1. DNA: The Genetic Material
Chapter 14

2. Griffith’s experiment with Streptococcus pneumoniae
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

3. 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

4. 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

5. 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

6. 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

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

8. The chain of nucleotides has a 5′- to-3′ orientation
Phosphodiester bond
Bond between adjacent nucleotides
Formed between the phosphate group on the 5’ carbon of one nucleotide and the 3′ —OH of the next nucleotide
The chain of nucleotides has a 5′- to-3′ orientation
Base
CH2 O 5´ 3´ P OH –O C
PO4
Phosphodiester
bond

9. 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)

10. 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

11. 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

12. Double helix 2 strands are polymers of nucleotides
Phosphodiester backbone – repeating sugar and phosphate units joined by phosphodiester bonds
Wrap around 1 axis
Antiparallel

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

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

15. 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
The semiconservative method was confirmed

16. DNA Replication Requires 3 things Something to copy
Parental DNA molecule
Something to do the copying
Enzymes
Building blocks to make copy
Nucleotide triphosphates

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

18. All have several common features 5´ 3´
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

19. 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

20. 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

21. Semidiscontinous Helicase opens the double helix
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

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

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

24. 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 5´ 3´
Primase
RNA primer
Okazaki fragment
made by DNA
polymerase III
Leading strand
(continuous)
DNA polymerase I
Lagging strand
(discontinuous)
DNA ligase

25. 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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27. 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

28. 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)

29. 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

30. 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

31. 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

32. 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

33. 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

34. 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