2Important Early Discoveries Fred Griffith (1928) – Experiments with pneumonia and bacterial transformation determined that there is a molecule that controls inheritance.Oswald T. Avery (1944) - Transformation experiment determined that DNA was the genetic material responsible for Griffith’s results (not RNA).Hershey-Chase Experiments (1952) – discovered that DNA from viruses can program bacteria to make new viruses.Erwin Chargaff (1947) – noted that the the amount of A=T and G=C and an overall regularity in the amounts of A,T,C and G within species.
3Frederick Griffith’s Transformation Experiment The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928Griffith worked with two strains of a bacterium, a pathogenic “S” strain and a harmless “R” strainWhen he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain, some living cells became pathogenicHe called this phenomenon transformation, now defined as a change in genotype and phenotype due to assimilation of foreign DNALiving S cells(control)Living R cellsHeat-killedS cells (control)Mixture of heat-killedS cells and living R cellsMouse diesare found inblood sampleMouse healthyRESULTS
4Oswald T. Avery’s Transformation Experiment In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance was DNATheir conclusion was based on experimental evidence that only DNA worked in transforming harmless bacteria into pathogenic bacteriaMany biologists remained skeptical, mainly because little was known about DNA
6Hershey-Chase Bacteriophage Experiment In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2To determine the source of genetic material in the phage, they designed an experiment showing that only one of the two components of T2 (DNA or protein) enters an E. coli cell during infection32P is discovered within the bacteria and progeny phages, whereas 35S is not found within the bacteria but released with phage ghosts.They concluded that the injected DNA of the phage provides the genetic informationBacterial cellPhageDNARadioactiveproteinEmptyprotein shellRadioactivity(phage protein)in liquidBatch 1:Sulfur (35S)CentrifugePellet (bacterialcells and contents)Pellet(phage DNA)in pelletBatch 2:Phosphorus (32P)
7Additional Evidence That DNA Is the Genetic Material In 1947, Erwin Chargaff reported that DNA composition varies from one species to the nextThis evidence of diversity made DNA a more credible candidate for the genetic materialBy the 1950s, it was already known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate groupFranklin’s X-ray crystallographic images of DNA enabled Watson to deduce that DNA was helicalThe X-ray images also enabled Watson to deduce the width of the helix and the spacing of the nitrogenous basesThe width suggested that the DNA molecule was made up of two strands, forming a double helix
8James D. Watson & Francis H. Crick In 1953 presented the double helix model of DNATwo primary sources of information:1. Chargaff Rule: #A#T and #G#C. “A strange but possibly meaningless phenomenon”.2. X-ray diffraction studies of Rosalind Franklin & Maurice H. F. Wilkins
9DNA StructureConclusion-DNA is a helical structure with distinctive regularities, 0.34 nm & 3.4 nm.
101962: Nobel Prize in Physiology and Medicine Watson, J.D. and F.H. Crick, “Molecular Structure of Nucleic Acids: A Structure for Deoxynucleic Acids”. Nature 171 (1953), p. 738.James D.WatsonFrancis H.CrickMaurice H. F.WilkinsWhat about?Rosalind Franklin
11The Structure of DNADNA is composed of four nucleotides, each containing: adenine, cytosine, thymine, or guanine.The amounts of A = T, G = C, and purines = pyrimidines [Chargaff’s Rule].DNA is a double-stranded helix with antiparallel strands [Watson and Crick].Nucleotides in each strand are linked by 5’-3’ phosphodiester bondsBases on opposite strands are linked by hydrogen bonding: A with T, and G with C.
12The Basic Principle: Base Pairing to a Template Strand The relationship between structure and function is manifest in the double helixSince the two strands of DNA are complementary each strand acts as a template for building a new strand in replicationIn DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules5 end3 endHydrogen bond0.34 nm3.4 nm1 nm
13DNA replicationThe parent molecule unwinds, and two new daughter strands are built based on base-pairing rules(a) The parent molecule has two complementary strands of DNA Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.(b) The first step in replication is separation of the two DNA strands.(c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.(d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands Each “daughter” DNA molecule consists of one parental strand and one new strand.ACTGTTGACACTGTGACACTGATGACACTGTGACACTGTGACGCATTACG
14DNA Replication DNA must replicate during each cell division 3 alternative models for DNA replication were hypothesized:Semiconservative replicationConservative replicationDispersive replicationSemi-conservativeConservativeDispersive
15Meselson-Stahl Experiments Labeled the nucleotides of old strands with a heavy isotope of nitrogen (15N), new nucleotides were indicated by a lighter isotope (14N).The first replication in the 14N medium produced a band of hybrid (15N-14N) DNA, eliminating the conservative model.A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model.Bacteriacultured inmediumcontaining15NDNA samplecentrifugedafter 20 min(after firstreplication)after 40 min(after secondtransferred to14NLessdenseMoreConservativemodelFirst replicationSemiconservativeSecond replicationDispersive
16DNA Replication is “Semi-conservative” Each 2-stranded daughter molecule is only half newOne original strand was used as a template to make the new strand
17DNA ReplicationThe copying of DNA is remarkable in its speed and accuracyInvolves unwinding the double helix and synthesizing two new strands.More than a dozen enzymes and other proteins participate in DNA replicationThe replication of a DNA molecule begins at special sites called origins of replication, where the two strands are separated
18Origins of Replication A eukaryotic chromosome may have hundreds or even thousands of replication originsReplication begins at specific siteswhere the two parental strandsseparate and form replicationbubbles.The bubbles expand laterally, asDNA replication proceeds in bothdirections.Eventually, the replicationbubbles fuse, and synthesis ofthe daughter strands iscomplete.123Origin of replicationBubbleParental (template) strandDaughter (new) strandReplication forkTwo daughter DNA moleculesIn eukaryotes, DNA replication begins at many sites along the giantDNA molecule of each chromosome.In this micrograph, three replicationbubbles are visible along the DNA ofa cultured Chinese hamster cell (TEM).(b)(a)0.25 µm
19Mechanism of DNA Replication DNA polymerase I degrades the RNA primer and replaces it with DNADNA polymerase III adds nucleotides to primerDNA replication is catalyzed by DNA polymerase III which needs an RNA primerDNA polymerase III cannot initiate the synthesis of a polynucleotide, they can only add nucleotides to the 3 endThe initial nucleotide strand is an RNA primerRNA primase synthesizes primer on DNA strandDNA polymerase adds nucleotides to the 3’ end of the growing strand
20Mechanism of DNA Replication Nucleotides are added by complementary base pairing with the template strandDNA always reads from 5’ end to 3’ end for transcription replicationDuring replication, new nucleotides are added to the free 3’ hydroxyl on the growing strandThe nucleotides (deoxyribonucleoside triphosphates) are hydrolyzed as added, releasing energy for DNA synthesis.The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cellsNew strand5¢ endPhosphateBaseSugarTemplate strand3¢ endNucleosidetriphosphateDNA polymerasePyrophosphate
21The Mechanism of DNA Replication DNA synthesis on the leading strand is continuousOnly one primer is needed for synthesis of the leading strandThe lagging strand grows the same general direction as the leading strand (in the same direction as the Replication Fork). However, DNA is made in the 5’-to-3’ directionTherefore, DNA synthesis on the lagging strand is discontinuousFor synthesis of the lagging strand, each fragment (Okazaki) must be primed separately, then DNA fragments are sythesized and subsequently ligated togetherParental DNA5¢3¢Leading strandOkazakifragmentsLagging strandDNA pol IIITemplatestrandDNA ligaseOverall direction of replication
22Mechanism of DNA Replication Many proteins assist in DNA replicationDNA helicases unwind the double helix, the template strands are stabilized by other proteinsSingle-stranded DNA binding proteins make the template availableRNA primase catalyzes the synthesis of short RNA primers, to which nucleotides are added.DNA polymerase III extends the strand in the 5’-to-3’ directionDNA polymerase I degrades the RNA primer and replaces it with DNADNA ligase joins the DNA fragments into a continuous daughter strand5¢3¢Parental DNAOverall direction of replicationDNA pol IIIReplication forkLeadingstrandDNA ligasePrimaseOVERVIEWPrimerDNA pol ILaggingOrigin of replication
23Enzymes in DNA replication Helicase unwindsparental double helixBinding proteinsstabilize separatestrandsPrimase addsshort primerto template strandDNA polymerase IIIbinds nucleotidesto form new strandsDNA polymerase I (Exonuclease) removes RNA primer and inserts the correct basesLigase joins Okazakifragments and sealsother nicks in sugar-phosphate backbone
24Replication Helicase protein binds to DNA sequences called 3’3’5’5’3’5’3’5’Helicase protein binds to DNA sequences calledorigins and unwinds DNA strands.Binding proteins prevent single strands from rewinding.Primase protein makes a short segment of RNAcomplementary to the DNA, a primer.
25Replication DNA polymerase III enzyme adds DNA nucleotides Overall directionof replication3’3’5’5’3’5’3’5’DNA polymerase III enzyme adds DNA nucleotidesto the RNA primer.
26Replication DNA polymerase proofreads bases added and Overall directionof replication3’3’5’5’3’5’3’5’DNA polymerase proofreads bases added andreplaces incorrect nucleotides.
27Replication Leading strand synthesis continues in a Overall directionof replication3’3’5’5’3’5’3’5’Leading strand synthesis continues in a5’ to 3’ direction.
28Replication Leading strand synthesis continues in a Overall directionof replication3’3’5’5’Okazaki fragment3’3’5’5’3’5’Leading strand synthesis continues in a5’ to 3’ direction.Discontinuous synthesis produces 5’ to 3’ DNAsegments called Okazaki fragments.
29Replication Leading strand synthesis continues in a Overall directionof replication3’3’5’5’Okazaki fragment3’3’5’5’3’5’Leading strand synthesis continues in a5’ to 3’ direction.Discontinuous synthesis produces 5’ to 3’ DNAsegments called Okazaki fragments.
30Replication Leading strand synthesis continues in a Overall directionof replication3’3’5’5’Okazaki fragment3’5’3’5’3’5’Leading strand synthesis continues in a5’ to 3’ direction.Discontinuous synthesis produces 5’ to 3’ DNAsegments called Okazaki fragments.
31Replication Leading strand synthesis continues in a 3’3’5’5’3’5’3’5’3’3’5’5’Leading strand synthesis continues in a5’ to 3’ direction.Discontinuous synthesis produces 5’ to 3’ DNAsegments called Okazaki fragments.
32Replication Leading strand synthesis continues in a 3’3’5’5’3’5’3’5’3’3’5’5’Leading strand synthesis continues in a5’ to 3’ direction.Discontinuous synthesis produces 5’ to 3’ DNAsegments called Okazaki fragments.
33Replication5’3’3’5’5’3’5’3’3’3’5’5’Exonuclease activity of DNA polymerase I removes RNA primers.
34Replication Polymerase activity of DNA polymerase I fills the gaps. 3’3’5’3’5’3’3’5’5’Polymerase activity of DNA polymerase I fills the gaps.Ligase forms bonds between sugar-phosphate backbone.
35Replication Fork Overview 5¢3¢Parental DNAOverall direction of replicationDNA pol IIIReplication forkLeadingstrandDNA ligasePrimaseOVERVIEWPrimerDNA pol ILaggingOrigin of replication
36Other Proteins That Assist DNA Replication Helicase, topoisomerase, single-strand binding protein are all proteins that assist DNA replication
37Proofreading DNA must be faithfully replicated…but mistakes occur DNA polymerase (DNA pol) inserts the wrong nucleotide base in 1/10,000 basesDNA pol has a proofreading capability and can correct errorsMismatch repair: ‘wrong’ inserted base can be removedExcision repair: DNA may be damaged by chemicals, radiation, etc. Mechanism to cut out and replace with correct bases
38MutationsA mismatching of base pairs, can occur at a rate of 1 per 100,000 bases.DNA polymerase proofreads and repairs accidental mismatched pairs.Chances of a mutation occurring at any one gene is over 1 in 10,000,000,000 (billion)Because the human genome is so large, even at this rate, mutations add up. Each of us probably inherited 3-4 mutations!
39Proofreading and Repairing DNA DNA polymerases proofread newly made DNA, replacing any incorrect nucleotidesIn mismatch repair of DNA, repair enzymes correct errors in base pairingIn nucleotide excision DNA repair nucleases cut out and replace damaged stretches of DNANucleaseDNApolymeraseligaseA thymine dimerdistorts the DNA molecule.1A nuclease enzyme cutsthe damaged DNA strandat two points and thedamaged section isremoved.2Repair synthesis bya DNA polymerasefills in the missingnucleotides.3DNA ligase seals theFree end of the new DNATo the old DNA, making thestrand complete.4
41Accuracy of DNA Replication The chromosome of E. coli bacteria contains about 5 million bases pairsCapable of copying this DNA in less than an hourThe 46 chromosomes of a human cell contain about 6 BILLION base pairs of DNA!!Printed one letter (A,C,T,G) at a time…would fill up over 900 volumes of Campbell.Takes a cell a few hours to copy this DNAWith amazing accuracy – an average of 1 per billion nucleotides
42Replicating the Ends of DNA Molecules The ends of eukaryotic chromosomal DNA get shorter with each round of replicationEnd of parentalDNA strandsLeading strandLagging strandLast fragmentPrevious fragmentRNA primerRemoval of primers andreplacement with DNAwhere a 3 end is availablePrimer removed butcannot be replacedwith DNA becauseno 3 end availablefor DNA polymeraseSecond roundof replicationNew leading strandNew lagging strand 5Further roundsShorter and shorterdaughter molecules53
43TelomeresEukaryotic chromosomal DNA molecules have at their ends nucleotide sequences, called telomeres, that postpone the erosion of genes near the ends of DNA molecules1 µm
44TelomerasesIf the chromosomes of germ cells became shorter in every cell cycle essential genes would eventually be missing from the gametes they produceAn enzyme called telomerase catalyzes the lengthening of telomeres in germ cells