DNA Replication is semi-conservative Minor Revision 1/12/2001 page 1DNA ReplicationEvery time a cell divides the genome must be duplicated and passed on to the offspring.That is:5'--- TGCT ----3'3'--- ACGA ----5'5'--- TGCT ----3'3'--- ACGA ----5'5'--- TGCT ----3'3'--- ACGA ----5'Original molecule yields 2 molecules following DNA replication.REPLICATIONOur topic in this section is how is this done?DNA replication must have high fidelity. Why? Well, if DNA replication was low fidelitythe consequences would be:1. dramatic and rapid random changes in the sequence of genes2. which would cause an extreme reduction in viabilityBecause of this, in a complex organism, evolution will select against low fidelity DNAreplication. The structure of DNA suggested a way that it could be replicated with highfidelity. Because the strands are complementary, one strand could specify the base on theopposite strand. This is actually what happens.During the 1950's, three theories were proposed for how DNA might be replicated.They went by the names Conservative, Dispersive and Semi-conservative DNA replication.They are illustrated below.Lowercase letters represent newly synthesized DNA and capital letters represent materialfrom the original parental molecule.ConservativeReplicationFollowingreplication, onedaughter moleculecontains both of theparental strands.The other daughtermolecule containstwo newlysynthesized DNAstrands.5'--- TGCT ----3'3'--- ACGA ----5'5'--- TGCT ----3'3'--- ACGA ----5'5'--- tgct ----3'3'--- acga ----5'This theory was proven to be incorrect.DispersiveReplicationBoth strands of eachdaughter moleculecontain nucleotidesderived from theparental molecule.5'--- TGCT ----3'3'--- ACGA ----5'5'--- tGcT ----3'3'--- AcGa ----5'5'--- TgCt ----3'3'--- aCgA ----5'This theory was proven to be incorrect.DNA Replication is semi-conservative Minor Revision 1/12/2001 page 2Semi-ConservativeReplicationEach parental strandacts as a template forthe synthesis of onenew daughter strand.Therefore, indaughter molecules anewly synthesizedstrand is base pairedto one of the originalparental strands.5'--- TGCT ----3'3'--- ACGA ----5'5'--- TGCT ----3'3'--- acga ----5'5'--- tgct ----3'3'--- ACGA ----5'The semiconservative theory is correct !How was it shown that DNA replication is Semi-Conservative?In 1958, the Meselson-Stahl experiment proved that in bacteria DNA replication was semi-conservative. In the early 1960's the Herbert Taylor Colchicine bean rootip experimentdemonstrated that a eukaryotic cell replicated by semiconservative replication.Meselson and Stahl ExperimentTo address this question this group used a way to isotopically label newly synthesized DNA.To do this they used two different isotopes of nitrogen (N).14N most common form of nitrogen15N less common form and has greater mass than 14NDNA made using 15N is about 1% denser than DNA made using 14N. These two forms canbe separated by equilibrium density gradient centrifugation (also called isopycniccentrifugation).What is equilibrium density gradient centrifugation?In this technique molecules mixed witha salt (CsCl2), dissolved in water, andcentrifuged at very high speed. The saltmolecules form a density gradient.Centrifuge tubes spinning in a centrifugePrior to centrifugation the salt molecules are evenly distributedthroughout the centrifuge tube. During centrifugation, the saltmolecules are forced towards the bottom of the tube and a gradient ofmolecules is established. More of the salt is found at the bottom of thetube and less is at the top. Therefore, the density of the solution issmaller at the top and greater at the bottom.Any DNA molecules in this centrifuge tube will also be forced towards the bottom of thetube. As the DNA travels down the tube the density of the surrounding salt solutiongradually increases. The DNA stops moving relative to the salt solution when their twodensities are equal. This is because of a physical property called buoyancy.An example:DNA Replication is semi-conservative Minor Revision 1/12/2001 page 3Prior tocentrifugation, theDNA is evenlydistributedthroughout thecentrifuge tubes.Centrifugation begins andthe salt and DNA beginsedimenting.After prolongedcentrifugation, the DNA hasformed discrete bandsreflecting its buoyant density.In the third panel, notice that the left tube has one band of DNA. This means that in thistube all of the DNA molecules have the same buoyant density. Also notice that the righttube has two bands. This means that the DNA in this tube is a mixture of DNAs with twodifferent buoyant densities.Back to the 15N, 14N stuff.E. coli can be grown in media in which the sole source of nitrogen is 15N or 14N. GenomicDNA from bacteria grown in 15N media will be about 1% denser than the DNA frombacteria grown in the 14N media. This difference in density means that the 15N and 14NDNA can be separated by equilibrium density gradient centrifugation.Previous experimentation had shown that:15N labeledDNAsedimentedat thisposition.14N labeledDNAsedimentedat thispositionIf the DNA is 50%15N and 50% 14Nthen it sedimentsat an intermediateposition.How was this used to determine which of the three DNA replication models is correct?Well, Meselson and Stahl grew bacteria in 15N media until all of the DNA was uniformlylabeled with 15N. They then synchronized these cultures (in this context synchrony meansthat all of the bacteria in the culture are replicating their DNA and performing cell divisionin unison) and grew them in 14N media. Here are the details.DNA Replication is semi-conservative Minor Revision 1/12/2001 page 4Grow bacteria in 15Nand synchronize theircell divisionsRemove a sampleand isolate DNA.Call it Sample 1.Transfer bacteria to 14N mediaFirst GenerationOne cell divisionhas occured.Remove a sampleand isolate DNA.Call it Sample 2.Second GenerationTwo cell divisionshave occured.Remove a sampleand isolate DNA.Call it Sample 3.Now samples 1, 2 and 3 are subjected to equilibrium density gradient centrifugation.The three DNA replication models make specific predictions about the result of thisexperiment. These predictions are presented below. In this table representsDNA synthesized with 15N and represents DNA synthesized with 14N.DNA Replication is semi-conservative Minor Revision 1/12/2001 page 5Predicted resultsfollowingcentrifugationReplicating DNAin samples1, 2 and 3Predictions based on theConservative