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Chapter 10 Notes.docx

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Robert Boulianne

Chapter 10 – DNA Replication Different Theoretical Modes of Replication • Semiconservative replication has each newly replicated DNA molecule consisting of one “old” strand and one “new” strand. • Conservative replication will have two DNA molecules, one molecule with both strands composed of “old” DNA and one molecule with both strands of “new” DNA • Dispersive replication has both strands composed of a mixture of “old” and “new” DNA Proof for Mode of DNA Replication • The Meselson-Stahl Experiment – Grew bacterial cells (E. coli) for many generations in medium supplemented with 15NH 4l as the only nitrogen source. • Remember the nitrogenous bases have nitrogen atoms, so the DNA will now be labelled15ith this “heavy” version of nitrogen 14 – N contains one more neutron than naturally occurring N. – Molecules containing N are more dense than molecules containing N 14 15 – Importantly,14NA containing N can be distinguished from DNA containing N. Sedimentation Equilibrium Centrifugation • Samples forced by centrifugation into a density gradient of a heavy metal salt solution – Usually CsCl (cesium chloride) • Molecules of DNA will migrate into the density gradient. – 15e DNA will stop moving when it reaches a region of CsCl that m14ches its own density – N-DNA will move further down the tube of CsCl compared to N-DNA. Semiconservative Replication in Eukaryotes • In 1957 Taylor, Woods and Hughes provided evidence of semiconservative replication in broad bean plants (Vicia faba). • Technique used was autoradiography – Can locate location of radioactive isotope in a cell – A photographic emulsion is placed over a histological section/sample and placed in the dark • Radioactivity exposes the emulsion just like light would expose a photographic film. • Exposure of emulsion results in tiny black dots – Locations of black dots = location of radioactive isotope in cell Taylor et al. Experiment • Labelled DNA with H thymidine for one generation – Then placed in “cold” medium – Arrested cells at metaphase with colchicine over two division cycles – Performed autoradiography. – Results, at first metaphase, both chromatids labelled, while in second metaphase only one chromatid labelled ...semiconservative replication Origins, Forks and Units of Replication • Origin of Replication – Location where DNA replication is initiated – Replication is bidirectional from the origin • Replication Fork is where DNA strands are being unwound – Initially appear at origin and then 2 forks move apart in opposite directions • A replicon represents how much DNA is replicated following one initiation event – Eg E. coli has only one origin of replication (oriC) for its entire 4.6 million base pairs (bp) of circular DNA DNA Polymerase I • Kornberg accomplished in vitro synthesis of E. coli DNA. His reaction mixture included: a. A DNA fragment (act as template). b. Radioactively labeled dNTPs (dATP, dGTP, dTTP, and dCTP). a. DNA Polymerase I (DNA Pol I). b. Enzyme originally called the Kornberg enzyme, now known as DNA polymerase I. Once isolated, its activity could be characterized, showing that the above components are 2+ required, along with Mg ions for maximum activity. Roles of DNA Polymerases All DNA polymerases link dNTPs into DNA chains. Main features of the reaction: – An incoming nucleotide is attached by its 5’-phosphate group to the 3’-OH of the growing DNA chain. Energy comes from the release of two phosphates from the dNTP. The DNA chain acts as a primer for the reaction. – The incoming nucleotide is selected by its ability to hydrogen bond with the complementary base in the template strand. The process is fast and accurate. – DNA polymerases synthesize only from 5’ to 3’. Initiation of Replication in Prokaryotes • Initiator proteins attach. E. coli’s initiator protein i
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