page 8.1 Revised 1/21/2000Solving the structure of DNAAs long as the structure of DNA was unknown there was no meaningful way to think aboutthe nature of the genetic code or how the genome is replicated. During the 1950's aconsiderable amount of evidence concerning the structure of DNA had accumulated anintense effort was underway to solve the structure of DNA. This was considered to be oneof the paramount problems in biology.Phosphodiester bondsBiochemists had shown that DNA was composed of nucleotides and that each nucleotidewas joined to the next by a phosphodiester bond.DensityThe density of the molecule much too high for DNA to be a single stranded molecule. Thedensity of DNA could be achieved, however, by a double stranded molecule.X-ray diffractionRosalind Franklin and Maurice Wilkins performed X ray diffraction experiments on pureDNA fibers. In these experiments, X rays are fired at the DNA and the molecule scattersthem. The scattered X rays then strike a piece of film and expose it, producing black spotswhere they hit. The angle of scatter represented by each spot provides information on theposition of atoms in the DNA.Their data indicated that DNA:1. formed a regular helix2. that the helix made one turn every 34Å (3.4 nm)3. that the diameter of the helix was about 20 Å (2 nm)4. that there was one nucleotide every 3.4 Å (0.34 nm) and therefore there mustbe 10 nucleotides/turn of the helixAT/GC ratioIn 1951, Chargaff analyzed the ratios of the different nitrogenous bases to one another in alarge number of different organisms. What he saw was that the amount of A essentiallyequal to amount of T and that the amount of G was equal to the amount of C. That is [A] =[T] & [G] = [C]. Furthermore, [A & T] does not necessarily equal [G & C].Here is a sample of Chargaff's data.Organism Tissue Adenine Thymine Guanine CytosineEscherichia coli 26.0 23.9 24.9 25.2Diplococcuspneumoniae29.8 31.6 20.5 18.0Mycobacteriumtuberculosis15.1 14.6 34.9 35.4Yeast 31.3 32.9 18.7 17.1Sea Urchin sperm 32.8 32.1 17.7 18.4Herring sperm 27.8 27.5 22.2 22.6Rat bonemarrow28.6 28.4 21.4 21.5Human thymus 30.9 29.4 19.9 19.8Human liver 30.3 30.3 19.5 19.9Human sperm 30.7 31.2 19.3 18.8Chargaff saw that DNA from different animals or plants had different AT/GC ratios but the[A]=[T] & [G]=[C] rule still held. Furthermore, in a single organism it didn't matter whichtissue you examined. All of the tissues, had the same AT/GC ratio.page 8.2 Revised 1/21/2000Watson & Crick's ModelAll of the pieces of the puzzle were in place and many were racing to solve DNA's structure.The structure was solved in 1953 by James Watson and Francis Crick. They assembled all ofthe data given above and built stick figure molecules until they found one that fit all of thedata.1) Based on the buoyant density Watson & Crick knew that DNA must be a doublehelix.2) Thermodynamically, they postulated that the most stable double helix would be onein which the sugar phosphate backbone interacted with water and the morehydrophobic nitrogenous bases were inside the helix hidden from the water.3) Chargaff's data suggested that the different bases interacted with one another and thatsomehow the amount of A determined the amount of T (& vice versa) and theamount of G determined the amount of C (& vice versa). They postulated that the Aon one strand H-bonded with T on the other and that the G on one strand H-bondedwith C on the other. This pairing would stabilize the helix.4) From Franklin and Wilkin's data they knew that there was one nucleotide every 3.4Å (0.34 nm), that there were 10 nucleotides/turn of the helix and that the helix was 20Å wide.5) In support of 3) Watson & Crick's modeling showed that only pyrimidine:purine basepairs could produce a helix of 20 Å. See belowPyrimidime:Pyrimidine base pairing wouldbe <20Å.pyrimidinepyrimidinePurine:Purine base pairing would producea helix that was > 20 Å.purinepurineBut Pyrimidine:Purine base pairing wouldproduce a double helix of about 20 Å.pyrimidinepurinepage 8.3 Revised 1/21/2000The only model that fit all of the data is the one with which you are alreadyfamiliar. I present to you the Watson & Crick Double Helical Model of DNA.34 Å3.4 Å20 ÅMinorGrooveMajorGrooveGCCGATTACGGCATTATAATGCCGGCStrands areantiparallelThey did this without performing any experiments. Theirs was a conceptualcontribution that is perhaps the most important scientific breakthrough of this century.Although, the model has been refined it is still basically correct. The structure suggested atheory that base-paired was used to both replicate and decode information.DNA 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?page 8.4 Revised 1/21/2000DNA 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.Semi-ConservativeReplicationEach