1. CHAPTER 14 LECTURE SLIDES
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2. DNA: The Genetic Material
Chapter 14

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

4. Griffith’s 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

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

8. 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

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

10. Bacteriophage DNA was labeled with radioactive phosphorus (32P)
Bacteriophage protein was labeled with radioactive sulfur (35S)
Radioactive molecules were tracked
Only the bacteriophage DNA (as indicated by the 32P) entered the bacteria and was used to produce more bacteriophage
Conclusion: DNA is the genetic material

12. DNA Structure DNA is a nucleic acid Composed of nucleotides
5-carbon sugar called deoxyribose
Phosphate group (PO4)
Attached to 5′ carbon of sugar
Nitrogenous base
Adenine, thymine, cytosine, guanine
Free hydroxyl group (—OH)
Attached at the 3′ carbon of sugar

13. NH2 N N N CH2 O Nitrogenous Base Nitrogenous base NH2 O N C N C C N C
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Nitrogenous Base
Nitrogenous base
NH2
NH2 O 7 N 6 5 N C N C C N C N H 1 H C H C
Phosphate group 8
Purines C C 2 N C H N C
NH2 N N O N 4 N H H 9 3
Adenine
Guanine –O P O
CH2 5´ O– O
NH2 O O 1´ C C C 4´ H C N
H3C C N H H C N H H C C O H C C O H C C O 3´ 2´
OH in RNA
Pyrimidines N N N OH H H H
H in DNA
Sugar
Cytosine
(both DNA and RNA)
Thymine
(DNA only)
Uracil
(RNA only)

14. The chain of nucleotides has a 5′-to-3′ orientation
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
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PO4
Base
CH2 O C O
Phosphodiester
bond –O P O O
Base
CH2 O OH 3´

15. 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)

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

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

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
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Phosphate group P 5´ O 4´ 1´
Phosphodiester bond 3´ 2´ P 5´ O 4´ 1´ 3´ 2´ P 5´ O 4´ 1´
5-carbon sugar 3´ 2´
Nitrogenous base P 5´ O 4´ 1´ 3´ 2´ OH 3´

20. Complementarity of bases A forms 2 hydrogen bonds with T
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Hydrogen
bond H
Complementarity of bases
A forms 2 hydrogen bonds with T
G forms 3 hydrogen bonds with C
Gives consistent diameter H N O H N H N G N H N C H
Sugar N N N H
Sugar H
Hydrogen
bond H H N N H O
CH3 N A N H N T H
Sugar N N H
Sugar

21. DNA Replication 3 possible models Conservative model
Semiconservative model
Dispersive model

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Conservative

23. Conservative Semiconservative 23
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Conservative
Semiconservative

24. Conservative Semiconservative Dispersive 24
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Conservative
Semiconservative
Dispersive

25. Meselson and Stahl – 1958
Bacterial cells were grown in a heavy isotope of nitrogen, 15N
All the DNA incorporated 15N
Cells were switched to media containing lighter 14N
DNA was extracted from the cells at various time intervals

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
1st round results consistent
2nd round – did not observe 1 band

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

29. DNA replication includes
Initiation – replication begins
Elongation – new strands of DNA are synthesized by DNA polymerase
Termination – replication is terminated

31. All have several common features
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RNA polymerase makes primer
DNA polymerase extends primer
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

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

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

35. Unwinding DNA causes torsional strain
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Supercoiling
Replisomes
No Supercoiling
Replisomes
DNA gyrase
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

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

38. Partial opening of helix forms replication fork
DNA primase – RNA polymerase that makes RNA primer
RNA will be removed and replaced with DNA

39. Leading-strand synthesis
Single priming event
Strand extended by DNA pol III
Processivity –  subunit forms “sliding clamp” to keep it attached

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

41. Leading strand (continuous)
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DNA ligase
Lagging strand
(discontinuous)
RNA primer
DNA polymerase I
Okazaki fragment
made by DNA
polymerase III
Primase
Leading strand
(continuous) 3´

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

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DNA polymerase III
Leading strand 5´ 3´
Helicase 5´ 3´
Clamp loader
DNA gyrase 5´ 3´ 5´ 3´
RNA primer
First Okazaki
fragment
RNA primer
β clamp 5´
Primase 3´ 5´
Second Okazaki
fragment nears
completion
Single-strand
binding proteins
(SSB)
Lagging strand 3´
Loop
grows
RNA primer
1. A DNA polymerase III enzyme is active on each strand. Primase
synthesizes new primers for the lagging strand.
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´
DNA polymerase III 3´
DNA polymerase I
Lagging
strand
releases 5´ 3´
β clamp
releases
3. When the polymerase III on the lagging strand hits the previously
synthesized fragment, it releases the β clamp and the template
strand. DNA polymerase I attaches to remove the primer.

44. 4. The clamp loader attaches the β clamp and transfers this
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Clamp loader 5´ 5´ 3´ 3´
DNA ligase
patches “nick”
Leading strand
replicates
continuously 5´ 3´
DNA polymerase I
detaches after
removing RNA primer 5´ 3´
Loop
grows
New bases 5´ 3´
4. The clamp loader attaches the β 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.
5. After the β clamp is loaded, the DNA polymerase III on
the lagging strand adds bases to the next Okazaki fragment.

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47. 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

48. Not sequence specific; can be adjusted
Multiple replicons – multiple origins of replications for each chromosome
Not sequence specific; can be adjusted
Initiation phase of replication requires more factors to assemble both helicase and primase complexes onto the template, then load the polymerase with its sliding clamp unit
Primase includes both DNA and RNA polymerase
Main replication polymerase is a complex of DNA polymerase epsilon (pol ε) and DNA polymerase delta (pol δ)

49. 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

51. Telomeres composed of short repeated sequences of DNA
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

53. 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

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

55. Photorepair Specific repair mechanism
For one particular form of damage caused by UV light
Thymine dimers
Covalent link of adjacent thymine bases in DNA
Photolyase
Absorbs light in visible range
Uses this energy to cleave thymine dimer

57. Excision repair Nonspecific repair
Damaged region is removed and replaced by DNA synthesis
3 steps
Recognition of damage
Removal of the damaged region
Resynthesis using the information on the undamaged strand as a template