bio325-lec14,15 - D NA Replication St ructural Overview...

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DNA Replication Structural Overview Existing DNA strands are templates for the synthesis of new strands. DNA replication relies on complementary DNA strands. Process: The two DNA strands come apart (parental strands). Each is a template strand for the synthesis of new strands (daughter strands). Experiment 11A – Three mechanisms described the result of DNA replication. Conservative : parental strands stay together Semiconservative model: double-stranded DNA has 1 HH LL HL HL HL HL HL HL parental and 1 daughter strand after replication. 50% HH Dispersive model: parental and daughter DNA is 50% LL 100% HL 100% HL interspersed in both strands following replication. (15N has an extra neutron) How to distinguish between daughter and parental strands: LH LL LL LH 8x HLLL Hypothesis : DNA replication is semiconservative Testing : Starting with E. coli thats grown for many generations with 15N; so all nitrogen in the DNA is labeled with 15N. Add excess of 14N so that all newly made DNA will contain 14N. Incubate for various lengths of time. Lyse cells by adding lysozyme and detergent. Load sample of lysate onto a CsCl gradient. Centrifuge until DNA molecules reach equilibrium densities. Observe under UV light. Interpreting the Data : After one generation, DNA is “half-heavy”; this relies on semi-conservative and dispersive models. After two generations, DNA is “light” and “half-heavy”; this is consistent with only the semi-conservative model. 25% HH 50% HL 100% HLLL 75% LL 50% LL Bacterial DNA Replication DNA synthesis begins at the origin of replication; each bacterial chromosome has only one.
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Synthesis proceeds bidirectionally around the chromosome. The replication forks eventually meet at the opposite side to end replication. Binding DnaA protein to origin initiates replication: In E. coli this is called oriC – origin of replication. 3 DNA sequences in oriC are significant: AT-rich region, DnaA boxes, and GATC methylation sites. DNA replication is begun by binding of DnaA proteins to the DnaA box sequences and to each other; this stimulates cooperative binding of an additional 20 to 40 DnaA proteins to form a large complex. Other proteins that cause the DNA to bend also bind; this causes the region to wrap around the DnaA proteins and separate the AT-rich region. The DnaA proteins, with DnaC protein, get DNA helicase enzymes (DnaB protein) to this site. When a DNA helicase encounters a double-stranded region, it breaks the hydrogen bonds between the two strands generating two single strands. Two DNA helicases begin strand separation in the oriC and continue to separate the DNA in both/ opposite directions creating 2 replication forks; this is bidirectional replication . Proteins required at replication fork:
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bio325-lec14,15 - D NA Replication St ructural Overview...

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