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Chapter 12: DNA Mrs. Lipenta Upper Moreland High School What is DNA? DNA stands for deoxyribonucleic acid. It was discovered by James Watson and Francis Crick The shape of DNA can be described as a double helix. ◦ Think about it as a spiral staircase, or a ladder. Watson and Crick DNA is composed of nucleotides Each nucleotide is made up of 3 parts; a sugar, a phosphate, and a nitogenous base. In DNA the P sugar is deoxyribose. S N.B. The sides of the ladder (called the DNA backbone) is composed of the sugar and phosphates. The rungs of the ladder contain the nitrogenous bases. Chargaff 1947 DNA composition: “Chargaff’s rules” ◦ varies from species to species humans: A = 30.9% T = 29.4% G = 19.9% C = 19.8% The bases pair up based on Chargoff’s Rules A=T & C=G A = Adenine T = Thymine C = Cytosine G = Guanine Pairing like nucleotides did not fit the uniform diameter indicated by the X-ray data. ◦ A purine-purine pair would be too wide and a pyrimidine-pyrimidine pairing would be too short. ◦ Only a pyrimidine- purine pairing would produce the 2-nm diameter indicated by the X-ray data. How does it fit together? We need to look at the numbered Carbons Pay close attention to the 3’ and 5’ carbons 5’ attaches to the P 3’ attaches to the next nucleotide Remember nucleotides are monomers How do we link monomers? Through bonding But where do we get the energy? We need energy! What carrier energy from place to place in the body? Show me the Money!!!! Yes it is our old friend ATP… And he has brought along some friends CTP GTP Notice anything? ATP TTP Nucleotides CTP GTP WOW Why? ATP TTP Energy B.Y.O.E. Process where we end up with 2 exact copies of DNA. Steps of Replication ◦ Step 1 – Unzip DNA ◦ Step 2 – Add free nucleotides to the original strands of DNA. These nucleotides will add together and form the new strand of DNA. ◦ Step 3 – End up with 2 exact copies Replication: 1st step Unwind DNA ◦ helicase enzyme unwinds part of DNA helix stabilized by single-stranded binding proteins single-stranded binding proteins Replication 5' 3' Step 2 energy DNA P III Adding bases energy ◦ can only add nucleotides to 3 end of a growing DNA strand energy ◦ strand grow 5'3’ energy 3' 5' leading strand 2005-2006 Thanks to Kim Foglia for the animation New nucleotides can only fit one way. Remember they bring their energy with them. One side is copied continuously… no problem there. What about the other side? Thanks to Kim Foglia for the animation 5' 3' 5' 3' ligase energy 3' 3' leading strand 5' lagging strand 5' 2005-2006 Copying DNA Replication of DNA ◦ base pairing allows each strand to serve as a pattern for a new strand Leading & Lagging strands Leading strand - continuous synthesis Okazaki Lagging strand - Okazaki fragments - joined by ligase - “spot welder” enzyme 2005-2006 Okazaki fragments Helicase comes in and unzips the DNA Single-Stranded Bonding Proteins hold it open One side – the leading strand gets copied continuously The other side – the lagging strand gets copied in little fragments called Okazaki fragments DNA polymerase III is responsible for adding the nucleotides to the DNA, but it can only add to the strand, it can not start copying. Primase starts the copying by adding a small amount of RNA to the DNA. That small section of RNA is called the Primer Priming DNA synthesis We can’t have RNA in our DNA… DNA polymerase I comes in and removes sections of RNA primer and replaces with DNA nucleotides Also proofreads and corrects the DNA Now we have little fragments of DNA, need to glue them together. This is a job for Ligase. Ligase acts like the glue and will link the Okazaki fragments together. Replication fork DNA polymerase III lagging strand DNA polymerase I Okazaki 3’ primase fragments 5’ 5’ ligase 3’ 5’ SSB 3’ helicase DNA polymerase III 5’ leading strand 3’ direction of replication You need to make… helicase DNA polymerase III primase DNA polymerase I ligase single-stranded binding proteins Show me DNA replication Also don’t forget to B.Y.O.E. If I asked you a question about something in the textbook, would you read the entire textbook? What would you do? Why? What is the complementary strand? DNA = ATCGCGCTAATCGATCG DNA = ? Chargoff’s Rules During what part of the cell cycle does DNA replication occur? And in the end… Ends of chromosomes are eroded with each replication ◦ an issue in aging? ◦ ends of chromosomes are protected by telomeres 2005-2006 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? 2005-2006 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 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 DNA Replication A Team Effort! Large team of enzymes coordinates replication Bonding in DNA hydrogen 5’ bonds 3’ phosphodiester bonds 3’ 5’ ….strong or weak bonds? How do the bonds fit the mechanism for copying DNA? 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 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?! 1928 The “Transforming Factor” Frederick Griffith ◦ Streptococcus pneumonia bacteria was working to find cure for pneumonia ◦ harmless live bacteria mixed with heat-killed infectious bacteria causes disease in mice ◦ substance passed from dead bacteria to live bacteria = “Transforming Factor” Transformation? something in heat-killed bacteria could still transmit disease-causing properties The “Transforming Factor” mix heat-killed pathogenic & live pathogenic live non-pathogenic heat-killed non-pathogenic strain of bacteria strain of bacteria pathogenic bacteria bacteria A. B. C. D. mice die mice live mice live mice die 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 What’s the conclusion? ◦ injected DNA into bacteria transformed harmless bacteria into virulent bacteria Avery, McCarty & MacLeod Oswald Avery Colin MacLeod Maclyn McCarty 1952 | 1969 Hershey & Chase ◦ classic “blender” experiment ◦ worked with bacteriophage viruses that infect bacteria ◦ grew phage viruses in 2 media, radioactively labeled with either 35S in their proteins 32P in their DNA ◦ infected bacteria with labeled phages Models of DNA Replication Alternative models ◦ so how is DNA copied? Semi-conservative replication 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 ” How to prove it! 1958 Semi-conservative replication Make predictions… ◦ 15N strands replicated in 14N medium ◦ 1st round of replication? ◦ 2nd round? Hershey & Chase Martha Chase Alfred Hershey Protein coat labeled DNA labeled with 32P with 35S Hershey T2 bacteriophages are labeled with & Chase radioactive isotopes S vs. P bacteriophages infect bacterial cells bacterial cells are agitated Which to remove viral protein coats radioactive marker is found inside the cell? Which molecule carries viral 32P 35Sradioactivity radioactivity found genetic info? found in the medium in the bacterial cells 2005-2006 Radioactive phage & bacteria in blender ◦ 35S phage radioactive proteins stayed in supernatant therefore protein did NOT enter bacteria ◦ 32P phage radioactive DNA stayed in pellet therefore DNA did enter bacteria ◦ Confirmed DNA is “transforming factor” Your DNA!
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