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
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
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
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
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.
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)
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.
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.
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.
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
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.
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.