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DNA Structure, Replication, and DNA Structure, Replication, and RecombinationRecombination

Chapter 6

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Sections to studySections to study

6.1 Experimental evidence for DNA as the genetic 6.1 Experimental evidence for DNA as the genetic materialmaterial

6.2 The Watson and Crick double helix model of DNA6.2 The Watson and Crick double helix model of DNA6.3 Genetic information in DNA base sequence6.3 Genetic information in DNA base sequence6.4 DNA replication6.4 DNA replication6.5 Recombination at the DNA level6.5 Recombination at the DNA level

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Storage of informationStorage of information Expression of information Expression of information ReplicationReplication Variation by mutationVariation by mutation

The genetic material must exhibit four major The genetic material must exhibit four major characteristicscharacteristics

Genes mutation Alleles new genes

Raw materialsfor evolutionTraits

Expression

Replication

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What are genes composed of ?What are genes composed of ? Protein ?Protein ?

20 different subunits 20 different subunits –– greater greater potential variety of combinationspotential variety of combinations

Chromosomes contain more Chromosomes contain more protein than DNA by weight.protein than DNA by weight.

DNA ?DNA ? Only four different subunits make Only four different subunits make

up DNA.up DNA. Chromosomes contain less DNA Chromosomes contain less DNA

than protein by weight.than protein by weight.

Other molecules ? Other molecules ?

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Proteins are abundant in the chromosomes.Proteins are abundant in the chromosomes. Proteins are well characterized chemically.Proteins are well characterized chemically.

6.1 Experimental evidence for DNA as6.1 Experimental evidence for DNA asthe genetic materialthe genetic material

Chemical staining Chemical staining revealed that revealed that DNADNA is is localized almost localized almost exclusively in the exclusively in the chromosomes.chromosomes.

Until 1944, observations favored protein as the genetic material.

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1869 1869 –– Friedrich Friedrich MiescherMiescherextracted a weakly acidic, extracted a weakly acidic, phosphorousphosphorous--rich rich material from nuclei of material from nuclei of human white blood cells human white blood cells which he named which he named nucleinnuclein..

Discovery of DNA:Discovery of DNA:

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DNADNA –– ddeoxyriboeoxyribonnucleic ucleic aacidcid Four nucleotide subunits Four nucleotide subunits

linked together by linked together by phosphodiesterphosphodiester bonds.bonds.

Nitrogenous Base

Fig. 6.2

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1928 1928 –– Frederick GriffithFrederick Griffith published his experiment with smooth published his experiment with smooth (S), virulent strain (S), virulent strain Streptococcus Streptococcus pneumoniaepneumoniae, and rough (R), , and rough (R), nonvirulentnonvirulent strain.strain.

Polysaccharidecapsule

Fig. 6.3

Transformation studies:

Evidence favoring DNA as the genetic material was first Evidence favoring DNA as the genetic material was first obtained during the study of bacteria and obtained during the study of bacteria and bacteriophagesbacteriophages

6-9Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or displayFig. 6.4

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Genetic information from dead bacterial cells could be transmitted to live cells.

Transformation – One of the mechanisms that bacteria transfer genes from one strain to another.

Transformation occurred in the animal body.

Conclusion from Griffith’s experiment:

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1931 1931 –– Oswald T. AveryOswald T. Avery found that bacterial found that bacterial transformation can occur in the culture medium transformation can occur in the culture medium without using animals.without using animals.

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Fig. 6.5 b

Purification of the “transforming principle”

Purification

Transforming principle

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Fig. 6.5 c

1944 1944 –– Oswald T. AveryOswald T. Avery, , Colin MacLeodColin MacLeod, and , and MaclynMaclynMcCartyMcCarty found that the transforming principle is DNA, found that the transforming principle is DNA, but not proteins, but not proteins, RNAsRNAs, lipids or polysaccharides., lipids or polysaccharides.

Transformingprinciple

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1952 1952 –– Alfred HersheyAlfred Hershey and and Martha ChaseMartha Chase tested tested whether the injected whether the injected material is composed of material is composed of protein or DNA.protein or DNA.

WaringWaring blender blender experimentexperiment using T2 using T2 bacteriophagebacteriophage and and bacteria.bacteria.

The HersheyThe Hershey--Chase experimentChase experiment

Martha Chase(1927-2003)

Alfred Hershey(1908-1997)

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Fig. 6.6

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Radioactive labels Radioactive labels 3232PP for DNA and for DNA and 3535S S for protein.for protein.

Fig. 6.7

6-17Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or displayFig. 6.7

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Other evidence that supports DNA asOther evidence that supports DNA asthe genetic materialthe genetic material

1. DNA exclusively localized in the chromosomes.2. The amount of DNA and the number of sets of

chromosomes are closely correlated.

OrganismOrganism nn ((picogramspicograms)) 22nn ((picogramspicograms))HumanHuman 3.253.25 7.307.30ChickenChicken 1.261.26 2.492.49TroutTrout 2.672.67 5.795.79CarpCarp 1.651.65 3.493.49ShadShad 0.910.91 1.971.97

SpermSperm Nucleated Nucleated precursors to red precursors to red blood cellsblood cells

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UV light is most mutagenic at 260 nm wavelength.

Action spectrum

240 250 260 270 280 290UV wavelength (-nm)

Mut

atio

n fr

eque

ncy

3. DNA, but not protein, absorbs UV light more strongly at 260 nm, the wavelength that has the strongest mutagenic effect.

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DNA absorb UV light most strongly at 260 nm but protein at 280 nm.

Absorption spectrum

240 250 260 270 280 290UV wavelength (-nm)

Abs

orpt

ion

ProteinNucleic acid

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6.2 The Watson and Crick double helix6.2 The Watson and Crick double helixmodel of DNAmodel of DNA

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X ray crystallography

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In 1938-1947, William Astbury detected a periodicity of 3.4 Åwithin DNA. The bases may stack on top of each other.

Rosalind Franklin（1920-1958）

Maurice Wilkins(1916-2004)

William Astbury(1898-1961)

In 1950-1953, Rosalind Franklin, who worked in Maurice Wilkins’s lab then, obtained improved X-ray data.

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According to the X-ray photograph of DNA,

The repeating units along the axis of helix is 3.4 Å.

DNA appears to be a helical structure

Helix undergoes one complete turn every 34 Å.

The diameter of the helix is 20 Å, roughly twice the width of a nucleotide.

Franklin R and Gosling RG (1953) Molecular Molecular configuration in sodium configuration in sodium thymonucleatethymonucleate..Nature 171:740-741

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James Watson and Francis CrickJames Watson and Francis Crick

Watson (25 yr old) and Crick (37 yr old) with their DNA model at the Cavendish Laboratories in 1953

Fig. 6.11 a

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Structurally, Structurally, purinespurines (A and G) pair best with (A and G) pair best with pyrimidinespyrimidines (T and C)(T and C)

Thus, A pairs with T and G pairs with C, also Thus, A pairs with T and G pairs with C, also explaining explaining ChargaffChargaff’’ss ratiosratios

Fig. 6.11 d

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ChargaffChargaff’’ss ratioratio

Erwin Chargaff(1905-2002)

In 1949In 1949--1953,1953, Erwin Erwin ChargaffChargaffmeasured the amount of A, G, T, measured the amount of A, G, T, and C in DNA samples from and C in DNA samples from various organisms.various organisms.

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Zamenhof S and Chargaff E (1950) Dissymmetry in nucleotide sequence of deoxypentose nucleic acids. J. Biol. Chem. 187:1-14

Conclusions: A = T, G = C

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Complementary base pairing by formation of Complementary base pairing by formation of hydrogen bonds explain hydrogen bonds explain ChargaffChargaff’’ss ratiosratios

Fig. 6.10

Watson JD and Crick FHC Watson JD and Crick FHC (1953) (1953) GeneticalGenetical implications implications of the structure of of the structure of deoxyribonucleic acid. deoxyribonucleic acid. NatureNature171:964171:964--967.967.

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The double helix The double helix model of DNA model of DNA structure proposed structure proposed by by James WatsonJames Watsonand and Francis CrickFrancis Crick

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DNA is double helix.DNA is double helix.

Strands are Strands are antiparallelantiparallel with with a sugara sugar--phosphate backbone phosphate backbone on outside and pairs of bases on outside and pairs of bases in the middle.in the middle.

Two strands wrap around Two strands wrap around each other every 30 each other every 30 ÅÅ, once , once every 10 base pairs.every 10 base pairs.

Two chains are held together Two chains are held together by hydrogen bonds between by hydrogen bonds between AA--T and GT and G--C base pairs.C base pairs.

Fig.

6.1

1 b

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The structure of DNA was proposed in 1953The structure of DNA was proposed in 1953

1.1. Watson JDWatson JD and and Crick FHCCrick FHC (1953, April 25) Molecular (1953, April 25) Molecular structure of nucleic acids. structure of nucleic acids. NatureNature 171:737171:737--738.738.

2.2. Wilkins MHFWilkins MHF, Stokes AR, and Wilson HR (1953, , Stokes AR, and Wilson HR (1953, April 25) Molecular structure of April 25) Molecular structure of deoxypentosedeoxypentose nucleic nucleic acids. acids. NatureNature 171:738171:738--740.740.

3.3. Franklin REFranklin RE and Gosling RG (1953, April 25) and Gosling RG (1953, April 25) Molecular configuration in sodium Molecular configuration in sodium thymonucleatethymonucleate. . NatureNature 171:740171:740--741.741.

4.4. Watson JDWatson JD and and Crick FHCCrick FHC (1953, May 30) (1953, May 30) GeneticalGeneticalimplications of the structure of deoxyribonucleic acid. implications of the structure of deoxyribonucleic acid. NatureNature 171:964171:964--967.967.

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Maurice Wilkins(1916-2004)

James Watson Francis Crick(1928- ) (1916-2004)

The 1962 Nobel Prize in Physiology or Medicine

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DNA double helix may assume alternative formsDNA double helix may assume alternative forms

AA BB ZZhelixhelix RightRight--handedhanded RightRight--handedhanded LeftLeft--handedhanded

diameterdiameter ~23~23ǺǺ ~20 ~20 ǺǺ ~18 ~18 ǺǺ

bpbp/turn/turn 1111 10.510.5 1212

conditioncondition HighHigh--saltsalt LowLow--saltsalt CGCGCGCGCGCG

Pitch

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Some DNA molecules are circular instead of linear.Some DNA molecules are circular instead of linear. ProkaryotesProkaryotes MitochondriaMitochondria ChloroplastsChloroplasts VirusesViruses

Some viruses carry single-stranded DNA. Bacteriophages

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6.4 DNA Replication

DNA replicationDNA replication: Copying genetic information for : Copying genetic information for transmission to the next generationtransmission to the next generation

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Three Three possible possible models of models of DNA DNA replicationreplication

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SemiconservativeSemiconservative replicationreplication Double helix unwinds.Double helix unwinds. Each strand acts as template.Each strand acts as template. Complementary base pairing Complementary base pairing

ensures that T signals addition of ensures that T signals addition of A on new strand, and G signals A on new strand, and G signals addition of C.addition of C.

Two daughter helices produced Two daughter helices produced after replication.after replication.

The model of DNA replication postulatedby Watson and Crick

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Experimental proof of Experimental proof of semiconservativesemiconservative replicationreplication

1958 – Matthew Meselson and Franklin Stahl’s experiment

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The mechanism of DNA replicationThe mechanism of DNA replication

Arthur Arthur KornbergKornberg, a Nobel prize winner , a Nobel prize winner and other biochemists deduced steps of and other biochemists deduced steps of DNA replication.DNA replication. InitiationInitiation

Proteins bind to DNA and open up double Proteins bind to DNA and open up double helix.helix.

Prepare DNA for complementary base Prepare DNA for complementary base pairing.pairing.

ElongationElongation Proteins connect the correct sequences of Proteins connect the correct sequences of

nucleotides into a continuous new strand of nucleotides into a continuous new strand of DNA.DNA.

1959 Nobel Prize in Medicine or Physiology

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6-46Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or displayFig. 6.20 b

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Enzymes involved in DNA replicationEnzymes involved in DNA replication

DNA polymerase IIIDNA polymerase III –– produces new stands of produces new stands of complementary DNAcomplementary DNA

DNA polymerase IDNA polymerase I –– fills in gaps between newly fills in gaps between newly synthesized Okazaki fragmentssynthesized Okazaki fragments

DNA DNA helicasehelicase –– unwinds double helixunwinds double helix SingleSingle--stranded binding proteinsstranded binding proteins –– keep helix openkeep helix open PrimasePrimase –– creates RNA primers to initiate synthesiscreates RNA primers to initiate synthesis DNA DNA ligaseligase –– welds together Okazaki fragmentswelds together Okazaki fragments

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DNA replication is bidirectionalDNA replication is bidirectional

Replication forksReplication forks move in opposite directions.move in opposite directions. In linear chromosomes, In linear chromosomes, telomerestelomeres ensure the ensure the

maintenance and accurate replication of chromosome maintenance and accurate replication of chromosome ends.ends.

In In circularcircular chromosomes, such as chromosomes, such as E. coliE. coli, there is only , there is only one one origin of replicationorigin of replication..

In circular chromosomes, unwinding and replication In circular chromosomes, unwinding and replication causes causes supercoilingsupercoiling, which may impede replication., which may impede replication.

TopoisomeraseTopoisomerase –– enzyme that relaxes enzyme that relaxes supercoilssupercoils by by nicking strands.nicking strands.

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The bidirectional replication of a circular The bidirectional replication of a circular chromosomechromosome

Fig. 6.21 a-b

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Cells must ensure accuracy of their genetic Cells must ensure accuracy of their genetic information during replication information during replication

Errors during replication are rare.Errors during replication are rare. RedundancyRedundancy

Basis for repair of errors that occur during replication or Basis for repair of errors that occur during replication or during storageduring storage

Enzymes repair chemical damage to DNA.Enzymes repair chemical damage to DNA.

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Human chromosomes

Chiasmata: Visible under the light microscope Mark the site where chromatids from homologous chromosomes

have crossed over, or exchanged parts.

6.5 Recombination at the DNA level

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MeselsonMeselson and and WeigleWeigle experimentexperiment

Fig. 6.22

Grow Grow bacteriophagebacteriophage lamdalamda with light (with light (1212C and C and 1414N) or heavy (N) or heavy (1313C C and and 1515N) isotope.N) isotope.

CoinfectedCoinfected E. coliE. coli strain.strain. Separate the progeny Separate the progeny bacteriophagebacteriophage on a on a CsClCsCl density gradient.density gradient. Conclusion: DNA breaks and rejoins during recombination.Conclusion: DNA breaks and rejoins during recombination.

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HeteroduplexesHeteroduplexes mark the spot of recombinationmark the spot of recombination

Fig .6.23 a b

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Region between break points is called Region between break points is called heteroduplexheteroduplex.. Products of recombination are always in exact register; Products of recombination are always in exact register;

not a single base pair is lost or gained.not a single base pair is lost or gained. Two strands do not break and rejoin at the same Two strands do not break and rejoin at the same

location; often they are hundreds of base pairs apart.location; often they are hundreds of base pairs apart.

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Gene conversionGene conversion

A deviation from expected 2:2 segregation of alleles deviation from expected 2:2 segregation of alleles due to mismatch repair.due to mismatch repair.

Studied most extensively in yeast where tetrad analysis Studied most extensively in yeast where tetrad analysis makes possible to follow products of meiosis.makes possible to follow products of meiosis.

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Gene conversion in Gene conversion in yeastyeast

Mismatch repair Mismatch repair changes the changes the A:aA:a ratio ratio from 2:2 to 3:1.from 2:2 to 3:1.

Fig. 6.23 c

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Mechanisms of DNA recombinationMechanisms of DNA recombination

DNA DNA homologshomologs physically break, exchange parts, and physically break, exchange parts, and rejoin.rejoin.

Breakage and repair create reciprocal products.Breakage and repair create reciprocal products. Recombination events can occur anywhere along the Recombination events can occur anywhere along the

DNA molecule.DNA molecule. Precision in the exchange prevents mutations from Precision in the exchange prevents mutations from

occurring during the process.occurring during the process.

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DoubleDouble--strandstrand--break repair break repair model of DNA recombinationmodel of DNA recombination

Proposed in 1983.Proposed in 1983. DNA recombination is induced by DNA recombination is induced by

the doublethe double--strand breakage in strand breakage in DNA.DNA.

A B

a b

5’3’

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A B

a b

5’3’

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5’3’

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