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 2

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 3

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 4

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 5

6. 6 Bacteriophage DNA was labeled with radioactive phosphorus ( 32 P) Bacteriophage protein was labeled with radioactive sulfur ( 35 S) Radioactive molecules were tracked Only the bacteriophage DNA (as indicated by the 32 P) entered the bacteria and was used to produce more bacteriophage Conclusion: DNA is the genetic material

7. 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) 7

8. 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 8

9. 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 9

10. DNA Structure DNA is a nucleic acid Composed of nucleotides ◦ 5-carbon sugar called deoxyribose ◦ Phosphate group (PO 4 )  Attached to 5 ′ carbon of sugar ◦ Nitrogenous base  Adenine, thymine, cytosine, guanine ◦ Free hydroxyl group (—OH)  Attached at the 3 ′ carbon of sugar 10

11. 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 11 Base CH 2 O 5´5´ 3´3´ O P O OH CH 2 –O–OO C Base O PO 4 Phosphodiester bond

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 12

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

14. ConservativeSemiconservativeDispersive DNA Replication 3 possible models 1.Conservative model 2.Semiconservative model 3.Dispersive model 14

15. Meselson and Stahl – 1958 Bacterial cells were grown in a heavy isotope of nitrogen, 15 N All the DNA incorporated 15 N Cells were switched to media containing lighter 14 N DNA was extracted from the cells at various time intervals The semiconservative method was confirmed 15

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 16

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

18. 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 18 5´5´ 3´3´ 5´5´ 5´5´ 5´5´ 3´3´ 3´3´ RNA polymerase makes primerDNA polymerase extends primer

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 19

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 20

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 21

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

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

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 24 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 25

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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 27

28. 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 ) 28

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 29

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 30

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 31

32. DNA Repair Falls into 2 general categories 1. Specific repair ◦ Targets a single kind of lesion in DNA and repairs only that damage 2. Nonspecific ◦ Use a single mechanism to repair multiple kinds of lesions in DNA 32

33. Photorepair 33 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 1.Recognition of damage 2.Removal of the damaged region 3.Resynthesis using the information on the undamaged strand as a template 34