1. DNA: The Genetic Material 1

2. 2 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

3. 3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Purines Pyrimidines Adenine Guanine NH 2 C C N N N C H N C C H O H H OC NC H N C H C H O O C N C H N C H3CH3C C H H O O C N C H N C H C H C C N N N C H N C C H H Nitrogenous Base 4′4′ 5′5′ 1′1′ 3′3′2′2′ 2 8 76 39 4 5 1 Phosphate group Sugar Nitrogenous base CH 2 N N O N NH 2 OH in RNA Cytosine (both DNA and RNA) Thymine (DNA only) Uracil (RNA only) OH H in DNA O P O–O– –O–O O

4. Phosphodiester bond –Bond between adjacent nucleotides –Formed between the phosphate group of one nucleotide and the 3′ —OH of the next nucleotide The chain of nucleotides has a 5′- to-3′ orientation 4 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Base CH 2 O 5′5′ 3′3′ O P O OH CH 2 –O–OO C Base O PO 4 Phosphodiester bond

5. 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) 5

6. 6 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 a. b. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Courtesy of Cold Spring Harbor Laboratory Archives

7. 7 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

8. 8 Watson & Crick with their DNA Model in 1953

9. Double helix 2 strands are polymers of nucleotides Phosphodiester backbone – repeating sugar and phosphate units joined by phosphodiester bonds Wrap around 1 axis Antiparallel 9 5´ 3  O O O O 4  5  1  3  2  4  5  1  3  2  4  5  1  3  2  4  5  1  3  2  5-carbon sugar Nitrogenous base Phosphodiester bond Phosphate group OH P P P P Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

10. C C C G G G G G T T T T A A A 2nm 5′5′3′3′ 3.4nm 0.34nm Minor groove Major groove 5′5′ 3′3′ Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Major groove Minor groove 10

11. Complementarity of bases A forms 2 hydrogen bonds with T G forms 3 hydrogen bonds with C Gives consistent diameter 11 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 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

12. “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 The following quote is from Watson & Crick’s paper on the structure of DNA:

13. 13 DNA Replication Requires 3 things –Something to copy Parental DNA molecule –Something to do the copying Enzymes –Building blocks to make copy Nucleotide triphosphates

14. 14 DNA replication includes –Initiation – replication begins –Elongation – new strands of DNA are synthesized by DNA polymerase –Termination – replication is terminated

15. 15 P P P P P P P P P P P Pyrophosphate 3′3′ 3′3′ 5′5′ 5′5′ New StrandTemplate Strand O HO OH O O O O O O O O O O C C T T T A A A G G A P P P P P P P P P P P P P P 3′3′ 3′3′ 5′5′ 5′5′ New StrandTemplate Strand O HO OH O O O O O O O O O C C T T A A A G G A Sugar– phosphate backbone DNA polymerase III T O P Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

16. 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 16 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5  3  5  5 5  5 5  3 3  3 3  RNA polymerase makes primerDNA polymerase extends primer

17. Prokaryotic Replication E. coli model Single circular molecule of DNA Replication begins at one origin of replication Proceeds in both directions around the chromosome 17

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

19. Replication is Semidiscontinous 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 19

20. 20 RNA primer Open helix and replicate First RNA primer Open helix and replicate further Lagging strand (discontinuous) Second RNA primer Leading strand (continuous) RNA primer 5′5′ 3′3′ 3′3′ 5′5′ 5′5′ 3′3′ 3′3′ 5′5′ 5′5′ 3′3′ 5′5′ 3′3′ 5′5′ 3′3′ Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

21. 21 Partial opening of helix forms replication fork DNA primase – RNA polymerase that makes RNA primer –RNA will be removed and replaced with DNA

22. Leading-strand synthesis –Single priming event –Strand extended by DNA pol III Processivity –  subunit forms “sliding clamp” to keep it attached 22 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. a-b: From Biochemistry by Stryer. © 1975, 1981, 1988, 1995 by Lupert Stryer. Used with permission of W.H. Freeman and Company a.b.

23. Lagging-strand synthesis –Discontinuous synthesis –RNA primer made by primase for each Okazaki fragment –All RNA primers removed and replaced by DNA –Backbone sealed DNA ligase Termination occurs at specific site –DNA gyrase unlinks 2 copies 23

24. 24 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5′ 3′ Primase RNA primer Okazaki fragment made by DNA polymerase III Leading strand (continuous) DNA polymerase I Lagging strand (discontinuous) DNA ligase

25. 25 Replication fork Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5  3  New bases β clamp (sliding clamp) Leading strand Single-strand binding proteins (SSB) DNA gyrase Parent DNA Primase Helicase 3  5  Clamp loader Open β clamp Lagging strand Okazaki fragment 5  3  DNA ligase polymerase I DNA RNA primer New bases polymerase III DNA

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27. 27 Eukaryotic Replication Complicated by –Larger amount of DNA in multiple chromosomes – Linear structure Multiple replicons – multiple origins of replications for each chromosome

28. 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 28

29. 29 Leading strand (no problem) Lagging strand (problem at the end) Last primer Replication first round Shortened template Origin 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ Removed primer cannot be replaced Leading strand Lagging strand Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Primer removal Replication second round

30. 30 Telomeres composed of short repeated sequences of DNA Telomerase – enzyme makes telomere section of lagging strand using an 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

31. 31 G GGGGG TT TTTT G T T G G G GG T TT T CCCCCAAAA CCCCCAAAA Telomere extended by telomerase Template RNA is part of enzyme Telomerase Now ready to synthesize next repeat 5 3 ́ 5 5 Synthesis by telomerase Telomerase moves and continues to extend telomere Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

32. 32 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 many repair systems that have been discovered

33. 33 DNA Repair Falls into 2 general categories 1. Specific repair –Targets a single kind of error in DNA and repairs only that damage 2. Nonspecific –Use a single mechanism to repair multiple kinds of lesions in DNA

34. 34 Photorepair Specific repair for 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

35. 35 T A T A AA AA T A T A T T T T Thymine dimer cleaved Photolyase Helix distorted by thymine dimer Thymine dimer DNA with adjacent thymines UV light Visible light Photolyase binds to damaged DNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

36. 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 36

37. 37 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Damaged or incorrect base Uvr A,B,C complex binds damaged DNA DNA polymerase Excision of damaged strand Resynthesis by DNA polymerase Excision repair enzymes recognize damaged DNA