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bio214i - Biology 214/414 2009 Packet#8 Omit sections 11.13...

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Biology 214/414 2009 Packet #8 Omit sections 11.13, 11.14 Suggested problems: Chapter 11: 2, 3, 16, 18, 19, 20, 21, 22, 25, 28, DNA replication I. DNA replication is “semi-conservative ”. ( Figures 11-1, 2 ) a. This follows directly from its double helical structure. b. Each parental single strand directs the synthesis of a new complementary single strand according to base pairing requirements. c. This yields two exact copies of the original double helix—each with one new and one original single strand d. This was proved in 1958 by the “Meselson-Stahl ” experiment. ( Figures 11-3,4 ) i. They grew E. coli on a heavy isotope of N, 15 N (the common isotope is 14 N) for many generations so that the DNA had a higher density than normal (“heavy” vs. “light” DNA) ii. They then transferred the cells to 14 N medium for various times. iii. Then they harvested the cells, isolated their DNA, and determined the DNA’s density by centrifugation on “CsCl equilibrium density gradients ”. iv. After one generation in 14 N medium, the DNA density was half way between light and heavy. v. After two generations half of the DNA was light and half of the DNA was half way between heavy and light. vi. These results are consistent only with semi-conservative replication. ( Figure 11-2 ) e. Fairly early on in the study of DNA replication semi- conservative replication was observed directly by electron microscopy due to the fact the semiconservative replication produces “replication forks ”. ( Figure 11-6 ) i. For circular chromosomes (such as those for E. coli or certain phage) one can actually see the parental strand
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separating at replication forks to form “Cairns ” or “ θ structures. ii. In E. coli , many phage, and eukaryotes, replication is “bidirectional ” from an “origin of replication ” (“O ”). iii. In a few cases replication is unidirectional starting at the origin. iv. Phage and bacteria have a single origin in each chromosome; multiple origins (probably 100’s or 1000’s) exist in eukaryotic chromosomes. ( Figure 11-14 ) II. DNA synthesis/mechanism/enzymology a. All cell processes are mediated by enzymes, which are usually proteins (and sometimes RNA’s). b. The first DNA synthesizing enzyme that was isolated and extensively characterized was E. coli “DNA polymerase I (also known as “pol I ”), which has the basic characteristics shared by all DNA polymerases. ( Figures 11-7, 8 ) i. As “substrates ”, they all require all four 5’ deoxynucleotide triphosphates (substrates are molecules
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