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Chapter 16. DNA The Genetic Material Replication Scientific History The march to understanding that DNA is the genetic material T.H. Morgan (1908) Frederick Griffith (1928) Avery, McCarty & MacLeod (1944) Hershey & Chase (1952) Watson & Crick (1953) Meselson & Stahl (1958) 1908 | 1933 Genes are on chromosomes T.H. Morgan working with Drosophila (fruit flies) genes are on chromosomes but 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?! Protein 20 different amino acids copious amounts and variations of proteins Found everywhere vs DNA 4 different bases (A, T, G, C) limited amounts in each cell found mostly in the nucleus The “Transforming Factor” Frederick Griffith 1918 – the world was struck by a pandemic (cross continents) an influenza that turned to pneumonia, killing an estimated 30 – 50 million people was working to find cure for pneumonia Streptococcus pneumonia bacteria He “stumbled upon” a substance that was passed from dead bacteria to live bacteria = “Transforming Factor” 1928 In Frederick Griffith’s 1920’s experiments: 1. What happened to the mice he injected with the R-Strain bacteria? 2. What happened to the mice he injected with the S-Strain bacteria that causes pneumonia? 3. What happened to the mice he injected with the Heat-killed S-Strain bacteria that causes pneumonia? 4. What happened to the mice he injected with the R-Strain bacteria AND the heat-killed S-Strain bacteria? Frederick Griffith’s conclusions: 1. The living bacteria recovered from the dead mice were the S (smooth) Strain. “Something” must have “Transformed” the R-Strain to become smooth. 2. He proposed that when the smooth bacteria were killed… “something” from inside the S-Strain bacteria must have been released into the media upon the death of the bacteria. 3. Whatever that transforming factor was must have instructed the non-pathogenic R-Strain bacteria to become pathogenic SStrain bacteria. The “Transforming Factor” live pathogenic strain of bacteria A. mice die live non-pathogenic heat-killed strain of bacteria pathogenic bacteria B. C. mice live mice live mix heat-killed pathogenic & non-pathogenic bacteria D. mice die Transformation? something in heat-killed bacteria could still transmit disease-causing properties 1944 DNA is the “Transforming Factor” Avery, McCarty & MacLeod purified both DNA & proteins from Streptococcus pneumonia bacteria which will transform non-pathogenic bacteria? injected protein into bacteria no effect injected DNA into bacteria transformed harmless bacteria into virulent bacteria What’s the conclusion? What was Oswald Avery’s conclusion when he used enzymes to break down specific bacterial components in this experiment? Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Conclusion – Griffith’s “Transforming Factor” was DNA, not RNA or protein Confirmation of DNA Hershey & Chase 1952 | 1969 classic “blender” experiment worked with bacteriophage viruses that infect bacteria Why use Sulfur vs. Phosphorus? grew phage viruses in 2 media, radioactively labeled with either 35S in their proteins 32P in their DNA infected bacteria with labeled phages Confirmed DNA is “transforming factor” Protein coat labeled with 35S Hershey & Chase DNA labeled with 32P T2 bacteriophages are labeled with radioactive isotopes S vs. P bacteriophages infect bacterial cells Watch video clip 03 Hershey and Chase bacterial cells are agitated to remove viral protein coats Which radioactive marker is found inside the cell? Which molecule carries viral genetic info? 35S radioactivity found in the medium 32P radioactivity found in the bacterial cells Blender experiment Radioactive phage & bacteria in blender 35S phage radioactive proteins stayed in supernatant therefore protein did NOT enter bacteria 32 P phage radioactive DNA stayed in pellet therefore DNA did enter bacteria Confirmed DNA is “transforming factor” Taaa-Daaa! Experiments Overview Griffith’s Experiments (Streptococcus pneumoniae) Experiment Results Conclusion 1 R-Strain bacteria S-Strain bacteria Mouse lived Mouse died Nonvirulent strain of bacteria Virulent/deadly strain 2 Heat-killed S-Strain Mouse lived Destroyed deadly bacteria 3 Heat-killed S-Strain + R-Strain Mouse died Some how the killed bacteria was able to pass “something” to the R-strain That Transformed it to become deadly Avery’s Experiments (enzymes & bacteria) Experiments Results Conclusion Enzymes to break down proteins, carbohydrates, lipids, RNA, & finally DNA Deadly in spite of all DNA is the transforming molecule that enzymes except one made Griffith’s R-Strain bacteria turn that broke apart DNA into an S-Strain bacteria Hershey & Chase Experiments (bacteria & a virus called bacteriophage) Experiments Radioactive Protein Radioactive DNA Results Conclusion New phages = no radioactivity Protein does not make new phages DNA makes new phages New phages = radioactive Structure of DNA 1953 James D. Watson/Francis H. Crick proposed the Double Helix Model based on two sources of information: 1. X-ray crystallography studies by: Rosalind Franklin 1920 - 1958 Maurice Wilkins 1916 - 2004 2. Chargaff Base-Pair Ruling 15 1947 Chargaff DNA composition: “Chargaff’s rules” varies from species to species all 4 bases not in equal quantity bases present in characteristic ratio humans: A = 30.9% T = 29.4% G = 19.9% C = 19.8% What do you notice?! Base pairing in DNA Purines adenine (A) guanine (G) Pyrimidines thymine (T) cytosine (C) Pairing A:T C G Memory technique: All Girls are Pure, Cut The Pie Critical Thinking: How do the number of purines compare to the number of pyrimidines? Double helix structure of DNA the structure of DNA suggested a mechanism for how DNA is copied by the cell Directionality of DNA You need to PO4 nucleotide number the carbons! it matters! N base 5 5 CH2 1 4 3 2 This will be IMPORTANT!! O 4 deoxyribose 3 OH 2 1 The DNA backbone Putting the DNA backbone together refer to the 3 and 5 ends of the DNA the last trailing carbon I mean it… This will be IMPORTANT!! 5 PO4 base CH2 O C O –O P O O CH2 base O OH 3 Anti-parallel strands Phosphate to sugar bond involves carbons in 3 & 5 positions DNA 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 Bonding in DNA 5’ hydrogen bonds 3’ phosphodiester bonds 3’ glycosidic bonds 5’ ….strong or weak bonds? How do the bonds fit the mechanism for copying DNA? Models of DNA Replication Alternative models so how is DNA copied? verify through experiments… Semi-conservative replication 1958 Meselson & Stahl label nucleotides of “parent” DNA strands with heavy nitrogen = 15N label new nucleotides with lighter isotope = 14N “The Most Beautiful Experiment in Biology” parent make predictions… replication Semi-conservative replication Make predictions… 15 N strands replicated in 14N medium 1st round of replication? 2nd round? 1958 Copying DNA Replication of DNA base pairing allows each strand to serve as a pattern for a new strand DNA Replication let’s meet the team… Large team of enzymes coordinates replication What’s it really look like? How many replication bubbles do you see? 1 2 3 4 Replication: 1st step Unwind DNA helicase enzyme unwinds part of DNA helix (replication fork) stabilized by single-stranded binding proteins single-stranded binding proteins Replication Fork 4th – DNA Polymerase I Replaces the RNA primer with a DNA nucleotide Lagging Strand 3rd – DNA Polymerase III adds DNA 2nd – nucleotides Primase attaches a RNA Primer Okazaki Fragments 5’ 3’ 5th – Ligase “glues” Okazaki fragments together on the lagging strand. 3’ 5’ 5’ 3’ Leading Strand 5’ 3’ direction of replication? 1st – Helicase unzips a portion of DNA Single-Stranded Binding Proteins Control the unzipping Leading & Lagging strands Leading strand - continuous synthesis Built across from 3’ end In the 5’ to 3’ direction Okazaki Lagging strand - Okazaki fragments - joined by ligase - “spot welder” enzyme Replication: 2nd step Bring in new nucleotides to match up to template strands But… the Where’s We’re missing ENERGY forsomething! the bonding! What? Energy of Replication Where does the energy for the bonding come from? energy You remember ATP! Is that the only energy molecule? CTP TTP GTP ATP AMP CMP TMP GMP ADP Energy of Replication The nucleotides arrive as nucleosides DNA bases with P–P–P DNA bases arrive with their own energy source for bonding bonded by DNA polymerase III ATP GTP TTP CTP 5' Replication energy DNA P III Adding bases can only add nucleotides across from the 3 end of a growing DNA strand!! New strand grows 5'3’ 3' energy DNA P III energy energy B.Y.O. ENERGY 3' leading strand 5' 5' 3' 5' 3' ligase energy 3' lagging strand 5' 3' leading strand 5' Modeling Replication Make a DNA molecule (paper strip) 5’ 3’ 3’ 5’ Then replicate it Animation Analysis: 1. Red/Orange = ____________ How do you know? ________ 2. Grey/White = ____________ How do you know? ________ 3. Green = ________________ How do you know? ________ 4. Yellow = How do you know? ________ 5. Blue = How do you know? ________ 6. Pink = How do you know? ________ 7. Bonds = How do you know? ________ 8. What breaks off? How do you know? ________ 9. Why? __________________ 10. What were the molecules? ___________ 11. What do they become? ______________ 12. Where is the 5’ and 3’ on the template & complementary strands? Regulating Gene Expression As shown in the diagram, a chromosome in the nucleus of a eukaryotic cell is comprised of DNA wound tightly around histone proteins, packaging the DNA molecule into highly compact form called chromatin. This makes DNA inaccessible to enzymes that would code for the genetic Information. This diagram shows acetyl groups attaching to the histones. This causes the tight compaction to unravel, now allowing DNA to be susceptible to activation (replication or transcription) Regulating Gene Expression A methyl group (CH3) can be attached to a cytosine base on DNA, as shown here. When a methyl group is attached to a base, enhancer, promoter, and activator proteins cannot access the base to build nucleotides Implications: What would you expect (in terms of gene expression) for a gene that contains many methyl groups? The presence of the methyl group would probably block transcription from occurring, so the gene would not be expressed. Okazaki fragments Priming DNA synthesis DNA polymerase III can only extend an existing DNA molecule cannot start new one cannot place first base short RNA primer is built first by primase starter sequences DNA polymerase III can now add nucleotides to RNA primer Cleaning up primers DNA polymerase I removes sections of RNA primer and replaces with DNA nucleotides Replication bubble Adds 1000 bases/second! Which direction does DNA build? List the enzymes & their role Draw & Label using Word Bank lagging strand leading strand Okazaki fragments DNA polymerase I DNA polymerase III (3x) helicase primase ligase SSBP 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ Replication fork direction of replication Replication enzymes: Helicase – unzip helix structure Single Strand Binding Proteins (SSBP) – control unzipping Primase – lay down RNA Primer DNA Polymerase III – extends existing DNA beyond the primer DNA Polymerase I – replaces RNA Primer with DNA Ligase – “welds”/glues/bonds the Okazaki Fragments Replication fork DNA polymerase I DNA polymerase III lagging strand Okazaki fragments 5’ 3’ ligase primase 5’ SSB 3’ DNA polymerase III 5’ 3’ leading strand direction of replication 3’ 5’ helicase And in the end… Ends of chromosomes are eroded with each replication an issue in aging? ends of chromosomes are protected by telomeres Telomeres Expendable, non-coding sequences at ends of DNA short sequence of bases repeated 1000s times TTAGGG in humans Telomerase enzyme in certain cells enzyme extends telomeres prevalent in cancers Why? DNA polymerases DNA polymerase III 1000 bases/second main DNA building enzyme DNA polymerase I 20 bases/second primer removal, editing, & repair DNA polymerase III enzyme RNA is temporary so nature has not invested in a proof-reader for it! Editing & proofreading DNA 1000 bases/second = lots of typos! DNA polymerase I proofreads & corrects typos repairs mismatched bases excises abnormal bases repairs damage throughout life reduces error rate from 1 in 10,000 to 1 in 100 million bases UV radiation can cause molecular lesions, where T’s or C’s form a covalent bond between themselves Fast & accurate! It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome divide to form 2 identical daughter cells Human cell copies its 6 billion bases & divide into daughter cells in only few hours remarkably accurate only ~1 error per 100 million bases ~30 errors per cell cycle The “Central Dogma” flow of gene genetic information within a cell transcription DNA replication RNA translation protein Check for understanding #1