1. 11 DNA & Its Role in Heredity

2. DNA: The Genetic Material 1859 – Friedrich Meischer discovered & named nucleic acids (DNA) 1900-1910 – Embryologist & geneticists associated traits with chromosomes  EB Wilson & Nettie Stevens – sex & chromosome make-up  TH Morgan – sex linked genes

3. DNA: The Genetic Material 1920s - Frederick Griffith - transformation experiment  Virulent vs avirulent pneumococcal bacterial strains 1940-44 - Oswald Avery & colleagues identified transforming substance

4. Oswald Avery's Isolation of the Transforming Substance

5. DNA: The Genetic Material 1941 – George Beadle & Edward Tatum  1 gene = 1 enzyme  Mutated Neurospora crassa (bread mold) w/ X-rays

6. DNA: The Genetic Material 1952 - Alfred Hershey & Martha Chase  DNA is genetic material of viruses  Bacteriophage - virus infecting bacteria  T2 - DNA phage of E. coli  DNA packed in protein coat

7. DNA: The Genetic Material Phage lifecycle  Virus infects host  Replicates particles  Lysis  Infects new host

8. The Hershey–Chase Experiment dissociate phage coat from bacteria after infection but before lysis pellet unlysed bacteria is radioactivity IN bacteria or OUTSIDE bacteria?

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 By 1940s known DNA was a polymer of nucleotides. DNA was assumed to be non-varying, repeating sequence of nucleotides unique to individual species 1950 - Erwin Chargaff  determined % A =%T & %G = %C  A:T / G:C ratio varies 1953 – Jim Watson & Francis Crick  modeled DNA double helical structure

11. Nitrogenous Bases

12. Ribose Sugars RNA Ribonucleic Acid DNA Deoxyribonucleic Acid

13. Adenosine monophosphate Cytodine monophosphate Guanosine monophosphate Uridine monophosphate Deoxyadenosine monophosphate Deoxycytodine monophosphate Deoxyguanosine monophosphate Deoxythymidine monophosphate

14. OH Deoxynucleoside triphosphate (dNTP) Deoxyadenosine triphosphate (dATP) dATP dGTP dCTP dTTP ATP GTP CTP UTP

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

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

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20. DNA Is a Double Helix

21. Models of A & B DNA A-DNAB-DNA

22. Base Pairing in DNA Is Complementary

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

24. Determining the DNA Replication Mechanism 1957 - Matthew Meselson & Franklin Stahl  demonstrated semiconservative DNA replication

25. The Meselson–Stahl Experiment

26. Theoretical Predictions Experimental Observation

27. Determining the DNA Replication Mechanism 1958 - Arthur Kornberg purified DNA polymerase & used it to replicate DNA in vitro  Polymerization only with nicked, ds DNA templates  DNA polymerase requires a priming 3’- OH to initiate synthesis

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

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

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 1000-2000 nt long DNA fragments Begin with ~12 nucleotides of RNA Priming

32. Mechanics of DNA Synthesis

33. Nucleus Cam DNA replication movie

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

35. A Replisome leading strand lagging strand primase helicase Origin of Replication Replisome replication fork Replicon

36. Completing The Lagging Strand DNA polymerase III

37. Figure 11.18 Telomeres & Telomerase

38. DNA Proofreading & Repair Proofreading by DNA polymerase minimizes errors Mutation rate of most eukaryotic DNA polymerases ~ 10 -8  1 error every 1x10 8 bp Mutation rate in prokaryotic cells is higher ~10 -6 – 10 -7 DNA damage  UV, free radicals, etc..  Repair mechanisms frequently excise damaged sequences & 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’ & 3 hydroxyl groups 3’ OH is site of addition of next nucleotide Like dNTPs, ddNTPs are picked up by DNA polymerase & added to a growing DNA chain But once added, chain elongation is terminated

40. Figure 11.21 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 1Cycle 2Cycle 3