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What were the three prevailing models of replication?
- Conservative: each replicated DNA molecule consists of two old strands and two new strands
- Semiconservative: each replicated DNA molecule consists of one new strand and one old strand
- Dispersive: each replicated DNA molecule consists of old and new strands interspersed together
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What did the Meselson-Stahl experiment entail, and how did it help to resolve that replication occurs by the semi-conservative mode?
- E. Coli grown in 15N medium is cultured in 14N medium. The DNA is extracted and centrifuged after each generation.
- Gen 0: 15N/15N (single band)
- Gen 1: 15N/14N (single band)
- Gen 2: 14N/14N 15N/14N (two bands, equal)
- Gen 3+: 14N/14N 15N/14N (two bands, 14/14 larger
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Explain origin of replication with regards to euk and pro
- The site of initial replication
- Pro: have only a single region
- Euk: have multiple points of origin
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Explain bi-directional replication with regards to euk and pro
- DNA replication that occurs in both directions at once.
- Pro: replication IS bi-directional and continues until the forks merge at the termination region
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Explain replication fork with regards to euk and pro
- The point at which the strands of the helix are unwound.
- Starts at origin and moves along strand as replication occurs.
- Two forks are present if replication is bidirectional.
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What are the critical enzymes involved in the DNA replication process (prokaryotes only)
- Polymerase III: 5' - 3' polymerization, with 3' - 5' exonuclease activity for proofreading
- Polymerase I: removing the RNA primer and filling gaps after removal, DNA repair (3' - 5' and 5' - 3' exonuclease activity)
- Polymerase II, IV, V: DNA repair from external damage
- DNA helicase: unwinds DNA double helix while single-stranded binding proteins stabalize the unwound DNA
- DNA gyrase: relieves the supercoiling caused by DNA helicase
- RNA primase: adds the short RNA primer that DNA Polymerase III attaches to
- DNA ligase: joins the Okazaki fragments together (creates the phosphodiester bond)
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What are telomere ends and what is their relation to replication?
Telomeres are present at the end of linear chromosomes to preserve the integrity of the chromosome.
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Explain leading and lagging strands
- Leading strand: the DNA strand that serves as the template for continuous DNA synthesis
- Lagging strand: the DNA strand that serves as the template for discontinuous DNA synthesis.
- Okazaki fragments: short fragments of DNA each with an RNA primer on the lagging strand.
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How are nucleotides added to the elongating DNA strand, and what is the directionality of the process?
DNA Polymerase III adds nucleotides in a 5'-3' direction, requiring a free 3'-OH for each addition. The inital 3'-OH comes an RNA primer inserted by RNA primase.
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What are the sequential steps involved in stitching together the separate Okazaki fragments?
- RNA primase lays down the primer
- DNA polymerase III adds nucleotides to the free 3'-OH
- DNA polymerase I removes the RNA primer and, with the help of DNA ligase joins the strands
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Why is there a shortening of the telomere ends of chromosomes with each cell division?
When the lagging strand reaches the end of the chromosome there is no further Okazaki fragment with a 3'-OH to join with, and a gap is created.
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How does the telomerase enzyme work to prevent shortening of telomere ends?
- Telomerase is able to add repeats of the telomere sequence to the 3' end of the G-rich strand using 5'-3' synthesis. It creates an RNA strand and then uses reverse transcription to create a DNA strand from this RNA template. The DNA template is then moved into place and the process is repeated.
- Not typically active in somatic eukaryotic cells.
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