Presentation on theme: "DNA The Genetic Material Replication"— Presentation transcript:
1 DNA The Genetic Material Replication Chapter 13.DNAThe Genetic MaterialReplicationBiology 114
2 Scientific HistoryThe march to understanding that DNA is the genetic materialT.H. Morgan (1908)Frederick Griffith (1928)Avery, McCarty & MacLeod (1944)Hershey & Chase (1952)Watson & Crick (1953)Meselson & Stahl (1958)
3 Genes are on chromosomes 1908 | 1933Genes are on chromosomesT.H. Morganworking with Drosophila (fruit flies)genes are on chromosomesbut is it the protein or the DNA of the chromosomes that are the genes?through 1940 proteins were thought to be genetic material… Why?What’s so impressive about proteins?!
4 The “Transforming Factor” 1928The “Transforming Factor”Frederick GriffithStreptococcus pneumonia bacteriawas working to find cure for pneumoniaharmless live bacteria mixed with heat-killed infectious bacteria causes disease in micesubstance passed from dead bacteria to live bacteria = “Transforming Factor”Fred Griffith, English microbiologist, dies in the Blitz in London in 1941
5 The “Transforming Factor” mix heat-killed pathogenic & non-pathogenicbacterialive pathogenicstrain of bacterialive non-pathogenicstrain of bacteriaheat-killed pathogenic bacteriaA.B.C.D.mice diemice livemice livemice dieTransformation?something in heat-killed bacteria could still transmit disease-causing properties
6 DNA is the “Transforming Factor” 1944DNA is the “Transforming Factor”Avery, McCarty & MacLeodpurified both DNA & proteins from Streptococcus pneumonia bacteriawhich will transform non-pathogenic bacteria?injected protein into bacteriano effectinjected DNA into bacteriatransformed harmless bacteria into virulent bacteriaWhat’s theconclusion?
8 Why use Sulfur vs. Phosphorus? 1952 | 1969Confirmation of DNAHershey & Chaseclassic “blender” experimentworked with bacteriophageviruses that infect bacteriagrew phage viruses in 2 media, radioactively labeled with either35S in their proteins32P in their DNAinfected bacteria with labeled phagesWhy use Sulfur vs. Phosphorus?
10 Hershey & Chase Which radioactive marker is found inside the cell? Protein coat labeledwith 35SDNA labeled with 32PHershey & ChaseT2 bacteriophagesare labeled withradioactive isotopesS vs. Pbacteriophages infectbacterial cellsbacterial cells are agitatedto remove viral protein coatsWhich radioactive marker is found inside the cell?Which molecule carries viral genetic info?35S radioactivityfound in the medium32P radioactivity found in the bacterial cells
12 Blender experiment Radioactive phage & bacteria in blender 35S phage radioactive proteins stayed in supernatanttherefore protein did NOT enter bacteria32P phageradioactive DNA stayed in pellettherefore DNA did enter bacteriaConfirmed DNA is “transforming factor”Taaa-Daaa!
13 Chargaff 1947 DNA composition: “Chargaff’s rules” varies from species to speciesall 4 bases not in equal quantitybases present in characteristic ratiohumans:A = 30.9%T = 29.4%G = 19.9%C = 19.8%
14 Structure of DNA 1953 | 1962 Watson & Crick developed double helix model of DNAother scientists working on question:Rosalind FranklinMaurice WilkinsLinus PaulingWatson & Crick’s model was inspired by 3 recent discoveries:Chargaff’s rulesPauling’s alpha helical structure of a proteinX-ray crystallography data from Franklin & WilkinsFranklinWilkinsPauling
16 Rosalind Franklin ( )A chemist by training, Franklin had made original and essential contributions to the understanding of the structure of graphite and other carbon compounds even before her appointment to King's College. Unfortunately, her reputation did not precede her. James Watson's unflattering portrayal of Franklin in his account of the discovery of DNA's structure, entitled "The Double Helix," depicts Franklin as an underling of Maurice Wilkins, when in fact Wilkins and Franklin were peers in the Randall laboratory. And it was Franklin alone whom Randall had given the task of elucidating DNA's structure. The technique with which Rosalind Franklin set out to do this is called X-ray crystallography. With this technique, the locations of atoms in any crystal can be precisely mapped by looking at the image of the crystal under an X-ray beam. By the early 1950s, scientists were just learning how to use this technique to study biological molecules. Rosalind Franklin applied her chemist's expertise to the unwieldy DNA molecule. After complicated analysis, she discovered (and was the first to state) that the sugar-phosphate backbone of DNA lies on the outside of the molecule. She also elucidated the basic helical structure of the molecule.After Randall presented Franklin's data and her unpublished conclusions at a routine seminar, her work was provided - without Randall's knowledge - to her competitors at Cambridge University, Watson and Crick. The scientists used her data and that of other scientists to build their ultimately correct and detailed description of DNA's structure in Franklin was not bitter, but pleased, and set out to publish a corroborating report of the Watson-Crick model. Her career was eventually cut short by illness. It is a tremendous shame that Franklin did not receive due credit for her essential role in this discovery, either during her lifetime or after her untimely death at age 37 due to cancer.
17 Double helix structure of DNA the structure of DNA suggested a mechanism for how DNA is copied by the cell
18 Directionality of DNA You need to number the carbons! nucleotide it matters!nucleotidePO4N base5CH2OThis will be IMPORTANT!!41ribose32OH
19 I mean it… This will be IMPORTANT!! 5The DNA backbonePO4Putting the DNA backbone togetherrefer to the 3 and 5 ends of the DNAthe last trailing carbonbaseCH2OCO–OPOObaseI mean it… This will be IMPORTANT!!CH2OOH3
20 Anti-parallel strands Phosphate to sugar bond involves carbons in 3 & 5 positionsDNA molecule has “direction”complementary strand runs in opposite direction“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”Watson & Crick
21 Bonding in DNA 5’ 3’ 3’ 5’ hydrogen bonds phosphodiester bonds ….strong or weak bonds?How do the bonds fit the mechanism for copying DNA?
22 Base pairing in DNA Purines Pyrimidines Pairing adenine (A) guanine (G)Pyrimidinesthymine (T)cytosine (C)PairingA : TC : G
23 Copying DNA Replication of DNA base pairing allows each strand to serve as a pattern for a new strand
24 Models of DNA Replication verify through experiments…Models of DNA ReplicationAlternative modelsso how is DNA copied?
25 Semi-conservative replication 1958Semi-conservative replicationMeselson & Stahllabel nucleotides of “parent” DNA strands with heavy nitrogen = 15Nlabel new nucleotides with lighter isotope = 14N“The Most Beautiful Experiment in Biology”parentreplicationmake predictions…
26 Semi-conservative replication 1958Semi-conservative replicationMake predictions…15N strands replicated in 14N medium1st round of replication?2nd round?
27 DNA Replication Large team of enzymes coordinates replication let’s meet the team…DNA ReplicationLarge team of enzymes coordinates replicationEnzymesmore than a dozen enzymes & other proteins participate in DNA replication
28 single-stranded binding proteins Replication: 1st stepUnwind DNAhelicase enzymeunwinds part of DNA helixstabilized by single-stranded binding proteinssingle-stranded binding proteins
29 Where’s the ENERGY for the bonding! We’re missing something! Replication: 2nd stepBring in new nucleotides to match up to template strandsWhere’s the ENERGY for the bonding!But…We’re missing something!What?
30 Is that the only energy molecule? Energy of ReplicationWhere does the energy for the bonding come from?energyYou remember ATP!Is that the only energy molecule?CTPGTPTTPATPCMPTMPAMPADPGMP
31 Energy of Replication ATP GTP TTP CTP The nucleotides arrive as nucleosidesDNA bases with P–P–PDNA bases arrive with their own energy source for bondingbonded by DNA polymerase IIIATPGTPTTPCTP
32 Replication Adding bases 5'3'ReplicationenergyDNAP IIIAdding basescan only add nucleotides to 3 end of a growing DNA strandstrand grow 5'3’energyenergyThe energy rules the process.energyB.Y.O. ENERGY3'5'leading strand
36 Priming DNA synthesisDNA polymerase III can only extend an existing DNA moleculecannot start new onecannot place first baseshort RNA primer is built first by primasestarter sequencesDNA polymerase III can now add nucleotides to RNA primer
37 Cleaning up primersDNA polymerase I removes sections of RNA primer and replaces with DNA nucleotides
38 direction of replication Draw this in your notes and label each of the structures and their functionReplication forkDNA polymerase IIIlagging strandDNA polymerase I3’Okazaki fragmentsprimase5’5’ligaseSSB3’5’3’helicaseDNA polymerase III5’leading strand3’direction of replication
39 And in the end… Ends of chromosomes are eroded with each replication an issue in aging?ends of chromosomes are protected by telomeres
40 Telomeres Expendable, non-coding sequences at ends of DNA short sequence of bases repeated 1000s timesTTAGGG in humansTelomerase enzyme in certain cellsenzyme extends telomeresprevalent in cancersWhy?
41 Replication bubble Adds 1000 bases/second! Which direction does DNA build? List the enzymes & their role
42 Replication enzymes helicase DNA polymerase III primase ligasesingle-stranded binding proteins
43 DNA polymerases DNA polymerase III DNA polymerase I 1000 bases/second main DNA building enzymeDNA polymerase I20 bases/secondediting, repair & primer removalDNA polymerase III enzyme
44 Editing & proofreading DNA 1000 bases/second = lots of typos!DNA polymerase Iproofreads & corrects typosrepairs mismatched basesexcises abnormal basesrepairs damage throughout lifereduces error rate from 1 in 10,000 to 1 in 100 million bases
45 Fast & accurate!It takes E. coli <1 hour to copy 5 million base pairs in its single chromosomedivide to form 2 identical daughter cellsHuman cell copies its 6 billion bases & divide into daughter cells in only few hoursremarkably accurateonly ~1 error per 100 million bases~30 errors per cell cycle