: Enzymatic Processes
2. “Proofreading”
Each nucleotide must be complementary
Exonucleases remove mismatched nucleotides as added
Now errors reduced to 1/10M bp

70. B. DNA Repair: Enzymatic Processes
3. Mismatch Repair
Occurs after synthesis
Proteins excise damage
Polymerases synthesize new strands
Error rate reduced to 1/10B bp

71. C. REPAIR ENZYMES
Often function after damage has been done
Over 130 known in humans
Photolyase: activated by absorbing visible light; breaks dimers apart
UV repair enzymes: uvrA, B, C
These are nucleases: Remove damaged sections of nucleotides

72. C. REPAIR ENZYMES Cancer Often caused by unrepaired DNA damage
Ex. Xeroderma pigmentosum

73. Xeroderma pigmentosum
Skin cells defective in excision repair enzymes
Can develop hundreds of skin cancers
“freckled”
Internal Cancers

74. TELOMERES Revisited
1972: James Watson noticed that the DNA polymerases could not start at the very tip of a DNA strand.
Analogy: Copy Machine
What could you do to be sure the important information on each page remains?

75. Definition/Information
TELOMERES: TTAGGG repeats
perhaps 2000 times!
2. In your body, shortening at the rate of about 31 bp/year

76. Definition/Information
80 year old person, telomeres are about 5/8 length at birth

77. How can this be remedied?
Egg and SPERM cells and other cells in other organisms have overcome this problem
TELOMERASE Enzyme that catalyzes the addition of lost DNA by using an RNA template

78. How can this be remedied?
Stopwatch = Telomere deterioration
Germ cells never start the watch.
Natural selection has built our telomeres so they can survive at most years.
People from long-lived families may have longer telomeres

79. How can this be remedied?
Remember the HeLa Cells?
Worldwide, weigh more than 400 times her own body weight
Oct. 11 is recognized as Henrietta Lacks Day in Atlanta
HeLa cells have excellent telomerase

80. Cancer Cells and Telomerase
Switching on of telomerase genes is an essential mutation that must occur if a cancer is to turn malignant

81. Figure 16.18 The end-replication problem

82. Figure 16.19a Telomeres and telomerase: Telomeres of mouse chromosomes

83. Figure 16.19b Telomeres and telomerase