Lecture4 - Synthesis of DNA DNA Replication 1 The double...

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1 Synthesis of DNA DNA Replication
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2 “Antiparallel” (opposite 5’-->3’ direction) “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material” Watson & Crick 1953 The double helical structure of DNA c.f. Figure3.9 c.f. Figure3.11
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3 1. selection of dNTPs based on Watson-Crick basepairing 2. nucleophilic attack of 3’-OH group on incoming nucleosidetriphosphate 3. hydrolysis of eliminated PPi by thepolymerase- ie. reaction is irreversiblein 5’to3’
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4 “copying mechanism for the genetic material” Prediction: replication should be semi-conservative Test this prediction: Meselson and Stahl
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5
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6 Meselson-Stahl 1958 Fractionation by density: ultracentrifugation
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7 Meselson-Stahl experiment demonstrating semi-conservative replication Lodish Fig. 4-32b 1. Make a gradient (e.g. sucrose gradient of 20-70%) 2. Add sample 3. High speed centrifuge 4. Sample will move up or down until density reaches buoyancy/density of sucrose 5. visualize bands under UV (DNA will fluoresce) and photograph. 6. Take measurements over many generations. Generations
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8 Meselson-Stahl experiment demonstrating semi-conservative replication Lodish Fig. 4-32b Start with H-H-->H-L detected--> then mostly LL… SC 1. Density gradient centrifugation after shift to 14 N-containing media 2. Bands visualized under UV (DNA will fluoresce) and photographed. 3. Take measurements over many generations.
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9 Replication bubble: extending on one direction or both directions? uni-directional vs. bidirectional one fork vs. two forks
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10 DNA Replication is bi-directional EM of replicating SV40 DNA Lodish Fig. 4-35 What is the distance between the center of the replication bubble and the end of the DNA?
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