bioc441 - 2008AnsProbSet3

bioc441 - 2008AnsProbSet3 - Biochemistry 441 Answers to...

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Unformatted text preview: Biochemistry 441 Answers to homework set #3: DNA replication and repair Ted Young Page1/4 1. Short, newly synthesized pieces of DNA were identified by Okazaki only after denaturing the DNA and centrifuging it to separate molecules of different sizes. Why was it necessary to denature the DNA? Denaturation was necessary to separate the short Okazaki fragments strands from the template to which it was hydrogen bonded. Without denaturation the nascent strands would sediment rapidly, being carried along with the template strand to which it was hydrogen bonded . 2. A very important experiment was done to prove that DNA and RNA were covalently linked in Okazaki fragments. Radioactive Okazaki fragments were made in vivo by adding 32 P-dNTP (labeled in the α-position ) to E. coli which had been treated (permeabilized) to allow dNTP to diffuse into the cells and be used as precursors for DNA synthesis. After purifying the Okazaki fragments they were treated with OH- to hydrolyze the suspected RNA portion. Acid insoluble material was removed and the resulting nucleotides were separated by chromatography and electrophoresis. Four radioactive spots were detected after autoradiography. What were they? Ribonucleotide monophosphates . It will help to draw out a diagram of the nucleotides that were used in the experiment, and what happens to the label in the α-position of dNTP when the phosphodiester bond is hydrolyzed. Are the labeled nucleotides ribo- or deoxyribonucleotides? Did addition of deoxyribonucleotides by Pol III begin after a particular base in the template? No How would you know? If polymerization of DNA had begun after a particular base in the template, the 3’ end of all of the Okazaki fragments would have the same base, which would be complementary to the base in the DNA template. In this case, a single ribonucleotide would have picked up the radioactivity from the DNA, and a single spot, representing that ribonucleotide, would have been detected by autoradiography. Since four spots were detected, it implies that polymerization of DNA does not begin after a particular base in the template ....
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This note was uploaded on 04/15/2010 for the course BIOC 441 taught by Professor Young&parson during the Spring '08 term at University of Washington.

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bioc441 - 2008AnsProbSet3 - Biochemistry 441 Answers to...

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