TheMolecularBasisTheMolecularBasisofInheritanceofInheritanceBIOL222

Ch.16

Overview:LifeOverview:Life’’sOpera:ngInstruc:onssOpera:ngInstruc:ons

• JamesWatsonandFrancisCrick

• 1953‐produceddouble‐helicalmodelforthestructureofDNA

TheSearchfortheGene:cMaterialTheSearchfortheGene:cMaterial

• T.H.Morgan

• Showedthatgenesarelocatedonchromosomes

Therefore…

• Twocomponentsofchromosomes—DNAand

protein—becamecandidatesforthegeneOcmaterial

• TheroleofDNAinheredity

• Firststudiedinbacteria/bacteriophages

EvidenceThatDNACanTransformBacteriaEvidenceThatDNACanTransformBacteria• FrederickGriffith‐1928

• twostrainsofabacterium

• onepathogenicandoneharmless

• heat‐killedpathogenicremains

• mixedwithlivingharmlessstrain

• somelivingcellsbecamepathogenic

• Calledtransforma:on

• Changeingenotypeandphenotype

• duetoassimilaOonofforeignDNA

Fig.Fig. 16‐216‐2

Living S cells(control)

Living R cells(control)

Heat-killedS cells (control)

Mixture ofheat-killedS cells andliving R cells

Mouse diesMouse dies Mouse healthy Mouse healthy

Living S cells

RESULTS

EXPERIMENT

• OswaldAvery,MaclynMcCarty,andColinMacLeod‐1944

• transformingsubstancewasDNA

• onlyDNAtransformedharmlessbacteriaintopathogens

• SequenOallydeacOvatedproteins,RNA,orDNA

EvidenceThatDNACanTransformBacteriaEvidenceThatDNACanTransformBacteria

EvidenceThatViralDNACanProgramCellsEvidenceThatViralDNACanProgramCells

• MoreevidenceforDNAasthegeneOcmaterialcamefromstudiesofvirusesthatinfectbacteria

• bacteriophages

• widelyusedinmoleculargeneOcsresearch

Bacterialcell

Phage head

Tail sheathTail fiberDNA

100

nm

• AlfredHersheyandMarthaChase‐1952

• showedDNAisgeneOcmaterial

• phageknownasT2

• LookedtoseeifviralDNAorprotein

• entersthebacterium

• concludedinjectedDNA

• providesgeneOcinformaOon

EvidenceThatViralDNACanProgramCellsEvidenceThatViralDNACanProgramCells

Fig.Fig. 16‐4‐116‐4‐1

EXPERIMENT

Phage

DNA

Bacterial cell

Radioactiveprotein

RadioactiveDNA

Batch 1:radioactivesulfur (35S)

Batch 2:radioactivephosphorus (32P)

Fig.Fig. 16‐4‐216‐4‐2

EXPERIMENT

Phage

DNA

Bacterial cell

Radioactiveprotein

RadioactiveDNA

Batch 1:radioactivesulfur (35S)

Batch 2:radioactivephosphorus (32P)

Emptyproteinshell

PhageDNA

Fig.Fig. 16‐4‐316‐4‐3

EXPERIMENT

Phage

DNA

Bacterial cell

Radioactiveprotein

RadioactiveDNA

Batch 1:radioactivesulfur (35S)

Batch 2:radioactivephosphorus (32P)

Emptyproteinshell

PhageDNA

Centrifuge

Centrifuge

Pellet

Pellet (bacterialcells and contents)

Radioactivity(phageprotein)in liquid

Radioactivity(phage DNA)in pellet

EvidenceThatDNAIstheGene:cMaterialEvidenceThatDNAIstheGene:cMaterial

• DNAisapolymerofnucleoOdes

• base,asugar,andaphosphategroup

• ErwinChargaff‐1950

• DNAcomposiOonvaries

• fromonespeciestothenext

• evidenceofdiversitypointstoDNA

• Chargaff’srules

• equalnumberofAandTbases

• andequalGandCbases

StructuralModelofDNAStructuralModelofDNA

• RosalindFranklinandMauriceWilkins

• X‐raycrystallography

• FranklinproducedapictureoftheDNA

(a) Rosalind Franklin (b) Franklin’s X-ray diffraction photograph of DNA

• WatsonandCrick

• basespairedlikewithlike

• resultedinanon‐uniformwidth

• purinewithapyrimidine

• uniformwidthconsistentwithX‐ray

StructuralModelofDNAStructuralModelofDNA

Purine + purine: toowide

Pyrimidine + pyrimidine: toonarrow

Purine + pyrimidine:width consistentwith X-ray data

• ThereforeWatsonandCrick…

• pairingmorespecific

• dictatedbythebasestructures

• Determinedadenine(A)withthymine(T)

• guanine(G)withcytosine(C)

• explainsChargaff’srule:

• inanyorganismtheamountofA=T,andthe

amountofG=C

StructuralModelofDNAStructuralModelofDNA

Fig.Fig. 16‐816‐8

Cytosine (C)

Adenine (A) Thymine (T)

Guanine (G)

Fig.Fig. 16‐716‐7

(c) Space-filling model

Hydrogen bond 3′ end

5′ end

3.4 nm

0.34 nm3′ end

5′ end

(b) Partial chemical structure(a) Key features of DNA structure

1 nm

• WatsonandCrick

• specificbasepairing

• suggestedapossiblecopyingmechanism

• twostrandsofDNAarecomplementary

• eachstrandactsasatemplate

DNAReplica:onDNAReplica:on

Fig.Fig. 16‐9‐116‐9‐1

A T

GC

T A

TA

G C

(a) Parent molecule

Fig.Fig. 16‐9‐216‐9‐2

A T

GC

T A

TA

G C

A T

GC

T A

TAG C

(a) Parent molecule (b) Separation ofstrands

Fig.Fig. 16‐9‐316‐9‐3

A T

GC

T A

TA

G C

(a) Parent molecule

A T

GC

T A

TAG C

(c) “Daughter” DNA molecules,each consisting of oneparental strand and onenew strand

(b) Separation ofstrands

A T

GC

T A

TA

G C

A T

GC

T A

TAG C

• Semiconserva:vemodel

• Eachdaughtermolecule

• oneold,onenewstrand

• CompeOngmodels

• conservaOvemodel

• thetwoparentstrandsrejoin

• dispersivemodel

• eachstrandisamixofoldandnew

DNAReplica:onDNAReplica:on

Fig.Fig. 16‐1016‐10

Parent cellFirstreplication

Secondreplication

(a) Conservativemodel

(b) Semiconserva-tive model

(c) Dispersivemodel

DNAReplica:onDNAReplica:on

• DNAreplicaOon

• remarkablespeedandaccuracy

• MorethanadozenenzymesandotherproteinsparOcipate

DNA Replication

• Originsofreplica:on

• SiteswheretwoDNAstrandsseparate

• opensreplicaOon“bubble”

• eukaryoOcchromosome

• hundreds‐thousandsoforiginsofreplicaOon

• ReplicaOonproceedsinbothdirecOonsfromeachorigin

• unOltheenOremoleculeiscopied

DNAReplica:onDNAReplica:on

Fig.Fig. 16‐1216‐12

Origin ofreplication Parental (template) strand

Daughter (new) strand

Replication forkReplicationbubble

TwodaughterDNAmolecules

(a) Origins of replication in E. coli

Origin of replication Double-stranded DNA molecule

Parental (template) strandDaughter (new) strand

Bubble Replication fork

Two daughter DNA molecules

(b) Origins of replication in eukaryotes

0.5 µm

0.25 µm

Double-strandedDNA molecule

• Replica:onfork

• Y‐shapedregion

• endofeachreplicaOonbubble

• wherenewDNAstrandsareelongaOng

• Helicases

• untwistDNAatreplicaOonforks

• Single‐strandbindingprotein

• Binds/stabilizessingle‐strandedDNA

• unOlitcanbeusedasatemplate

• Topoisomerase

• corrects“overwinding”aheadofreplicaOonforks

• bybreaking,swiveling,andrejoiningDNAstrands

DNAReplica:onDNAReplica:on

Fig.Fig. 16‐1316‐13

Topoisomerase

Helicase

PrimaseSingle-strand bindingproteins

RNAprimer

5′5′

5′ 3′

3′

3′

• Primer

• ShortRNAstrandrequiredforiniOaOon

• Primase

• EnzymethatstartsanRNAchainfromscratch

• addsRNAnucleoOdes

• UsesDNAasatemplate

• 5–10nucleoOdeslong

• 3′endstarOngpointfornewDNA

DNAReplica:onDNAReplica:on

• DNApolymerases

• catalyzeelongaOonofnewDNA

• atareplicaOonfork

• 500nucleoOdespersecondinbacteria

• 50persecondinhumancells

DNAReplica:onDNAReplica:on