1. DNA and Its Role in Heredity

2. DNA: The Genetic Material
– Embryologist & geneticists had associated traits (genes) with chromosomes
EB Wilson & Nettie Stevens – sex & chromosome make-up
TH Morgan – sex linked traits (genes)

3. DNA: The Genetic Material
1941 – George Beadle & Edward Tatum demonstrated that a single gene corresponded to a single enzyme

4. DNA: The Genetic Material
1920s - Frederick Griffith’s transformation experiment
Virulent vs avirulent pneumococcal bacterial strains
Oswald Avery and colleagues identified the transforming substance of Griffith’s pneumococcal strain

5. Oswald Avery's Isolation of the Transforming Substance
Figure: 10-03a
Caption:
Summary of Avery, MacLeod, and McCarty’s experiment, which demonstrated that DNA is the transforming principle.

6. DNA: The Genetic Material
Alfred Hershey & Martha Chase confirmed DNA is genetic material of viruses
Bacteriophage - a virus infecting bacteria
T2 a DNA phage of E. coli
A DNA core packed in a protein coat

7. DNA: The Genetic Material
Hershey-Chase experiment determined whether viral protein or DNA entered the bacterium & directed the synthesis of further viral particles

8. Figure 11.3 The Hershey–Chase Experiment

9. Questions Remaining About of DNA
How does DNA cause the synthesis of specific proteins?
How is DNA replicated between nuclear divisions?
Structure of DNA ultimately provided insight to the answers

10. The Chemical Constituents of DNA
1859 – Friedrich Meidscher discovered and named nucleic acids (DNA)
By 1940s known DNA was a polymer of nucleotides.
DNA was assumed to be non-varying, repeating sequence of nucleotides unique to individual species
Erwin Chargaff carefully determined that individual percentages of A & T as well as G & C are equal and the A:T / G:C ratio varies among organisms

11. Nitrogenous Bases Figure: 10-07a Caption:
(a) Chemical structures of the pyrimidines and purines that serve as the nitrogenous bases in RNA and DNA. (b) Chemical ring structures of ribose and 2-deoxyribose, which serve as the pentose sugars in RNA and DNA, respectively.

12. Deoxyribonucleic Acid
Ribose Sugars
Figure: 10-07b
Caption:
(a) Chemical structures of the pyrimidines and purines that serve as the nitrogenous bases in RNA and DNA. (b) Chemical ring structures of ribose and 2-deoxyribose, which serve as the pentose sugars in RNA and DNA, respectively.
RNA
Ribonucleic Acid
DNA
Deoxyribonucleic Acid

13. Figure: 10-08
Caption:
Structures and names of the nucleosides and nucleotides of RNA and DNA.

14. Figure: 10-09
Caption:
Basic structures of nucleoside diphosphates and triphosphates, as illustrated by thymidine diphosphate and adenosine triphosphate.

15. Figure: 10-10
Caption:
(a) Linkage of two nucleotides by the formation of a C-3’–C-5’ (3’–5’) phosphodiester bond, producing a dinucleotide. (b) Shorthand notation for a polynucleotide chain.

16. DNA Structure Determination
1953
James Watson & Francis Crick
Rosalind Franklin & R Gosling
Maurice Wilkins
Jerry Donohue
Linus Pauling

17. Figure 11.4 X-Ray Crystallography Revealed the Basic Helical Structure of the DNA Molecule

18. Copyright © The McGraw-Hill Companies, Inc
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

19. Figure 11.6 (b) DNA Is a Double Helix

20. Models of A & B DNA
A-DNA
B-DNA

21. Figure 11.7 Base Pairing in DNA Is Complementary

22. In 1953 DNA Structure Suggests Function
Complementary Base Pairing
Replication mechanisms
Sequence of nucleotides
Code corresponding to proteins in some way

23. Determining the DNA Replication Mechanism
Matthew Meselson and Franklin Stahl demonstrated DNA replication is semiconservative

24. Figure 11.9 The Meselson–Stahl Experiment

25. Theoretical Predictions
Experimental Observation

26. Determining the DNA Replication Mechanism
Arthur Kornberg purified DNA polymerase & used it to replicate DNA in vitro
Polymerization only worked with nicked, ds DNA templates
Found that DNA polymerase requires a priming 3’- OH from which to initiate synthesis

27. The Mechanisms Overview of DNA Replication
H-bonds between strands are broken, making each strand available to base pair with new nucleotide
Sequence of new strand is directed by the sequence of the template strand – complementary base pairing
Nucleotides are attached to the 3’ end of each growing strand

28. Figure 11.10 Each New DNA Strand Grows from its 5¢ End to its 3¢ End

29. DNA Synthesis is Bidirectional
Two nascent, labeled strands at each fork means both parent strands serve as templates

30. Implication of Bidirectional Synthesis
RULE: Polymerization can only happen in 5'3' direction
Starting at 1 spot
only 1 strand can serve as template
but both strands do
therefore, one strand synthesized continuously
leading strand
the other strand made discontinuously
lagging stand

31. Okazaki Fragments
If model is correct, should be able to find lagging strand fragments
Discovered by Reiji & Tuneko Okazaki
nt long DNA fragments
Begin with ~12 nucleotides of RNA
Figure: 11-11
Caption:
Opposite polarity of DNA synthesis along the two strands, necessary because the two strands of DNA run antiparallel to one another and DNA polymerase III synthesizes only in one direction (5’ to 3’). On the lagging strand, synthesis must be discontinuous, resulting in the production of Okazaki fragments. On the leading strand, synthesis is continuous. RNA primers initiate synthesis on both strands.
Priming

32. Figure 11.15 Many Proteins Collaborate at the Replication Fork

33. Mechanics of DNA Synthesis
Figure: 11-13
Caption:
Summary of DNA synthesis at a single replication fork. Various enzymes and proteins essential to the process are shown.

34. The Molecular Mechanisms of DNA Replication
Enzymology
DNA polymerase
Primase
Helicase
Topisomerase
replication complex recognizes an origin of replication on a chromosome.

35. The Molecular Mechanisms of DNA Replication
Replication occurs from many origins simultaneously
Large chromosomes can have hundreds of origins of replication
The region replicated from a single origin is called a replicon
The complex of enzymes is the replisome

36. Figure 11.17 The Lagging Strand Story (Part 2)

37. Figure 11.18 Telomeres and Telomerase

38. DNA Proofreading and Repair
Proofreading by DNA polymerase minimizes errors
Mutation rate of most eukaryotic DNA polymerases ~ 10-8
1 error every 1x108 bp
Mutation rate in prokaryotic cells is higher ~10-6 – 10-7
DNA damage
UV, free radicals, etc..
Repair mechanisms frequently excise damaged sequences and resynthesize DNA to repair damage

39. Practical Applications of DNA Replication
The technique of DNA sequencing hinges on the use of modified nucleosides called dideoxynucleotides (ddNTPs).
ddNTPs lack both 2’ and 3¢ hydroxyl groups
3’ OH is site of addition of next nucleotide
Like dNTPs, ddNTPs are picked up by DNA polymerase and added to a growing DNA chain
But once added, chain elongation is terminated

40. Figure Sequencing DNA

41. DNA Sequencing
Primer annealed

42. DNA Sequencing
Primer extended

43. Practical Applications of DNA Replication
The polymerase chain reaction (PCR) technique for making multiple copies of a DNA sequence.
PCR cycles through three steps:
Double-stranded fragments of DNA are heated to denature them into single strands.
A short primer is annealed
DNA polymerase catalyzes the production of new DNA strands

44. Figure 11.20 The Polymerase Chain Reaction
Cycle 1
Cycle 2
Cycle 3