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DNA: The Genetic Material Chapter 14 2

Frederick Griffith – 1928 Studied Streptococcus pneumoniae, a pathogenic bacterium causing pneumonia 2 strains of Streptococcus –S strain is virulent –R strain is nonvirulent Griffith infected mice with these strains hoping to understand the difference between the strains 3

4 Griffith’s results –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

5 Live Nonvirulent Strain of S. pneumoniae Mice live b. Live Virulent Strain of S. pneumoniae Mice die Polysaccharide coat a. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

6 Heat-killed Virulent Strain of S. pneumoniae Mice live c. + Mixture of Heat-killed Virulent and Live Nonvirulent Strains of S. pneumoniae Their lungs contain live pathogenic strain of S. pneumoniae Mice die d. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

7 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

8 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

9 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

10 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

Phage grown in radioactive 35 S, which is incorporated into phage coat Virus infect bacteria Blender separates phage coat from bacteria Centrifuge forms bacterial pellet 35 S in supernatant 35 S-Labeled Bacteriophages Phage grown in radioactive 32 P. which is incorporated into phage DNA Virus infect bacteria Blender separates phage coat from bacteria Centrifuge forms bacterial pellet 32 P in bacteria pellet 32 P-Labeled Bacteriophages Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

12 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

13 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′ 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

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 14 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

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) 15

16 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

17 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

Double helix 2 strands are polymers of nucleotides Phosphodiester backbone – repeating sugar and phosphate units joined by phosphodiester bonds Wrap around 1 axis Antiparallel 18 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.

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 19

Complementarity of bases A forms 2 hydrogen bonds with T G forms 3 hydrogen bonds with C Gives consistent diameter 20 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

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

22 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Conservative

23 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. ConservativeSemiconservative

24 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. ConservativeSemiconservativeDispersive

25 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

Meselson and Stahl’s Results Conservative model = rejected –2 densities were not observed after round 1 Semiconservative model = supported –Consistent with all observations –1 band after round 1 –2 bands after round 2 Dispersive model = rejected –1 st round results consistent –2 nd round – did not observe 1 band 26

27 Samples are centrifuged E. coli rounds1 round2 rounds Bottom 15 N medium 14 N medium E. coli cells grown in 15 N medium Cells shifted to 14 N medium and allowed to grow DNA Samples taken at three time points and suspended in cesium chloride solution Rounds of replication Top 0 min 0 rounds 20 min 1 round 40 min 2 rounds From M. Meselson and F.W. Stahl/PNAS 44(1958):671 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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

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

30 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.

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 31 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

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 32

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

34 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 –All 3 have 3′-to-5′ exonuclease activity – proofreading –DNA pol I has 5′-to-3′ exonuclase activity

Unwinding DNA causes torsional strain –Helicases – use energy from ATP to unwind DNA –Single-strand-binding proteins (SSBs) coat strands to keep them apart –Topoisomerase prevent supercoiling DNA gyrase is used in replication 35 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Supercoiling Replisomes No Supercoiling Replisomes DNA gyrase

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 36

37 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.

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

Leading-strand synthesis –Single priming event –Strand extended by DNA pol III Processivity –  subunit forms “sliding clamp” to keep it attached 39 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.

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 40

41 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

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 42

43 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

44 Leading strand Lagging strand Primase Clamp loader Helicase DNA polymerase III DNA gyrase RNA primer Single-strand binding proteins (SSB) RNA primer β clamp 1. A DNA polymerase III enzyme is active on each strand. Primase synthesizes new primers for the lagging strand. 5´ 3´ 5´ 3´ 5´ 3´ RNA primer Loop grows Second Okazaki fragment nears completion First Okazaki fragment 2. The “loop” in the lagging-strand template allows replication to occur 5´-to- 3´ on both strands, with the complex moving to the left. 5´ 3´ 5´ 3´ 5´ 3´ Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´ 3´ 3. When the polymerase III on the lagging strand hits the previously synthesized fragment, it releases the β clamp and the template strand. DNA polymerase I attaches to remove the primer. β clamp releases Lagging strand releases DNA polymerase III DNA polymerase I 5´ 3´ 5´ 3´

45 Clamp loader 4. The clamp loader attaches the β clamp and transfers this to polymerase III, creating a new loop in the lagging-strand template. DNA ligase joins the fragments after DNA polymerase I removes the primers. DNA ligase patches “nick” DNA polymerase I detaches after removing RNA primer 5´ 3´ 5´ 3´ 5´ 3´ New bases 5. After the β clamp is loaded, the DNA polymerase III on the lagging strand adds bases to the next Okazaki fragment. Leading strand replicates continuously Loop grows 5´ 3´ 5´ 3´ 5´ 3´ Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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47 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

48 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)

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 49

50 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

51 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

52 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.

53 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

54 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

55 Photorepair 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

56 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.

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 57

58 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