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DNA: The Carrier of Genetic Information Chapter 12 Learning Objective 1 • What evidence was accumulated during the 1940s and early 1950s demonstrating that DNA is the genetic material? The Mystery of Genes • Many early geneticists thought genes were proteins • • Proteins are complex and variable Nucleic acids are simple molecules Evidence for DNA • • DNA (deoxyribonucleic acid) Transformation experiments • DNA of one strain of bacteria can transfer genetic characteristics to related bacteria Bacteriophage Experiments • Bacteriophage (virus) infects bacterium • • only DNA from virus enters the cell virus reproduces and forms new viral particles from DNA alone KEY CONCEPTS • Beginning in the 1920s, evidence began to accumulate that DNA is the hereditary material Learning Objective 2 • What questions did these classic experiments address? • • • Griffith’s transformation experiment Avery’s contribution to Griffith’s work Hershey–Chase experiments Griffith’s Transformation Experiment • • Can a genetic trait be transmitted from one bacterial strain to another? Answer: Yes Griffith’s Transformation Experiment Experiment 1 Experiment 2 Experiment 3 Experiment 4 R cells injected S cells injected Heat-killed S cells injected R cells and heatkilled S cells injected Mouse lives Mouse dies Mouse lives Mouse dies Fig. 12-1, p. 261 Animation: Griffith’s Experiment CLICK TO PLAY Avery’s Experiments • • What molecule is responsible for bacterial transformation? Answer: DNA Hershey–Chase Experiments • • Is DNA or protein the genetic material in bacterial viruses (phages)? Answer: DNA Hershey–Chase Experiments 35S 1 32 P Bacterial viruses grown in 35S to label protein coat or 32P to label DNA 2 Viruses infect bacteria Fig. 12-2, p. 262 3 Agitate cells in blender Agitate cells in blender 4 Separate by centrifugation Separate by centrifugation 32 P 35S 5 Bacteria in pellet contain 32Plabeled DNA 35S-labeled protein in supernatant Fig. 12-2, p. 262 6 Viral reproduction inside bacterial cells from pellet 7 Cell lysis 32P 5 6 7 Fig. 12-2, p. 262 Learning Objective 3 • How do nucleotide subunits link to form a single DNA strand? Watson and Crick • • DNA Model Demonstrated • • how information is stored in molecule’s structure how DNA molecules are templates for their own replication Nucleotides • • DNA is a polymer of nucleotides Each nucleotide subunit contains • a nitrogenous base • purines (adenine or guanine) • pyrimidines (thymine or cytosine) • • a pentose sugar (deoxyribose) a phosphate group Forming DNA Chains • Backbone • • • alternating sugar and phosphate groups joined by covalent phosphodiester linkages Phosphate group attaches to • • 5′ carbon of one deoxyribose 3′ carbon of the next deoxyribose DNA Nucleotides Thymine Adenine Phosphate group Nucleotide Cytosine Guanine Phosphodiester linkage Deoxyribose (sugar) Fig. 12-3, p. 264 Animation: Subunits of DNA CLICK TO PLAY KEY CONCEPTS • The DNA building blocks consist of four nucleotide subunits: T, C, A, and G Learning Objective 4 • How are the two strands of DNA oriented with respect to each other? DNA Molecule • 2 polynucleotide chains • associated as double helix DNA Molecule Sugar–phosphate backbone Minor groove Major groove 3.4 nm 0.34 nm 2.0 nm = hydrogen = atoms in base pairs = carbon = oxygen = phosphorus Fig. 12-5, p. 266 Double Helix • Antiparallel • • 5′ end • • chains run in opposite directions phosphate attached to 5′ deoxyribose carbon 3′ end • hydroxyl attached to 3′ deoxyribose carbon KEY CONCEPTS • • The DNA molecule consists of two strands that wrap around each other to form a double helix The order of its building blocks stores genetic information Animation: DNA Close Up CLICK TO PLAY Learning Objective 5 • • What are the base-pairing rules for DNA? How do complementary bases bind to each other? Base Pairs • Hydrogen bonding • • • Adenine (A) with thymine (T) • • between specific base pairs binds two chains of helix forms two hydrogen bonds Guanine (G) with cytosine (C) • forms three hydrogen bonds Base Pairs and Hydrogen Bonds Fig. 12-6a, p. 267 Adenine Deoxyribose Guanine Deoxyribose Thymine Deoxyribose Cytosine Deoxyribose Fig. 12-6b, p. 267 Chargaff’s Rules • Complementary base pairing • • • between A and T; G and C therefore A = T; G = C If base sequence of 1 strand is known • base sequence of other strand can be predicted KEY CONCEPTS • • Nucleotide subunits pair, based on precise pairing rules: T pairs with A, and C pairs with G Hydrogen bonding between base pairs holds two strands of DNA together Learning Objective 6 • What evidence from Meselson and Stahl’s experiment enabled scientists to differentiate between semiconservative replication of DNA and alternative models? Models of DNA Replication (a) Hypothesis 1: Semiconservative replication Parental DNA First generation Second generation Fig. 12-7a, p. 268 (b) Hypothesis 2: Conservative replication Parental DNA First generation Second generation Fig. 12-7b, p. 268 (c) Hypothesis 3: Dispersive replication Parental DNA First generation Second generation Fig. 12-7c, p. 268 Meselson-Stahl Experiment • E. coli • • • grown in medium containing heavy nitrogen (15N) incorporated 15N into DNA Transferred from 15N to 14N medium • after one or two generations, DNA density supported semiconservative replication Meselson-Stahl Experiment Bacteria are grown in (heavy) medium. All DNA is heavy. 15N Some cells are transferred to 14N (light) medium. Some cells continue to grow in 14N medium. First generation Second generation Cesium chloride (CsCl) High Low density density DNA DNA is mixed with CsCl solution, placed in an ultracentrifuge, and centrifuged at very high speed for about 48 hours. The greater concentration of CsCl at the bottom of the tube is due to sedimentation under centrifigal force. 15N (heavy) 14N (light) 14N – 15N DNA DNA hybrid DNA DNA molecules move to positions where their density equals that of the CsCl solution. Fig. 12-8a, p. 269 14N (light) DNA – 15N hybrid DNA 14N – 15N hybrid DNA 14N 15N (heavy) DNA Before transfer to 14N One cell generation after transfer to 14N Two cell generations after transfer to 14N The location of DNA molecules within the centrifuge tube can be determined by UV optics. DNA solutions absorb strongly at 260 nm. Fig. 12-8b, p. 269 Semiconservative Replication • Each daughter double helix consists of • • 1 original strand from parent molecule 1 new complementary strand Learning Objective 7 • • How does DNA replicate? What are some unique features of the process? DNA Replication • 2 strands of double helix unwind • • Replication is initiated • • each is template for complementary strand DNA primase synthesizes RNA primer DNA strand grows • DNA polymerase adds nucleotide subunits DNA Replication Base Nucleotide joined to growing chain by DNA polymerase Phosphates released Fig. 12-10, p. 271 Other Enzymes • DNA helicases • • open the double helix Topoisomerases • prevent tangling and knotting KEY CONCEPTS • DNA replication results in two identical double-stranded DNA molecules • molecular mechanism passes genetic information from one generation to the next Learning Objective 8 • What makes DNA replication (a) bidirectional and (b) continuous in one strand and discontinuous in the other? Bidirectional Replication • Starting at origin of replication • • proceeding in both directions Eukaryotic chromosome • • may have multiple origins of replication may replicate at many points at same time Bidirectional Replication DNA polymerase 3’ 5’ Origin of replication on DNA molecule 3’ 5’ Fig. 12-11a, p. 272 Twist introduced into the helix by unwinding RNA primer Single-strand binding proteins DNA polymerase 3’ 5’ 3’ DNA helicase 3’ 5’ 3’ RNA primer Direction of replication Fig. 12-11b, p. 272 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ Fig. 12-11c, p. 272 DNA Synthesis • • Always proceeds in 5′ → 3′ direction Leading strand • • synthesized continuously Lagging strand • • • • synthesized discontinuously forms short Okazaki fragments DNA primase synthesizes RNA primers DNA ligase links Okazaki fragments DNA Synthesis 3’ 5’ Leading strand DNA helix RNA primer DNA polymerase 3’ 5’ 3’ 5’ 3’ 5’ Replication fork Lagging strand (first Okazaki fragment) Direction of replication Fig. 12-12a, p. 273 3’ 5’ Leading strand 3’ RNA primers 3’ 5’ 5’ 5’3’ 3’ 5’ Two Okazaki fragments Fig. 12-12b, p. 273 3’ 5’ Leading strand 3’ 3’ DNA ligase 5’ 3’ 5’ 5’ Third Okazaki fragment Lagging strand 3’ 5’ Fig. 12-12c, p. 273 Replication in Bacteria and Eukaryotes Template DNA (light blue) New DNA (dark blue) 3’ 5’ 5’ 3’ Fig. 12-13a, p. 274 340 nm Fig. 12-13b, p. 274 5’ Replication “bubbles” 3’ Single replication bubble formed from two merged bubbles 3’ 5’ Replication fork Fig. 12-13c, p. 274 Animation: Overview of DNA replication and base pairing CLICK TO PLAY Learning Objective 9 • How do enzymes proofread and repair errors in DNA? DNA Polymerases • Proofread each new nucleotide • • against template nucleotide Find errors in base pairing • • remove incorrect nucleotide insert correct one DNA Mutation Mutation Fig. 12-9, p. 270 Mutation Stepped Art Fig. 12-9, p. 270 Mismatch Repair • • Enzymes recognize incorrectly paired nucleotides and remove them DNA polymerases fill in missing nucleotides Nucleotide Excision Repair • Repairs DNA lesions • • caused by sun or harmful chemicals 3 enzymes • • • nuclease cuts out damaged DNA DNA polymerase adds correct nucleotides DNA ligase closes breaks in sugar–phosphate backbone Nucleotide Excision Repair Nuclease enzyme bound to DNA DNA lesion 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ DNA polymerase DNA ligase 5’ 3’ New DNA 3’ 5’ Fig. 12-14, p. 275 Learning Objective 10 • • What is a telomere? What are the possible connections between telomerase and cell aging, and between telomerase and cancer? Telomeres • Eukaryotic chromosome ends • • • noncoding, repetitive DNA sequences Shorten slightly with each cell cycle Can be extended by telomerase Replication at Telomeres 5’ 3’ 3’ 5’ DNA replication 5’ 3’ 3’ 5’ + RNA primer RNA primer 5’ 3’ 3’ 5’ Removal of primer 3’ 5’ 3’ 5’ + 5’ 3’ 3’ 5’ Fig. 12-15a, p. 276 3’ 5’ Fig. 12-15b, p. 276 Cell Aging • • May be caused by absence of telomerase activity Cells lose ability to divide • after a limited number of cell divisions Cancer Cells • Have telomerase • • to maintain telomere length and possibly resist apoptosis Including human cancers • breast, lung, colon, prostate gland, pancreas