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DNA Replication - DNA Replication Structural Overview of...

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1 DNA Replication Structural Overview of DNA Replication Existing DNA strands act as templates for the synthesis of new strands. DNA replication relies on the complementarity of DNA strands. The process can be summarized as follows (see fig. 11.1): The two DNA strands come apart (parental strands). Each serves as a template strand for the synthesis of new strands (daughter strands). Experiment 11A – Three different models were proposed that described the net result of DNA replication. In the late 1950s, three different mechanisms were proposed for the replication of DNA (see fig. 11.2): Conservative model: both parental strands stay together after DNA replication. Semiconservative model: the double-stranded DNA contains one parental and one daughter strand following replication. Dispersive model: parental and daughter DNA is interspersed in both strands following replication. In 1958, Matthew Meselson and Franklin Stahl devised a method to investigate these models; they found a way to experimentally distinguish between daughter and parental strands. The hypothesis. Based on Watson’s and Crick’s ideas, the hypothesis was that DNA replication is semiconservative (see fig. 11.2b). Testing the hypothesis (see fig. 11.3). The starting material is a strain of E. coli that has been grown for many generations in the presence of 15 N; therefore, all of the nitrogen in the DNA is labeled with 15 N. Add an excess of 14 N-containing compounds to the bacterial cells so that all of the newly made DNA will contain 14 N. Incubate the cells for various lengths of time.
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2 Lyse the cells by adding lysozyme and detergent. Load a sample of the lysate onto a CsCl gradient. Centrifuge the gradients until the DNA molecules reach their equilibrium densities. Observe DNA within each gradient under UV light. The data (see fig. 11.3). Interpreting the data. After one generation, the DNA is “half-heavy”; this is consistent with both semi-conservative and dispersive models. After two generations, the DNA is of two types: “light” and “half-heavy”; this is consistent with only the semi-conservative model. Bacterial DNA Replication Bacterial chromosomes contain a single origin of replication (see fig. 11.4). 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. Replication is initiated by the binding of DnaA protein to the origin of replication (see figs. 11.5 and 11.6). 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.
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