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DNA Replication

DNA Replication. Part 1 Principle Features. Mechanistic Overview. Identical base sequences. Figure 11.1. More than 1 way to replicate a cat . Figure 11.2. How does it really happen?. 1958 - Matthew Meselson and Franklin Stahl

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DNA Replication

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  1. DNA Replication Part 1 Principle Features

  2. Mechanistic Overview Identical base sequences Figure 11.1

  3. More than 1 way to replicate a cat Figure 11.2

  4. How does it really happen? • 1958 - Matthew Meselson and Franklin Stahl • Experimentally distinguished between daughter and parental strands

  5. Figure 11.3 11-9

  6. DNA Figure 11.3 11-10

  7. Interpreting The Data After one generation, DNA is “half-heavy” After ~ two generations, DNA is of two types: “light” and “half-heavy” consistent with both semi-conservative and dispersive models consistent with only the semi-conservative model

  8. Bacterial DNA Replication • DNA synthesis begins at a site termed the origin of replication • Each bacterial chromosome has only one • Synthesis of DNA proceeds bidirectionally around the bacterial chromosome • The replication forks eventually meet at the opposite side of the bacterial chromosome • This ends replication

  9. Theta Mode of Replication of Circular DNA Figure 11.4

  10. Theta Mode of Replication of Circular DNA

  11. Initiation of Replication • The origin of replication in E. coli is termed oriC • origin of Chromosomal replication • Three types of DNA sequences in oriC are functionally significant • AT-rich region • DnaA boxes • GATC methylation sites

  12. OriC of E. coli Figure 11.5

  13. How the Ori Works: What happens at the Ori doesn’t stay at the Ori • HU and IHF also bind causing the region to wrap around the DnaA proteins and melts the AT-rich region • Replication is initiated by binding of DnaAproteins to DnaA box sequences Figure 11.6

  14. Replication Initiation • Bidirectional replication Figure 11.6

  15. Unwinding DNA • DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them • This generates positive supercoiling ahead of each replication fork • DNA gyrase travels ahead of the helicase and alleviates these supercoils • Single-strand binding proteins bind to the separated DNA strands to keep them apart

  16. DNA Synthesis is Bidirectional Two nascent, labeled strands at each fork means both parent strands serve as templates

  17. Demonstration of Bi-Directional Synthesis

  18. Nucleotide Polymerization Reaction

  19. Implication of Bidirectional Synthesis • RULE: Polymerization can only happen in 5'3' direction • Starting at 1 spot • only 1 strand can serve as template • but both strands do • therefore, one strand synthesized continuously • leading strand • the other strand made discontinuously • lagging stand

  20. Okazaki Fragments Priming • If model is correct, should be able to find lagging strand fragments • 1000-2000 nt long DNA fragments • Discovered by Reiji & Tuneko Okazaki

  21. Okazaki Fragments Begin with ~15nt RNA Primer

  22. Origins, Replicons & Replisomes • Origin of replication • 1 per bacterial chromosome or plasmid • Many per eukaryotic chromosomes • Replicon • Region of DNA replicated from a single origin • Consists of 2 replisomes moving in opposite directions • Replisome • 2 DNA pol holoenzymes and all associated proteins (primase, helicase, clamploader & clamps)

  23. Multiple Origins of Replication Figure 11.20 Bidrectional DNA synthesis Replication forks will merge

  24. Eukaryotic Replicons In Action

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