2. AP Biology 2006-2007 DNA The Genetic Material Genetics Lecture #5

3. AP Biology Objectives  Explain how the structure of DNA was discovered.  Explain how and why DNA replicates itself.

4. AP Biology Scientific History  The march to understanding that DNA is the genetic material  T.H. Morgan (1908)  Frederick Griffith (1928)  Avery, McCarty & MacLeod (1944)  Erwin Chargaff (1947)  Hershey & Chase (1952)  Watson & Crick (1953)  Meselson & Stahl (1958)

5. AP Biology Chromosomes related to phenotype  T.H. Morgan  working with Drosophila  fruit flies  associated phenotype with specific chromosome  white-eyed male had specific X chromosome 1908 | 1933

6. AP Biology Genes are on chromosomes  Morgan’s conclusions  genes are on chromosomes  but is it the protein or the DNA of the chromosomes that are the genes?  initially proteins were thought to be genetic material… Why? 1908 | 1933 What ’ s so impressive about proteins?!

7. AP Biology The “Transforming Principle” 1928  Frederick Griffith  Streptococcus pneumonia bacteria  was working to find cure for pneumonia  harmless live bacteria (“rough”) mixed with heat-killed pathogenic bacteria (“smooth”) causes fatal disease in mice  a substance passed from dead bacteria to live bacteria to change their phenotype  “Transforming Principle”

8. AP Biology The “Transforming Principle” Transformation = change in phenotype something in heat-killed bacteria could still transmit disease-causing properties live pathogenic strain of bacteria live non-pathogenic strain of bacteria mice diemice live heat-killed pathogenic bacteria mix heat-killed pathogenic & non-pathogenic bacteria mice livemice die A.B. C. D.

9. AP Biology DNA is the “Transforming Principle”  Avery, McCarty & MacLeod  purified both DNA & proteins separately from Streptococcus pneumonia bacteria  which will transform non-pathogenic bacteria?  injected protein into bacteria  no effect  injected DNA into bacteria  transformed harmless bacteria into virulent bacteria 1944 What ’ s the conclusion? mice die

10. AP Biology Oswald AveryMaclyn McCartyColin MacLeod Avery, McCarty & MacLeod  Conclusion  first experimental evidence that DNA was the genetic material 1944 | ??!!

11. AP Biology Confirmation of DNA  Hershey & Chase  classic “blender” experiment  worked with bacteriophage  viruses that infect bacteria  grew phage viruses in 2 media, radioactively labeled with either  35 S in their proteins  32 P in their DNA  infected bacteria with labeled phages 1952 | 1969 Hershey Why use S ulfur vs. P hosphorus?

12. AP Biology Protein coat labeled with 35 S DNA labeled with 32 P bacteriophages infect bacterial cells T2 bacteriophages are labeled with radioactive isotopes S vs. P bacterial cells are agitated to remove viral protein coats 35 S radioactivity found in the medium 32 P radioactivity found in the bacterial cells Which radioactive marker is found inside the cell? Which molecule carries viral genetic info? Hershey & Chase

13. AP Biology

14. Blender experiment  Radioactive phage & bacteria in blender  35 S phage  radioactive proteins stayed in supernatant  therefore viral protein did NOT enter bacteria  32 P phage  radioactive DNA stayed in pellet  therefore viral DNA did enter bacteria  Confirmed DNA is “transforming factor” Taaa-Daaa!

15. AP Biology Hershey & Chase Alfred HersheyMartha Chase 1952 | 1969 Hershey

16. AP Biology Chargaff  DNA composition: “Chargaff’s rules”Chargaff’s rules  varies from species to species  all 4 bases not in equal quantity  bases present in characteristic ratio  humans: A = 30.9% T = 29.4% G = 19.9% C = 19.8% 1947 That ’ s interesting! What do you notice? Rules A = T C = G

17. AP Biology Structure of DNA  Watson & Crick  developed double helix model of DNA  other leading scientists working on question:  Rosalind Franklin  Maurice Wilkins  Linus Pauling 1953 | 1962 Franklin WilkinsPauling

18. AP Biology Watson and Crick Watson Explains Base pairing CrickWatson

19. AP Biology Rosalind Franklin (1920-1958)

20. AP Biology But how is DNA copied?  Replication of DNA  base pairing suggests that it will allow each side to serve as a template for a new strand “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”— Watson & Crick

21. AP Biology Models of DNA Replication  Alternative models  become experimental predictions conservativesemiconservative Can you design a nifty experiment to verify? dispersive 1 2 P

22. AP Biology Semiconservative replication  Meselson & Stahl  label “parent” nucleotides in DNA strands with heavy nitrogen = 15 N  label new nucleotides with lighter isotope = 14 N “The Most Beautiful Experiment in Biology” 1958 parentreplication Make predictions … 15 N parent strands 15 N/ 15 N

23. AP Biology Predictions 1st round of replication conservative 15 N/ 15 N 14 N/ 14 N semi- conservative 15 N/ 14 N dispersive 15 N/ 14 N conservative 15 N/ 15 N 14 N/ 14 N semi- conservative 15 N/ 14 N dispersive 15 N/ 14 N 2nd round of replication 14 N/ 14 N 15 N parent strands 15 N/ 15 N    1 2 P

24. AP Biology Franklin Stahl Matthew Meselson Franklin Stahl Meselson & Stahl

25. AP Biology Scientific History  March to understanding that DNA is the genetic material  T.H. Morgan (1908)  genes are on chromosomes  Frederick Griffith (1928)  a transforming factor can change phenotype  Avery, McCarty & MacLeod (1944)  transforming factor is DNA  Erwin Chargaff (1947)  Chargaff rules: A = T, C = G  Hershey & Chase (1952)  confirmation that DNA is genetic material  Watson & Crick (1953)  determined double helix structure of DNA  Meselson & Stahl (1958)  semi-conservative replication

26. AP Biology proteinRNA The “Central Dogma” DNA transcriptiontranslation replication  Flow of genetic information in a cell

27. AP Biology 2007-2008 DNA Replication

28. AP Biology Double helix structure of DNA “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”Watson & Crick

29. AP Biology Directionality of DNA  You need to number the carbons!  it matters! OH CH 2 O 4 5 3 2 1 PO 4 N base ribose nucleotide This will be IMPORTANT!!

30. AP Biology The DNA backbone  Putting the DNA backbone together  refer to the 3 and 5 ends of the DNA  the last trailing carbon OH O 3 PO 4 base CH 2 O base O P O C O –O–O CH 2 1 2 4 5 1 2 3 3 4 5 5 Sounds trivial, but… this will be IMPORTANT!!

31. AP Biology Anti-parallel strands  Nucleotides in DNA backbone are bonded from phosphate to sugar between 3 & 5 carbons  DNA molecule has “direction”  complementary strand runs in opposite direction 3 5 5 3

32. AP Biology Bonding in DNA ….strong or weak bonds? How do the bonds fit the mechanism for copying DNA? 3 5 3 5 covalent phosphodiester bonds hydrogen bonds

33. AP Biology Base pairing in DNA  Purines  adenine (A)  guanine (G)  Pyrimidines  thymine (T)  cytosine (C)  Pairing  A : T  2 bonds  C : G  3 bonds

34. AP Biology Copying DNA  Replication of DNA  base pairing allows each strand to serve as a template for a new strand  new strand is 1/2 parent template & 1/2 new DNA  semi-conservative copy process

35. AP Biology DNA Replication  Large team of enzymes coordinates replication Let’s meet the team…

36. AP Biology Replication: 1st step  Unwind DNA  helicase enzyme  unwinds part of DNA helix  stabilized by single-stranded binding proteins single-stranded binding proteins replication fork helicase I’d love to be helicase & unzip your genes…

37. AP Biology DNA Polymerase III Replication: 2nd step But… We’re missing something! What? Where’s the ENERGY for the bonding!  Build daughter DNA strand  add new complementary bases  DNA polymerase III

38. AP Biology energy ATP GTPTTPCTP Energy of Replication Where does energy for bonding usually come from? ADPAMPGMPTMPCMP modified nucleotide energy We come with our own energy! And we leave behind a nucleotide! You remember ATP! Are there other ways to get energy out of it? Are there other energy nucleotides? You bet!

39. AP Biology Energy of Replication  The nucleotides arrive as nucleosides  DNA bases with P–P–P  P-P-P = energy for bonding  DNA bases arrive with their own energy source for bonding  bonded by enzyme: DNA polymerase III ATPGTPTTPCTP

40. AP Biology  Adding bases  can only add nucleotides to 3 end of a growing DNA strand  need a “starter” nucleotide to bond to  strand only grows 5  3 DNA Polymerase III DNA Polymerase III DNA Polymerase III DNA Polymerase III energy Replication energy 3 3 5 B.Y.O. ENERGY! The energy rules the process 5

41. AP Biology Let’s Replicate!!!! DNA Video