DNA: DNA: The Carrier of Genetic The Carrier of Genetic

InformationInformation

Chapter 12 Chapter 12

Learning Objective 1Learning Objective 1

• What evidence was accumulated during What evidence was accumulated during the 1940s and early 1950s demonstrating the 1940s and early 1950s demonstrating that DNA is the genetic material?that DNA is the genetic material?

The Mystery of GenesThe Mystery of Genes

• Many early geneticists thought genes were Many early geneticists thought genes were proteinsproteins• Proteins are complex and variableProteins are complex and variable• Nucleic acids are simple moleculesNucleic acids are simple molecules

Evidence for DNAEvidence for DNA

• DNA (deoxyribonucleic acid)DNA (deoxyribonucleic acid)• TransformationTransformation experiments experiments

• DNA of one strain of bacteria can transfer DNA of one strain of bacteria can transfer genetic characteristics to related bacteriagenetic characteristics to related bacteria

Bacteriophage ExperimentsBacteriophage Experiments

• BacteriophageBacteriophage (virus) infects bacterium (virus) infects bacterium• only DNA from virus enters the cellonly DNA from virus enters the cell• virus reproduces and forms new viral particles virus reproduces and forms new viral particles

from DNA alonefrom DNA alone

KEY CONCEPTSKEY CONCEPTS

• Beginning in the 1920s, evidence began to Beginning in the 1920s, evidence began to accumulate that DNA is the hereditary accumulate that DNA is the hereditary materialmaterial

Learning Objective 2Learning Objective 2

• What questions did these classic What questions did these classic experiments address? experiments address? • Griffith’s transformation experimentGriffith’s transformation experiment• Avery’s contribution to Griffith’s workAvery’s contribution to Griffith’s work• Hershey–Chase experimentsHershey–Chase experiments

Griffith’s Transformation ExperimentGriffith’s Transformation Experiment

• Can a genetic trait be transmitted from one Can a genetic trait be transmitted from one bacterial strain to another? bacterial strain to another?

• Answer: YesAnswer: Yes

Griffith’s Transformation ExperimentGriffith’s Transformation Experiment

Fig. 12-1, p. 261

Experiment 1 Experiment 2 Experiment 3 Experiment 4

R cells injected

S cells injected

Heat-killed S cells injected

R cells and heat-killed S cells injected

Mouse lives Mouse dies Mouse lives Mouse dies

Animation: Griffith’s ExperimentAnimation: Griffith’s Experiment

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Avery’s ExperimentsAvery’s Experiments

• What molecule is responsible for bacterial What molecule is responsible for bacterial transformation? transformation?

• Answer: DNAAnswer: DNA

Hershey–Chase ExperimentsHershey–Chase Experiments

• Is DNA or protein the genetic material in Is DNA or protein the genetic material in bacterial viruses (phages)? bacterial viruses (phages)?

• Answer: DNAAnswer: DNA

Hershey–Chase Hershey–Chase ExperimentsExperiments

Fig. 12-2, p. 262

35S

Bacterial viruses grown in 35S to

label protein coat or 32P to label DNA

32 P

Viruses infect bacteria

1

2

Fig. 12-2, p. 262

Agitate cells in blender

Agitate cells in blender

Separate by centrifugation

Separate by centrifugation

35S32 P

Bacteria in pellet contain 32P-labeled DNA

35S-labeled protein in supernatant

3

4

5

Fig. 12-2, p. 262

Viral reproduction inside bacterial cells

from pellet

7

32PCell lysis

6

5

6

7

Learning Objective 3Learning Objective 3

• How do How do nucleotidenucleotide subunits link to form a subunits link to form a single DNA strand?single DNA strand?

Watson and CrickWatson and Crick

• DNA ModelDNA Model • DemonstratedDemonstrated

• how information is stored in molecule’s how information is stored in molecule’s structurestructure

• how DNA molecules are how DNA molecules are templatestemplates for their for their own replication own replication

NucleotidesNucleotides

• DNA is a polymer of DNA is a polymer of nucleotides nucleotides • Each nucleotide subunit containsEach nucleotide subunit contains

• a nitrogenous basea nitrogenous base• purinespurines ( (adenineadenine or or guanineguanine))• pyrimidinespyrimidines ( (thyminethymine or or cytosinecytosine) )

• a pentose sugar (a pentose sugar (deoxyribosedeoxyribose))• a phosphate groupa phosphate group

Forming DNA ChainsForming DNA Chains

• BackboneBackbone• alternating sugar and phosphate groupsalternating sugar and phosphate groups• joined by covalent joined by covalent phosphodiester linkagesphosphodiester linkages

• Phosphate group attaches toPhosphate group attaches to• 55′′ carbon of one deoxyribose carbon of one deoxyribose• 33′′ carbon of the next deoxyribose carbon of the next deoxyribose

DNA DNA NucleotidesNucleotides

Fig. 12-3, p. 264

Thymine

Adenine Nucleotide

CytosinePhosphate group

Phosphodiester linkage

Guanine

Deoxyribose (sugar)

Animation: Subunits of DNAAnimation: Subunits of DNA

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KEY CONCEPTSKEY CONCEPTS

• The DNA building blocks consist of four The DNA building blocks consist of four nucleotide subunits: T, C, A, and Gnucleotide subunits: T, C, A, and G

Learning Objective 4Learning Objective 4

• How are the two strands of DNA oriented How are the two strands of DNA oriented with respect to each other?with respect to each other?

DNA MoleculeDNA Molecule

• 2 polynucleotide chains2 polynucleotide chains• associated as associated as double helixdouble helix

DNA MoleculeDNA Molecule

Fig. 12-5, p. 266

Sugar–phosphate backbone

Minor groove

3.4 nm Major groove

0.34 nm

2.0 nm

= hydrogen= oxygen

= carbon

= atoms in base pairs = phosphorus

Double HelixDouble Helix

• AntiparallelAntiparallel • chains run in opposite directionschains run in opposite directions

• 55′′ end end • phosphate attached to 5phosphate attached to 5′′ deoxyribose carbon deoxyribose carbon

• 33′′ end end • hydroxyl attached to 3hydroxyl attached to 3′′ deoxyribose carbon deoxyribose carbon

KEY CONCEPTSKEY CONCEPTS

• The DNA molecule consists of two strands The DNA molecule consists of two strands that wrap around each other to form a that wrap around each other to form a double helix double helix

• The order of its building blocks stores The order of its building blocks stores genetic informationgenetic information

Animation: DNA Close UpAnimation: DNA Close Up

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Learning Objective 5Learning Objective 5

• What are the base-pairing rules for DNA?What are the base-pairing rules for DNA?• How do complementary bases bind to How do complementary bases bind to

each other?each other?

Base PairsBase Pairs

• Hydrogen bondingHydrogen bonding• between specific base pairsbetween specific base pairs• binds two chains of helix binds two chains of helix

• Adenine (Adenine (AA) with thymine () with thymine (TT))• forms two hydrogen bondsforms two hydrogen bonds

• Guanine (Guanine (GG) with cytosine () with cytosine (CC))• forms three hydrogen bondsforms three hydrogen bonds

Base Pairs and Hydrogen BondsBase Pairs and Hydrogen Bonds

Fig. 12-6a, p. 267

Fig. 12-6b, p. 267

Adenine Thymine

Deoxyribose Deoxyribose

Guanine Cytosine

Deoxyribose Deoxyribose

Chargaff’s RulesChargaff’s Rules

• Complementary base pairingComplementary base pairing • between A and T; G and Cbetween A and T; G and C• therefore A = T; G = Ctherefore A = T; G = C

• If base sequence of 1 strand is knownIf base sequence of 1 strand is known• base sequence of other strand can be base sequence of other strand can be

predictedpredicted

KEY CONCEPTSKEY CONCEPTS

• Nucleotide subunits pair, based on precise Nucleotide subunits pair, based on precise pairing rules: T pairs with A, and C pairs pairing rules: T pairs with A, and C pairs with G with G

• Hydrogen bonding between base pairs Hydrogen bonding between base pairs holds two strands of DNA togetherholds two strands of DNA together

Learning Objective 6Learning Objective 6

• What evidence from Meselson and Stahl’s What evidence from Meselson and Stahl’s experiment enabled scientists to experiment enabled scientists to differentiate between differentiate between semiconservative semiconservative replicationreplication of DNA and alternative models? of DNA and alternative models?

Models of DNA Models of DNA ReplicationReplication

Fig. 12-7a, p. 268

(a) Hypothesis 1: Semiconservative replication

Parental DNA First generation Second generation

Fig. 12-7b, p. 268

(b) Hypothesis 2: Conservative replication

Parental DNA First generation Second generation

Fig. 12-7c, p. 268

(c) Hypothesis 3: Dispersive replication

Parental DNA First generation Second generation

Meselson-StahlMeselson-Stahl ExperimentExperiment

• E. coli E. coli • grown in medium containing heavy nitrogen grown in medium containing heavy nitrogen

((1515N)N)• incorporated incorporated 1515N into DNAN into DNA

• Transferred from Transferred from 1515N to N to 1414N mediumN medium• after one or two generations, DNA density after one or two generations, DNA density

supported supported semiconservative replication semiconservative replication

Meselson-StahlMeselson-Stahl ExperimentExperiment

Fig. 12-8a, p. 269

Bacteria are grown in 15N (heavy) medium. All

DNA is heavy.

Some cells are transferred to

14N (light) medium.

Some cells continue to grow in 14N medium.

First generation Second generation

Cesium chloride (CsCl)

High density

Low density

DNA

DNA is mixed with CsCl solution, placed in an ultracentrifuge, and centrifuged at very high speed for about 48 hours. 14N (light)

DNA

14N – 15N hybrid DNA

15N (heavy) DNA

DNA molecules move to positions where their density equals that of the CsCl solution.

The greater concentration of CsCl at the bottom of the tube is due to sedimentation under centrifigal force.

Fig. 12-8b, p. 269

14N (light) DNA

14N – 15N hybrid DNA

14N – 15N hybrid DNA

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.

Semiconservative ReplicationSemiconservative Replication

• Each daughter double helix consists ofEach daughter double helix consists of• 1 original strand from parent molecule1 original strand from parent molecule• 1 new complementary strand1 new complementary strand

Learning Objective 7Learning Objective 7

• How does DNA replicate?How does DNA replicate?• What are some unique features of the What are some unique features of the

process?process?

DNA ReplicationDNA Replication

• 2 strands of double helix unwind2 strands of double helix unwind• each is template for complementary strandeach is template for complementary strand

• Replication is initiatedReplication is initiated• DNA primaseDNA primase synthesizes synthesizes RNA primerRNA primer

• DNA strandDNA strand growsgrows• DNA polymeraseDNA polymerase adds nucleotide subunits adds nucleotide subunits

DNA ReplicationDNA Replication

Fig. 12-10, p. 271

Nucleotide joined to growing chain by DNA polymerase

Phosphates released

Base

Other EnzymesOther Enzymes

• DNA helicasesDNA helicases• open the double helixopen the double helix

• TopoisomerasesTopoisomerases • prevent tangling and knottingprevent tangling and knotting

KEY CONCEPTSKEY CONCEPTS

• DNA replication results in two identical DNA replication results in two identical double-stranded DNA moleculesdouble-stranded DNA molecules• molecular mechanism passes genetic molecular mechanism passes genetic

information from one generation to the nextinformation from one generation to the next

Learning Objective 8Learning Objective 8

• What makes What makes DNA replicationDNA replication (a) (a) bidirectional and (b) continuous in one bidirectional and (b) continuous in one strand and discontinuous in the other?strand and discontinuous in the other?

Bidirectional ReplicationBidirectional Replication

• Starting at Starting at origin of replicationorigin of replication• proceeding in both directionsproceeding in both directions

• Eukaryotic chromosomeEukaryotic chromosome• may have multiple origins of replicationmay have multiple origins of replication• may replicate at many points at same timemay replicate at many points at same time

Bidirectional ReplicationBidirectional Replication

Fig. 12-11a, p. 272

DNA polymerase

Origin of replication on DNA molecule

3’

5’3’

5’

Fig. 12-11b, p. 272

Twist introduced into the helix by unwinding

Single-strand binding proteinsRNA primer

DNA polymerase

DNA helicase

RNA primer

Direction of replication

3’

5’

3’

5’3’3’

Fig. 12-11c, p. 272

3’

5’

3’

5’

3’

5’

3’

5’

DNA SynthesisDNA Synthesis

• Always proceeds in 5Always proceeds in 5′′ →→ 3 3′′ direction direction• Leading strandLeading strand

• synthesized continuouslysynthesized continuously

• Lagging strandLagging strand• synthesized discontinuouslysynthesized discontinuously• forms short forms short Okazaki fragmentsOkazaki fragments• DNA primaseDNA primase synthesizes RNA primers synthesizes RNA primers• DNA ligaseDNA ligase links Okazaki fragments links Okazaki fragments

DNA SynthesisDNA Synthesis

Fig. 12-12a, p. 273

DNA helixRNA primer

Leading strand

DNA polymerase

Lagging strand (first Okazaki fragment)

Direction of replication

Replication fork

3’

5’

3’

5’

3’5’

3’5’

Fig. 12-12b, p. 273

Leading strand

RNA primers

Two Okazaki fragments

3’5’

3’5’

5’3’

3’

5’3’

5’

Fig. 12-12c, p. 273

Leading strand

DNA ligase Third Okazaki fragment

Lagging strand

3’

5’

3’5’

3’

5’3’5’

3’5’

Replication in Bacteria and Replication in Bacteria and EukaryotesEukaryotes

Fig. 12-13a, p. 274

Template DNA (light blue)

New DNA (dark blue)

3’5’

3’

5’

Fig. 12-13b, p. 274

340 nm

Fig. 12-13c, p. 274

Replication “bubbles”

Single replication bubble formed from two merged bubbles

Replication fork

3’

5’

3’5’

Animation: Overview of DNA Animation: Overview of DNA replication and base pairingreplication and base pairing

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Learning Objective 9Learning Objective 9

• How do How do enzymesenzymes proofread and repair proofread and repair errors in DNA?errors in DNA?

DNA PolymerasesDNA Polymerases

• Proofread each new nucleotideProofread each new nucleotide• against template nucleotide against template nucleotide

• Find errors in base pairing Find errors in base pairing • remove incorrect nucleotideremove incorrect nucleotide• insert correct oneinsert correct one

DNA MutationDNA Mutation

Fig. 12-9, p. 270

Mutation

Stepped Art

Fig. 12-9, p. 270

Mutation

Mismatch RepairMismatch Repair

• Enzymes recognize incorrectly paired Enzymes recognize incorrectly paired nucleotides and remove themnucleotides and remove them

• DNA polymerases fill in missing DNA polymerases fill in missing nucleotidesnucleotides

Nucleotide Excision RepairNucleotide Excision Repair

• Repairs DNA lesionsRepairs DNA lesions• caused by sun or harmful chemicalscaused by sun or harmful chemicals

• 3 enzymes3 enzymes• nucleasenuclease cuts out damaged DNA cuts out damaged DNA• DNA polymeraseDNA polymerase adds correct nucleotides adds correct nucleotides• DNA ligaseDNA ligase closes breaks in sugar–phosphate closes breaks in sugar–phosphate

backbonebackbone

Nucleotide Excision RepairNucleotide Excision Repair

Fig. 12-14, p. 275

Nuclease enzyme bound to DNA DNA lesion

DNA polymeraseDNA ligase

New DNA

3’ 5’

3’ 5’

3’ 5’

3’ 5’

3’ 5’

3’ 5’

Learning Objective 10Learning Objective 10

• What is a What is a telomeretelomere?? • What are the possible connections What are the possible connections

between between telomerasetelomerase and cell aging, and and cell aging, and between telomerase and cancer? between telomerase and cancer?

TelomeresTelomeres

• Eukaryotic chromosome endsEukaryotic chromosome ends• noncoding, repetitive DNA sequences noncoding, repetitive DNA sequences

• Shorten slightly with each cell cycleShorten slightly with each cell cycle• Can be extended by Can be extended by telomerasetelomerase

Replication at TelomeresReplication at Telomeres

Fig. 12-15a, p. 276

DNA replication

RNA primerRNA primer

Removal of primer

3’

3’

3’

3’

3’

3’

3’

3’

3’

3’

5’

5’

5’

5’

5’

5’

5’

5’

5’

5’

+

+

Fig. 12-15b, p. 276

3’

5’

Cell AgingCell Aging

• May be caused by absence of telomerase May be caused by absence of telomerase activity activity

• Cells lose ability to divideCells lose ability to divide• after a limited number of cell divisionsafter a limited number of cell divisions

Cancer CellsCancer Cells

• Have Have telomerasetelomerase• to maintain telomere length and possibly to maintain telomere length and possibly

resist apoptosisresist apoptosis

• Including human cancersIncluding human cancers• breast, lung, colon, prostate gland, pancreasbreast, lung, colon, prostate gland, pancreas