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Chapter 14 Lecture Outline See separate PowerPoint slides for all figures and tables pre-inserted into PowerPoint without notes and animations. 1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 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 • 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 4 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Live Nonvirulent Strain of S. pneumoniae Live Virulent Strain of S. pneumoniae Polysaccharide coat Mice die a. Mice live b. 5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Mixture of Heat-killed Virulent and Live Nonvirulent Strains of S. pneumoniae Heat-killed Virulent Strain of S. pneumoniae + Mice die Their lungs contain live pathogenic strain of S. pneumoniae Mice live c. d. 6 • 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 7 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 8 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 9 • Bacteriophage DNA was labeled with radioactive phosphorus (32P) • Bacteriophage protein was labeled with radioactive sulfur (35S) • Radioactive molecules were tracked • Only the bacteriophage DNA (as indicated by the 32P) entered the bacteria and was used to produce more bacteriophage • Conclusion: DNA is the genetic material 10 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 35S-Labeled Bacteriophages + Phage grown in radioactive 35S, which is incorporated into phage coat Virus infect bacteria 32P-Labeled Blender separates phage coat from bacteria Centrifuge forms bacterial pellet 35S in supernatant Bacteriophages + Phage grown in radioactive 32P. which is incorporated into phage DNA Virus infect bacteria Blender separates Centrifuge forms phage coat from bacteria bacterial pellet 32P in bacteria pellet 11 DNA Structure • DNA is a nucleic acid • Composed of nucleotides – 5-carbon sugar called deoxyribose – Phosphate group (PO4) • Attached to 5′ carbon of sugar – Nitrogenous base • Adenine, thymine, cytosine, guanine – Free hydroxyl group (—OH) • Attached at the 3′ carbon of sugar 12 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Nitrogenous Base Nitrogenous base NH2 1 8 Phosphate group 2 O N 9 P O 4 N N C C H N C H N C C N C C N H N C C NH2 C H N H 3 H Adenine CH2 N Guanine 5′ O– 1′ 4′ 3′ 2′ OH in RNA OH Sugar O NH2 O H in DNA Pyrimidines –O 5 O NH2 6 Purines 7N H C H C C N N C O H Cytosine (both DNA and RNA) H3C C H C C N O N H H C C O H C H Thymine (DNA only) C N N H C O H Uracil (RNA only) 13 • 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 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5′ PO4 Base CH2 O C O Phosphodiester bond –O O P O Base CH2 O OH 3′ 14 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 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 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. Courtesy of Cold Spring Harbor Laboratory Archives 16 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 17 Double helix Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´ Phosphate group P • 2 strands are polymers of nucleotides • Phosphodiester backbone – repeating sugar and phosphate units joined by phosphodiester bonds • Wrap around 1 axis • Antiparallel 5 O 1 4 3 Phosphodiester bond 2 P 5 O 4 1 3 P 2 5 O 1 4 5-carbon sugar 3 2 Nitrogenous base P 5 O 1 4 3 2 OH 3 18 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2nm 5′ A T G Minor groove 3′ 3.4nm C T G A T 0.34nm C G Major groove G A G T C Major groove Minor groove 19 3′ 5′ Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Hydrogen bond • Complementarity of bases • A forms 2 hydrogen bonds with T • G forms 3 hydrogen bonds with C • Gives consistent diameter O N H N G N H H H N N H N Sugar H C N N H Sugar H Hydrogen bond H N H N N Sugar A N CH3 O H H N H T N N H Sugar 20 DNA Replication 3 possible models 1. Conservative model 2. Semiconservative model 3. Dispersive model 21 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Conservative 22 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Conservative Semiconservative 23 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Conservative Semiconservative Dispersive 24 Meselson and Stahl – 1958 • Bacterial cells were grown in a heavy isotope of nitrogen, 15N • All the DNA incorporated 15N • Cells were switched to media containing lighter 14N • DNA was extracted from the cells at various time intervals 25 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 – 1st round results consistent – 2nd round – did not observe 1 band 26 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA E. coli 15N medium 14N medium 0 min 0 rounds E. coli cells grown in 15N medium Cells shifted to 14N medium and allowed to grow 20 min 1 round 40 min 2 rounds Samples taken at three time points and suspended in cesium chloride solution Samples are centrifuged 0 rounds 1 round 2 rounds 0 1 2 27 Top Bottom Rounds of replication From M. Meselson and F.W. Stahl/PNAS 44(1958):671 DNA Replication • Requires 3 things – Something to copy • Parental DNA molecule – Something to do the copying • Enzymes – Building blocks to make copy • Nucleotide triphosphates 28 • DNA replication includes – Initiation – replication begins – Elongation – new strands of DNA are synthesized by DNA polymerase – Termination – replication is terminated 29 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Template Strand HO New Strand 3′ Template Strand HO 5′ G 5′ P C G O O Sugar– phosphate backbone 3′ P C New Strand O O P P P T A P T A O O O O P P P A DNA polymerase III O T O P A P P P C O G P C O G O O P P 3′ P OH A A O P T P P O P O T A OH O OH P P Pyrophosphate A O 5′ P P O 3′ P O T O 5′ 30 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5 3 5 RNA polymerase makes primer 5 3 3 5 DNA polymerase extends primer • 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 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 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Replisome Origin Termination Termination Replisome Origin Termination Origin Termination Termination Origin Origin 33 • 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 34 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Supercoiling Replisomes No Supercoiling Replisomes DNA gyrase • 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 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 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5′ 3′ Lagging strand (discontinuous) First RNA primer 5′ 3′ Open helix and replicate further Open helix and replicate Second RNA primer 5′ 3′ 3′ 3′ 5′ 5′ RNA primer 5′ 3′ Leading strand (continuous) RNA primer 5′ 3′ 37 • Partial opening of helix forms replication fork • DNA primase – RNA polymerase that makes RNA primer – RNA will be removed and replaced with DNA 38 Leading-strand synthesis – Single priming event – Strand extended by DNA pol III • Processivity – subunit forms “sliding clamp” to keep it attached Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. a. b. a-b: From Biochemistry by Stryer. © 1975, 1981, 1988, 1995 by Lupert Stryer. Used with permission of W.H. Freeman and Company 39 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 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5′ DNA ligase Lagging strand (discontinuous) RNA primer DNA polymerase I Okazaki fragment made by DNA polymerase III Primase Leading strand (continuous) 3′ 41 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 Replication fork Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. New bases β clamp (sliding clamp) Leading strand 3 5 Single-strand binding proteins (SSB) Clamp loader DNA gyrase Open β clamp 5 3 Parent DNA Helicase Primase DNA polymerase III Lagging strand Okazaki fragment New bases 3 5 DNA polymerase I DNA ligase RNA primer 43 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA polymerase III Leading strand Helicase DNA gyrase 5´ 3´ 5´ 3´ Clamp loader 5´ 3´ 5´ 3´ First Okazaki fragment RNA primer RNA primer Primase Single-strand binding proteins (SSB) β clamp 5´ 3´ Lagging strand Loop grows Second Okazaki fragment nears completion 5´ 3´ RNA primer 1. A DNA polymerase III enzyme is active on each strand. Primase synthesizes new primers for the lagging strand. 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´ DNA polymerase III DNA polymerase I Lagging strand releases 5´ 3´ β clamp releases 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. 44 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´ 3´ Clamp loader 5´ 3´ 5´ 3´ DNA ligase patches “nick” Leading strand replicates continuously 5´ 3´ DNA polymerase I detaches after removing RNA primer 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. 5´ 3´ Loop grows New bases 5´ 3´ 5. After the β clamp is loaded, the DNA polymerase III on the lagging strand adds bases to the next Okazaki fragment. 45 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 46 • 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) 47 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 48 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Replication first round 5´ 3´ 3´ 5´ Leading strand (no problem) Lagging strand (problem at the end) 5´ 3´ 3´ 5´ Last primer Origin Leading strand Primer removal 3´ 5´ 5´ Lagging strand 3´ Replication second round Removed primer cannot be replaced 5´ 3´ 3´ 5´ 5´ 3´ 3´ 5´ Shortened template 49 • 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 50 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5́ T T G 3́ Synthesis by telomerase Telomerase 5́ 3́ T A T A G C G G C Telomere extended by telomerase G C C A A C Template RNA is part of enzyme Telomerase moves and continues to extend telomere 5́ 3́ T T G G G Now ready to synthesize next repeat G T T G A A C C C C A A C 51 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 52 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 53 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 54 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA with adjacent thymines T A T A UV light Helix distorted by thymine dimer Thymine dimer A A Photolyase binds to damaged DNA Photolyase A A Visible light Thymine dimer cleaved T A T A 55 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 56 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Damaged or incorrect base Excision repair enzymes recognize damaged DNA Uvr A,B,C complex binds damaged DNA Excision of damaged strand Resynthesis by DNA polymerase DNA polymerase 57