Chapter 12 - DNA Structure & Replication

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1 History of DNA 1869 Miescher Chapter 12 - DNA Structure & Replication Analyzed the contents of the nucleus & discovered a substance (containing nitrogen & phosphorus) which he called nuclein 1909 Garrod Linked protein deficiencies to unusual phenotypes. Could proteins be the genetic (hereditary) material? 1929 Griffith Experimented with two phenotypically different bateria: R (rough coated) which caused no disease & S (smooth coated) which would cause pneumonia Mice injected with the R bacteria did not dies of pneumonia Mice injected with the S bacteria died of pneumonia When S bacteria were heat treated (to kill them), they no longer caused pneumonia Makes sense, but here s the kicker!

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5 1929 Griffith (continued) Chapter 12 - DNA Structure & Replication When heat treated S bacteria was co-injected with live R bacteria, the mice DIED of pneumonia & LIVE S bacteria was found in the dead mice! What HAPPPENED?

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7 1930s Avery, MacLeod, McCarty Something in the heat-treated S bacteria transformed the R bacteria into live S bacteria Mice were injected with a mixture of heat-treated S bacteria, live R bacteria & either: 1. A protease which destroys proteins 2. A DNAse which destroys DNA Mice given the protease mixture died of pneumonia & live S were recovered Mice given the DNAse mixture lived Conclusion: S type DNA is capable of transforming (changing) live R into S type Confirmation: They isolated DNA from the heat treated S bacteria & co-injected it with live R into mice = mice died & contained living S bacteria

8 1950 Hershey & Chase Chapter 12 - DNA Structure & Replication Confirmed that DNA & not protein is the genetic material Used viruses because: Virus structure is simple: nucleic acid core surrounded by a protein coat Once a virus infects a cell it causes the host cell to produce more viruses (this is the phenotypic change that will indicate a transformation of the host cell s genotype) What part of the virus gets into the host cell to induce this transformation? Experiment You can differentially mark proteins & nucleic acids using radioactive isotopes of sulfur & phosphorus. These isotopes give off radiation which can be detected. Proteins were labeled with S35 which made them glow Think Homer Simpson Nucleic acids were labeled with P32 which made them glow

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11 1950 Hershey & Chase Chapter 12 - DNA Structure & Replication They incubated their host bacteria cells with either S35 labeled viruses or P32 labeled viruses & here are the results Both sets of bacteria cells were transformed into cells that produced more of the viruses In the S35 labeled experiment the radioactivity was localized outside the bacteria cells. Proteins were not injected into the bacteria cells & the cells were still transformed In the P32 labeled experiment the radioactivity was localized inside the bacteria cells. Nucleic acids were injected into the bacteria cells & the cells were transformed Conclusion: Injection of nucleic acids induces transformation

12 Structure of DNA The basic building block of a DNA molecule is the nucleotide 1. Deoxyribose 5 carbon sugar forms part of the backbone structure 2. Nitrogenous base Information is coded in the sequence of these bases A. Purines Double ring structure Adenine (A) & Guanine (G) B. Pyrimidines Single ring structure Thymine (T) & Cytosine (C) 3. Phosphate group forms part of the backbone structure

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16 Structure of DNA Chapter 12 - DNA Structure & Replication Nucleotides are arranged end-to-end in a very specific orientation. The backbone of the DNA molecule is formed by the phosphate group & the sugar, while the nitrogenous bases extend perpendicular to the backbone Each DNA molecule is actually made up of two strands of DNA. The two strands associate with each other via their nitrogenous bases. Hydrogen bonds form between them, stabilizing the molecule into its familiar double helix form Adenine always forms hydrogen bonds with thymine Guanine always form hydrogen bonds with cytosine These introduces us to Chargaff s rule Regardless of species 1. The number of purines in DNA always equals the number of pyrimidines 2. The amount of adenine always equals the amount of thymine, while the amount of guanine always equals the amount of cytosine

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19 Structure of DNA Chapter 12 - DNA Structure & Replication In order for the nitrogenous bases to form these hydrogen bonds, the orientation of the backbones must be specific. DNA strands must be anti-parallel to each other, meaning that the sugar-phosphate backbones must be arranged in opposite directions to each other The specific directionality is determined by the position of specific carbons & is referred to as 5 to 3 Why is this important? Important for understanding the intricacies of DNA replication Why is DNA replication necessary? When does it occur?

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23 Overhead diagram of replication simple version

24 DNA Replication Chapter 12 - DNA Structure & Replication Origin of replication is where it all begins Helicases bind to the origin & begin unraveling the strands Single-strand binding proteins keep the strands separated Topoisomerases relieve tension placed on the DNA molecule caused by this unraveling, by creating transient cuts which allow the DNA to untwist a little & then repair the cuts Once the strands have separated, the strands become templates for replication Do you remember how the bases are matched up? New DNA daughter strands cannot be synthesized without some priming. As soon as the parental strands have separated, RNA primase adds a short sequence of RNA nucleotides to begin the replication process Once the RNA primer sequence has been laid down, DNA polymerase will begin adding DNA nucleotides to the new daughter strand of DNA until the strand is completed

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27 DNA Replication Chapter 12 - DNA Structure & Replication Since each of the parental strands is used as a template for replication, the process is referred to as semi-conservative replication How do we know that this process works in this fashion? 1957 Meselson & Stahl

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32 DNA Replication DETAILS! Chapter 12 - DNA Structure & Replication DNA replication must occur in a specific direction: 5 to 3 Since DNA replication must occur in this direction, a problem arises due to the antiparallelism of the parental strands DNA replication is continuous on one parental strand & discontinuous on the other DNA polymerase reads & lays down the new DNA on the daughter strand DNA ligase is important for joining the discontinuous DNA fragments on the lagging daughter strand & well as the entire DNA molecule as you will see Eukaryotic DNAs have multiple origins of replication which will reduce the time it takes for replication to occur

33 Overhead diagram of replication leading & lagging strand version

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36 PRACTICE QUESTIONS Chapter 12 - DNA Structure & Replication 1. How did the following individuals contribute to our knowledge of the importance of DNA as the hereditary material: Miescher, Griffith, Avery et al, Hershey & Chase? 2. What is Chargaff s rule? 3. What 3 components make up a DNA nucleotide? RNA nucleotide? 4. What is the difference between DNA & RNA? 5. What enzymes are used during the steps of replication?