12 DNA Structure and Replication...

DNASTRUCTUREANDREPLICATION–CHAPTER12

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CHAPTER12–DNASTRUCTUREANDREPLICATION

Figure1.The scientists responsible fordetermining the structure ofDNA are Rosalind Franklin,Maurice Wilkins, Francis Crickand James Watson (left toright). Although the work waspublished in 1953, Wilkins,Crick,andWatsonreceivedtheNobel Prize in Physiology orMedicine in 1962, afterFranklin had died in 1958 ofovariancancer.

Fromlefttoright(Wikipedia-Unknown-PD)(Wikipedia-Unknown-PD)(Wikiepdia-MarcLieberman-CCBY2.5)(Wikipeida-Cold Spring HarborLaborator-PD)Bottom:(Wikipedia-Michael Ströck-CC BY-SA3.0)

INTRODUCTIONOne of the fundamental things to know whenstudyinggeneticsisthebasicstructureofDNAandhow it is replicated. DNA is the “blueprint” thatcontainsalltheinstructionsformakingtheproteinsthat each cell needs, whether it is a single celledbacteriumoramulticellularorganismlikehumans.J. Watson, F. Crick, and M. Wilkins received theNobelPrize (1962) fordiscoveringthestructureofDNA.(R.Franklinmighthavealsoreceivedtheprizeforthisdiscovery,butshediedin1958.)

ThebasicstructureofDNAprovidesinsightintoitsfunction.Themainfeaturesofitsstructurearethatit can reliably: (1) reproduce exact copies of thisinformationtopassontodescendantcells,and(2)use the information to create proteins thatproduceandregulatethebiochemistryof thecell.Remember however, DNA within the cell is morethan just a loose strand within the nucleus. DNAinteractswithproteinsandispackagedintohigherorder structures (chromosomes) that will bediscussedlaterinthetextbookinChapter7.These

proteins also regulate the expression of genes(informationintheDNA).

Thischapterwillbegoingthroughthecomponentsof the DNA molecule, how the double helixstructurewasdiscovered,andhowthemethodofreplicationwasdiscovered.

1. DNASTRUCTURE-DOUBLEHELIX

1.1. NUCLEICACIDSANDPHOSPHATESUGARBACKBONEIn 1869 Johannes Friedrich Miesher, a Swissphysician and biologist, first isolated a substancehe called ‘nuclein’ from the nuclei of a humanwhitebloodcell.Heidentifiedthissubstancetobeweakly acidic with a high amount of phosphorus.This substance, after being further purified andstudiedwas later calleddeoxyribonucleic acid, orDNA. Its name describes the three characteristicsof themolecule,namely ithasaribosesugarwithonlyonehydroxylgroupcalleddeoxyribose(Figure2), it is found in the nucleus of a cell, and it isacidic.


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After purifying the ‘nuclein’ to DNA they found itcontainedfourdifferentsubunitsthatarelinkedina chain. Those subunits were identified asnucleotides. A nucleotide contains threecomponents, a phosphate group (PO4

3-), adeoxyribose sugar, and one of four nitrogenousbases.Thosebasesfit intotwogroupsbasedupontheir structure. Purines have a double ringstructure and include adenine and guanine.Pyrimidines have a single ring structure andinclude cytosine and thymine (Figure 2). Thenature of the phosphate group and the singleoxygen containing groupof thedeoxyribose sugarallows each nucleotide to chain together, formingthelongDNAstrand.

IfyounoticeinFigure2,oneofthenucleotideshaseach carbon of the ribose labeledwith a numberfollowedbyaprime,1’-5’.The1’positioniswherethebase is attached. The2’ position iswhere theriboseismissingahydroxylgroup.The5’positionisattachedtothephosphategroup.Whenlinkedinachain,thatphosphategroupcanthenlinktothe3’oxygen of the next nucleotide using aphosphodiester bond. When a chain is formed,there will always be two termini, the free 5’phosphate and the free 3’ oxygen on the ribose.These are known as the 5’ and 3’ ends,respectively,oftheDNAstrand.

Ribonucleicacid(RNA)islikeDNA,inthatitformschainssimilarly,andhasthebasesattachedtothesame carbon. The extra hydroxyl group at the 2’positioncausesittoformadifferentconformationthan DNA, becoming a more flexible molecule(DNA’sconformationwillbedescribedlater inthischapter). There are also dideoxynucleotides thataremissingthehydroxylgroupatboththe2’and3’position. Because of this, a chain cannot form atthe3’carbon,terminatingthechain.Thisfeatureofdideoxynucleotides is used in Sanger sequencing,whichwillbedescribedinChapter33.

Figure2.Molecular models of the components of DNA. The topshows the three different types of ribose sugar found innucleicacids.Thebottomshowsthepurineandpyrimidinenucleotide monophosphates. Carbon numbers (1’-5’) arelabeledon thesugarsanddAMP. (Original- L.Canham-CCBY-NC3.0)

1.2. CHARGAFF’SRULESWhenWatson and Crick set out in the 1940’s todeterminethestructureofDNA,theyalreadyknewthat DNA ismade up of a series nucleotideswithfour different bases: adenine (A), cytosine (C),thymine(T),guanine(G).ForDNA,thenucleotidesare abbreviated as dNTPs (deoxyribonucleotidetriphosphates), which include dATP, dCTP, dGTP,and dTTP. For RNA they are abbreviated asNTPs,whichincludeATP,CTP,GTP,andUTP.WatsonandCrickalsoknewofChargaff’s Rules,whichwereaset of observations about the relative amount ofeach nucleotide that was present in almost anyextractofDNA.Chargaffhadobservedthatforanygivenspecies,theabundanceofAwasthesameasT,andGwas the sameasC.Thiswasessential toWatson&Crick’smodel.

Ribose Deoxyribose Dideoxyribose

deoxyadenosine 5'-monophasphate (dAMP)

deoxyguanosine 5'-monophasphate (dGMP)

deoxycytidine 5'-monophasphate (dCMP)

deoxythymidine 5'-monophasphate (dTMP)

Purine nucleotides

Pyrimidine nucleotides



Figure3.Chemical structure of two pairs of nucleotides in afragment of double-stranded DNA. Sugar, phosphate, andbasesA,C,G,Tare labeled.Hydrogenbondsbetweenbasesonopposite strandsare shownbydashed lines.Note thatthe G-C pair has more hydrogen bonds than A-T. Thepolarityofeachstrandisindicatedbythelabels5’and3’.(Wikipedia-MadeleinePriceBall-CC01.0)

1.3. THEDOUBLEHELIXUsing proportionalmetalmodels of the individualnucleotides,WatsonandCrickdeducedastructureforDNA thatwas consistentwithChargaff’s Rulesand with x-ray crystallography data that wasobtained (with some controversy) from anotherresearchernamedRosalindFranklin.InWatsonandCrick’s famous double helix, each of the twostrands contains DNA bases connected throughcovalent bonds to a sugar-phosphate backbone(Figure 1 and Figure 3) Because one side of eachsugarmoleculeisalwaysconnectedtotheoppositeside of the next sugar molecule, each strand ofDNAhaspolarity:thesearecalledthe5’(5-prime)end and the 3’ (3-prime) end. The two strands ofthedoublehelix run inanti-parallel (i.e.opposite)directions,with the5’ endofone strandadjacenttothe3’endoftheotherstrand.Thedoublehelixhas a right-handed twist, (rather than the left-

handed twist that is often represented incorrectlyinpopularmedia).TheDNAbasesextendfromthebackbone towards the center of the helix, with apair of bases from each strand forming hydrogenbonds thathelp tohold the twostrands together.Because of the structure of the bases, A can onlyformhydrogenbondswithT,andGcanonlyformhydrogen bonds with C (remember Chargaff’sRules). Each strand is therefore said to becomplementary to the other, and so each strandalso contains enough information to act as atemplate for the synthesis of the other. Thiscomplementary redundancy is important in DNAreplicationandrepair.

Under most conditions, the two strands in thedouble helix are slightly offset, which creates amajorgrooveononeface,andaminorgrooveontheother.InFigure1,noticehowifyoulookalongthebottomedgeofthefigureyouitmakesawavepattern, with a large dip followed by a small dip,followedbya largedip.That iswhereyoucanseethe major and minor grooves. These groovesprovideaccessfortranscriptionregulatingproteins(transcription factors), which bind to specificsequencesofbasesalongtheDNA.

2. SEMI-CONSERVATIVEREPLICATION(VS.CONSERVATIVE,DISPERSIVE)

From the complementary strands model of DNA,proposedbyWatsonandCrickin1953,therewerethree straightforward possible mechanisms forDNA replication: (1) semi-conservative, (2)conservative,and(3)dispersive(Figure4).

The semi-conservative model proposes the twostrands of a DNA molecule separate duringreplication and then strandacts as a template forsynthesisofanew,complementarystrand.

The conservative model proposes that the entireDNA duplex acts as a single template for thesynthesisofanentirelynewduplex.

The dispersive model has the two strands of thedoublehelixbreakingintounitsthatwhicharethenreplicatedandreassembled,withthenewduplexescontainingalternatingsegmentsfromonestrandtotheother.



Figure4.The three models of DNA replication possible from thedoublehelixmodelofDNAstructure.(Wikipedia-Adenosine-CCBY-SA2.5)

Each of these three models makes a differentprediction about the how DNA strands should bedistributed following two rounds of replication.These predictions can be tested in the followingexperiment by following the nitrogen componentinDNAinE. colias itgoesthroughseveralroundsof replication. Two scientists,Meselson and Stahlin1958,useddifferentisotopesofNitrogen,whichisamajorcomponentinDNA.Nitrogen-14 (14N) isthe most abundant natural isotope, whileNitrogen-15(15N)israre,butalsodenser.Neitherisradioactive;eachcanbefollowedbyadifferenceindensity–“light”14vs“heavy”15atomicweightinaCsCldensitygradientultra-centrifugationofDNA.

TheexperimentstartswithE.coligrownforseveralgenerationsonmediumcontainingonly15N. Itwillhave denser DNA.When extracted and separatedin a CsCl density gradient tube, this “heavy” DNAwillmove to a position nearer the bottom of thetubeinthemoredensesolutionofCsCl(leftsideinFigure 5). DNA extracted from E. coli grown onnormal, 14N containingmediumwillmigratemoretowardsthelessdensetopofthetube.

If these E. coli cells are transferred to a mediumcontainingonly14N,the“light”isotope,andgrownfor one generation, then their DNA will be

composedof one-half 15N andone-half 14N. If thethis DNA is extracted and applied to a CsClgradient, the observed result is that one bandappearsatthepointmidwaybetweenthelocationspredicted forwholly 15NDNAandwholly 14NDNA(Figure 5). This “single-band” observation isinconsistentwith thepredictedoutcome from theconservative model of DNA replication (disprovesthis model), but is consistent with both thatexpected for the semi-conservative anddispersivemodels.

If the E. coli is permitted to go through anotherround of replication in the 14N medium, and theDNA extracted and separated on a CsCl gradienttube, thentwobandswereseenbyMeselsonandShahl:oneatthe14N-15Nintermediatepositionandoneatthewholly14Nposition(Figure5).Thisresultis inconsistentwith the dispersivemodel (a singlebandbetweenthe14N-15Npositionandthewholly14N position) and thus disproves this model. Thetwobandobservation is consistentwith the semi-conservativemodelwhichpredictsonewholly 14N

duplex and one 14N-15N duplex. Additional roundsof replication also support the semi-conservativemodel/hypothesis of DNA replication. Thus, thesemi-conservativemodel is thecurrentlyacceptedmechanism for DNA replication. Note however,that we now also know from more recentexperiments that whole chromosomes, which canbe millions of bases in length, are also semi-conservativelyreplicated.

These experiments, published in 1958, are awonderful example of how science works.Researchersstartwiththreeclearlydefinedmodels(hypotheses). Thesemodelswere tested, and two(conservative and dispersive) were found to beinconsistent with the observations and thusdisproven.Thethirdhypothesis,semi-conservative,was consistentwith the observations and therebysupported and accepted as mechanism of DNAreplication. Note, however, this is not “proof” ofthemodel, just strongevidence for it; hypothesesarenot“proven”,onlydisprovenorsupported



Figure5.Thepositionsof the 14Nand 15Ncontaining DNA in the densitygradient tube on the left.(Wikipedia-LadyofHats-PD)

3. CHROMOSOMEREPLICATION(E.COLI)-CAIRNSEXPERIMENT

IftheresultsofMeselsonandStahlweretrueandthere was semi-conservative replication, then thetwostrandsofDNAhavetoseparatetoprovidethetemplate for copying. This should be seen as a‘fork’ in a linearmodel if youmanage to see theDNA just as it’s replicating. John Cairns in 1963chosetotestthis.

TodothishetookE.colicellsgrowinginanormalenvironment,andthenallowedthemtogrowandreplicate in the presence of radioactive 3H-thymidine. The hypothesis is that if the E. coli’sDNA or chromosome is semi-conservativelyreplicated then after the first roundof replicationthere should be one newly made strand that isradioactive, or “hot”, and the other strand that istheparentaltemplatestrandwithnoradioactivity,so is “cold”. The original parental DNA will havetwostrands,eachnotradioactive.AfterreplicationthedaughterDNAwillhavetwostrands,onethatis

radioactiveandonethatisnot.AfterathirdroundofreplicationtherewillbeatwotypesofdaughterDNA,one that has a non-radioactive strandandaradioactive strand, and one that has tworadioactivestrands.

Aftergrowth in the 3H-thymidine,Cairns lysed thebacteria and collected the contents onto amicroscopeslide.Hethencoveredtheslidewithaphotographic emulsion and allowed exposure tofilm for 2 months. As the 3H-thymidine decays itemits anelectronwith a lot of energy and speed,knownasabetaparticle.Theemulsionreactswiththebetaparticlecreatingablacksilvergrainonthefilm. The density of grains should be indicative ofwhetheroneortwostrandsareradioactive.

Afterthefirstreplicationcycle,thefilmhadathincircular ring of grains (Figure 6). This wasinterpreted to be a daughter chromosome withonestrandthatishotandonestrandcold.Thisalsoprovided physical evidence that the E. colichromosome is circular, something that has onlypreviouslybeenshowngenetically.



In thesecondreplicationcycle thereplication forkwas seen.HereCairns saw the typical thin ringofgrainsmuchlikethefirstreplicationcycle,butwitha branch in the middle that had a thicker strand(Figure6).Thismeansthatthebranchseenwasanactively replicating chromosome, using theradioactive strand of DNA as a template, andaddingmore radioactive thymidine as the DNA isbeing synthesized. Because of the shape thesecreated on the film this replicating structure wascalledatheta(Θ)structure.Cairnsobservedmanydifferent molecules corresponding to theprogression from starting replication to thecompletionofreplication.

Figure6.In his experiment, Cairns looked at DNA with radioactivethymidineonanautoradiograph film,with the radioactivethymidineleavingdotsonthefilm.Thisfigureshowswhatthe autoradiograph film would look like, and below whatthe interpretationofwhat theautoradiograph shows. Theblue line represents the ‘cold’ DNA that has noradioactivity, while the red shows the ‘hot’ radioactiveDNA.Thedensityof thedotsontheautoradiograph implywhether there is one strand or both strands of hot DNA.During the second round of replication, a theta structurecanbeseen,asthecircularE.coliDNAis intheprocessofbeingreplicated.(Original-L.Canham-CCBY-NC3.0)

Here Cairns’ results were able to further supportthe semi-conservative replication theory, showingthe existence of replication forks, as well as thehypothesis thatE.colihasacircularchromosome.WhatCairnsdidnotrealizeisthatreplicationgoesinbothdirectionsatthereplicationfork,wherehe

thoughtoneforkwasstaticwhiletheotherstrandwent around the chromosome replicating.Scientists laterwentontoshowthatreplication isin-factbidirectional.

4. ORIGINSOFREPLICATION(PROKARYOTE-SINGLEORGIN),REPLICATIONFORK

WhenthecellentersS-phaseinthecellcycle(SeeChapter 14) the entire chromosomal DNA isreplicated. This is done by enzymes called DNApolymerases.AllDNApolymerasessynthesizenewstrands by adding nucleotides to the 3'OH grouppresentonthepreviousnucleotide.Forthisreasonthey are said towork in a 5' to 3' direction. DNApolymerases use a single strand of DNA as atemplate upon which it will synthesize thecomplementary sequence. This works fine for themiddle of chromosomes. DNA-directed DNApolymerases travel along theoriginalDNA strandsmakingcomplementarystrands(Figure7a).

Figure7.DNApolymerasesmakenewstrandsina5'to3'direction.(a) Regular DNA polymerases are proteins or proteincomplexes that use a single strand ofDNA as a template.For example, the main human DNA polymerase, Pol α, islarge protein complex made of four polypeptides. (b)TelomerasesusetheirownRNAasatemplate.ThehumantelomeraseisacomplexmadeofonepolypeptideandoneRNAmolecule.(Original-Harrington-CCBY-NC3.0)

DNA replication in both prokaryotes andeukaryotesbeginsatanOriginofReplication(Ori).Originsarespecificsequencesonspecificpositionson the chromosome. In E. coli, theOriC origin is~245 bp in size. Chromosome replication beginswiththebindingoftheDnaAinitiatorproteintoanAT-rich 9-mer inOriC and melts the two strands.Then DnaC loader protein helps DnaB helicaseprotein extend the single stranded regions suchthattheDnaGprimasecaninitiatethesynthesisofan RNAprimer, fromwhich theDNApolymerases

One round ofreplication

Two rounds ofreplication

Autoradiograph

Interpretation



can begin DNA synthesis at the two replicationforks. The forks continue in opposite directionsuntil they meet another fork or the end of thechromosome(Figure8).

Figure8.Anoriginofreplication.ThesequencespecificDNAduplexismelted then the primase synthesizes RNAprimers fromwhich bidirectional DNA replication begins as the tworeplication forks head off in opposite directions. Theleading and lagging strands are shown alongwithOkazakifragments.Notethe5’and3’orientationofallstrands.(Original-Locke-CCBY-NC3.0)

5. EUKARYOTECHROMOSOMEREPLICATION-MULTIPLEORIGINS

In prokaryotes, with a small, simple, circularchromosome, only one origin of replication isneeded to replicate the whole genome. Forexample, E. coli has a ~4.5 Mb genome(chromosome) that can be duplicated in ~40minutes assuming a single origin, bi-directionalreplication, and a speed of ~1000bases/second/forkforthepolymerase.

However, in larger,more complicated eukaryotes,withmultiple linearchromosomes,morethanoneoriginofreplicationisrequiredperchromosometoduplicate the whole chromosome set in the 8-hoursofthereplicativephase(S-phase)ofthecellcycle.Forexample,thehumandiploidgenomehas46chromosomes (6 x109basepairs). The shortestchromosomesare~50Mbp longandsocouldnotpossiblybereplicatedfromoneorigin.Additionally,the rate of replication fork movement is slower,

only ~100 base/second. Thus, eukaryotes containmultipleoriginsof replicationdistributedover thelength of each chromosome to enable theduplication of each chromosome within theobservedtimeofS-phase(Figure9).

Figure9.PartofaeukaryotechromosomeshowingmultipleOrigins(1, 2, 3) of Replication, each defining a replicon (1, 2, 3).ReplicationmaystartatdifferenttimesinS-phase.Here#1and#2beginfirstthen#3.Asthereplicationforksproceedbi-directionally, they create what are referred to as“replication bubbles” thatmeet and form larger bubbles.Theendresultistwosemi-conservativelyreplicatedduplexDNAstrands.(Original-Locke-CCBY-NC3.0)

6. TELOMERESTheendsoflinearchromosomespresentaproblem– at each end one strand cannot be completelyreplicated because there is no primer to extend.Although the lossof sucha small sequencewouldnot be a problem, the continued rounds ofreplication would result in the continued loss ofsequence from the chromosome end to a pointwere it would begin to loose essential genesequences. Thus, this DNA must be replicated.Mosteukaryotessolvetheproblemofsynthesizingthis unreplicated DNA with a specialized DNApolymerasecalledtelomerase,incombinationwitha regular polymerase. Telomerases are RNA-directedDNApolymerases.Theyareariboprotein,astheyarecomposedofbothproteinandRNA.AsFigure 7b shows, these enzymes contain a small



pieceofRNAthatservesasaportableandreusabletemplate from which the complementary DNA issynthesized. The RNA in human telomerases usesthe sequence 3-AAUCCC-5' as the template, andthus our telomere DNA has the complementarysequence 5'-TTAGGG-3' repeated over and over1000’softimes.Afterthetelomerasehasmadethefirst strand a primase synthesizes an RNA primerand a regular DNA polymerase can then make acomplementary strand so that the telomere DNAwill ultimately be double stranded to the originallength (Figure 10). Note: the number of repeats,and thus the size of the telomere, is not set. Itfluctuates after each round of the cell cycle.Because there are many repeats at the end, thisfluctuationmaintainsa lengthbuffer– sometimes

it’slonger,sometimesit’sshorter–buttheaveragelengthwill bemaintained over the generations ofcellreplication.

In the absence of telomerase, as is the case inhumansomaticcells,repeatedcelldivisionleadstothe “Hayflick limit”,where the telomeres shortento a critical limit and then the cells enter asenescencephaseofnon-growth.Theactivationoftelomerase expression permits a cell and itsdescendants to become immortal and bypass theHayflick limit. This happens in cancer cells, whichcanformtumoursaswellasincellsinculture,suchasHeLacells,whichcanbepropagatedessentiallyindefinitely. HeLa cells have been kept in culturesince1951(SeeChapter41).

Figure10.Telomerereplicationshowingthecompletionofthe leadingstrandand incompletereplicationofthe laggingstrand.Thegap isreplicatedbytheextensionofthe3’endbytelomeraseandthenfilledinbyextensionofanRNAprimer.(Original-Locke-CCBY-NC3.0)



___________________________________________________________________________SUMMARY:

• DNAisadoublehelixmadeoftwoanti-parallelstrandsofbasesonasugar-phosphatebackbone.

• Specific bases on opposite strands pair through hydrogen bonding (A=T and G=C), ensuringcomplementarityofthestrands.

• ThehereditaryinformationispresentasthesequenceofbasesalongtheDNAstrand.

• ChromosomereplicationbeginsatanoriginandproceedsbyDNApolymerasesatareplicationfork.

• Replicationproceedsbi-directionally.

• Typicallyeukaryoteshavemultipleoriginsalongeachchromosome,whileprokaryoteshaveonlyone.

• Eukaryoteshavetelomerasetocompletethereplicationoftheendsofchromosomes.

KEYTERMS:deoxyribonucleicacidnucleotidespurineadenineguaninepyrimidinecytosinethyminephosphodiesterbondribonucleicaciddideoxynucleotideWatsonandCrickChargaff’sRulesdoublehelixanti-parallelright-handedmajorgrooveminorgroovesemi-conservativeconservativedispersive

E.coliMeselsonandStahlNitrogen-14Nitrogen-15lightheavyCsClgradientJohnCairns3H-thymidinephotographicemulsionsilvergrainthetastructurebidirectionalDNApolymerasesOriginofreplicationrepliconreplicationbubbletelomeraseriboproteinHayflicklimitHeLacells



STUDYQUESTIONS:1) Compare Watson and Crick’s discovery with

Avery,MacLeodandMcCarty’sdiscovery.a)What did each discover, and what was theimpactofthesediscoveriesonbiology?b) How did Watson and Crick’s approachgenerally differ from Avery, MacLeod andMcCarty’s?c) Briefly research Rosalind Franklin on theinternet. Why is her contribution to thestructureofDNAcontroversial?

2) ListtheinformationthatWatsonandCrickusedtodeducethestructureofDNA.

3) RefertoWatsonandCrick’a) List the defining characteristics of the

structureofaDNAmolecule.b) Which of these characteristics are most

importanttoreplication?

c) WhichcharacteristicsaremostimportanttotheCentralDogma?

4) RefertoFigure3.a) Identify the part of the DNAmolecule thatwould be radioactively labeled in the mannerusedbyHershey&Chaseb) DNA helices that are rich in G-C base pairsarehardertoseparate(e.g.byheating)thanA-Trichhelices.Why?

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