1. Chapter 11 DNA: The Carrier of Genetic Information

2. Experiments in DNA ???Protein as the genetic material 20 AA – many different combinations = unique properties Genes control protein synthesis DNA and RNA – only 4 nucleotides = dull

3. Experiments in DNA Frederick Griffith – 1928 – Bacteria – pneumococcus – 2 strains – (S) smooth strain – virulent (lethal) Mice – pneumonia - death – (R) rough strain – avirulent Mice survive – Heat killed (S) strain Mice survive – Heat killed (S) + live (R) Mice died Found living (S) in dead mice

4. Griffith continued –  transformation - type of permanent genetic change where the properties of 1 strain of dead cells are conferred on a different strain of living cells – “transforming principle” was transferred from dead to living cells

5. Fig. 16-2 Living S cells (control) Living R cells (control) Heat-killed S cells (control) Mixture of heat-killed S cells and living R cells Mouse dies Mouse healthy Living S cells RESULTS EXPERIMENT

6. Avery, MacLeod, McCarty - 1944 – Identified Griffith’s transforming principle as DNA – Live (R) + purified DNA from (S)  R cells transformed – R + (S) DNA  die – R = (S) protein  live – DNA responsible for transformation – Really?

7. Hershey and Chase – 1952 – Bacteriophages – Radioactive labels Viral protein – sulfur Viral DNA - phosphorus – infect bacteria, agitate in blender, centrifuge – Found Sulfur sample – all radioactivity in supernatant (not cells) Phosphorus sample – radioactivity in pellet (inside cells) – SO – bacteriophages inject DNA into bacteria, leaving protein on outside – DNA = hereditary material

8. Fig. 16-3 Bacterial cell Phage head Tail sheath Tail fiber DNA 100 nm

9. Fig. 16-4-3 EXPERIMENT Phage DNA Bacterial cell Radioactive protein Radioactive DNA Batch 1: radioactive sulfur ( 35 S) Batch 2: radioactive phosphorus ( 32 P) Empty protein shell Phage DNA Centrifuge Pellet Pellet (bacterial cells and contents) Radioactivity (phage protein) in liquid Radioactivity (phage DNA) in pellet

10. Rosalind Franklin (in lab of Wilkins) – X-ray diffraction on crystals of purified DNA – (X-ray crystallography) – Determine distance between atoms of molecules arranged in a regular, repeating crystalline structure Helix structure Nucleotide bases like rungs on ladder

11. Fig. 16-6 (a) Rosalind Franklin (b) Franklin’s X-ray diffraction photograph of DNA

12. James Watson and Francis Crick – 1953 – Model for DNA structure = double helixdouble helix – DNA now widely accepted as genetic material – Took all available info on DNA and put together – Showed – DNA can carry info for proteins Serve as own template for replication

13. Structure of DNA Nucleotides – Deoxyribose – Phosphate – Nitrogenous base (ATCG) Purines – adenine, guanine – 2 rings Pyrimidines – thymine, cytosine – 1 ring – covalent bonds link = sugar-phosphate backbone 3’ C of sugar bonded to 5’ phosphate = phophodiester linkage 5’ end – 5’ C attached to phosphate 3’ end – 3’ C attached to hydroxyl

14. Chargaff - 1950 # purines = # pyrimidines – #A = #T – #C = # G Each cross rung of ladder – 1 purine + 1 pyrimidine

15. Fig. 16-UN1 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data

16. Hydrogen bonding between N bases A-T = 2 H bonds G-C = 3 H bonds Complementary base pairs # possible sequences virtually unlimited  many genes, much info

17. Fig. 16-8 Cytosine (C) Adenine (A)Thymine (T) Guanine (G)

18. Fig. 16-5 Sugar–phosphate backbone 5 end Nitrogenous bases Thymine (T) Adenine (A) Cytosine (C) Guanine (G) DNA nucleotide Sugar (deoxyribose) 3 end Phosphate

19. Fig. 16-7a Hydrogen bond 3 end 5 end 3.4 nm 0.34 nm 3 end 5 end (b) Partial chemical structure(a) Key features of DNA structure 1 nm

20. DNA Replication Semiconservative – each strand of DNA is template to make opposite new strand Meselson and Stahl – E. coli and isotopes of N – 15N – heavy/dense; 14N “normal” – Bacteria with 15N in DNA replicated with medium having 14N – Centrifuge  – Supports semiconservative model Explains how mutagens can be passed on

21. Fig. 16-11a EXPERIMENT RESULTS 1 3 2 4 Bacteria cultured in medium containing 15 N Bacteria transferred to medium containing 14 N DNA sample centrifuged after 20 min (after first application) DNA sample centrifuged after 20 min (after second replication) Less dense More dense

22. Fig. 16-9-3 A T G C TA TA G C (a) Parent molecule AT GC T A T A GC (c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand (b) Separation of strands A T G C TA TA G C A T G C T A T A G C

23. Fig. 16-10 Parent cell First replication Second replication (a) Conservative model (b) Semiconserva- tive model (c) Dispersive model

24. Steps of DNA Replication 1. DNA helicase – 2. Helix-destabilizing proteins – 3. Topoisomerases – 4. RNA primer – 5. DNA polymerase – 6. Origin of replication – – Leading strand Leading strand – Lagging strand Lagging strand 7. DNA Ligase

25. Leading Strand

27. Fig. 16-12b 0.25 µm Origin of replicationDouble-stranded DNA molecule Parental (template) strand Daughter (new) strand Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes

28. Fig. 16-14 A C T G G G GC CC C C A A A T T T New strand 5 end Template strand 3 end 5 end 3 end 5 end 3 end Base Sugar Phosphate Nucleoside triphosphate Pyrophosphate DNA polymerase

29. Fig. 16-15a Overview Leading strand Lagging strand Origin of replication Primer Overall directions of replication

30. Fig. 16-17 Overview Origin of replication Leading strand Lagging strand Overall directions of replication Leading strand Lagging strand Helicase Parental DNA DNA pol III PrimerPrimase DNA ligase DNA pol III DNA pol I Single- strand binding protein 5 3 5 5 5 5 3 3 3 3 1 3 2 4

32. Telomeres Telomeres – caps end of chromosome; short non-coding sequences repeated many times Cell can divide many times before losing crucial info Lagging strand is discontinuous, so DNA polymerase unable to complete replication, leaving small part unreplicated  small part lost with each cycle

33. Fig. 16-19 Ends of parental DNA strands Leading strand Lagging strand Last fragment Previous fragment Parental strand RNA primer Removal of primers and replacement with DNA where a 3 end is available Second round of replication New leading strand New lagging strand Further rounds of replication Shorter and shorter daughter molecules 5 3 3 3 3 3 5 5 5 5