Chapter Three Flashcards
What was the purpose of the Messelson-Stahl experiment?
The purpose was to figure out the mechanism of DNA replication - whether it was conservative, semi-conservative, or dispersive.
Explain how they set up the Messelson-Stahl experiment and how the conservative model was ruled out.
Experiment:
They grew ecoli (with normal Nitrogen) using radioactive N15. So when the first generation reproduced, the offspring would have N15 (more dense) in their bases.
Then, they put the offspring in light N14 solution, and let the DNA replicate.
Then they analyzed the DNA using centrifuges.
If the DNA was replicated conservatively, they would see a difference between the copied N15 DNA (which was conserved) and the newly copied N14 DNA.
If it was not they would see a single clump of DNA in the middle.
Explain how the experiment determined semi-conservative vs. dispersive.
The ecoli were kept in the light N-14 solution to replicate once more for the third generation.
If the DNA was replicated in a semi-conservative manner, they knew they would see two distinct bands show up: one lighter one from the N-14 strand being replicated to create a fully N-14 double strand, and a dense band from the original dense which was half dense and half light.
If the DNA was replicated in a dispersive manner, they knew they would see a single band that got lighter and lighter as replications continued.
What is the function of the cell during the G1 and G2 phases?
To make stuff - synthesize enzymes etc. that are important for cell function and division.
What is the packed-nature of the DNA in the different phases of the cell cycle?
G1 phase: has regions that are packed and regions that are unpacked.
S phase: DNA is completely unpacked
G2 phase: DNA is getting packed up
Mitosis: DNA is completely packed up
What are ARS’s? What is their purpose?
They are called autonomously replicating sequences. They are specific DNA sequences that act as the origin of replication for ORC’s.
What are ORC’s?
They are origin of replication complexes. They are proteins (enzymes) that are made in G1.
They randomly run around looking for a part of the DNA that fits their active site.
DNA - A is the first enzyme in the ORC. What does it do?
DNA - A kinks the ARS which allows the denaturing of the region that was kinked.
What is DNA helicase C?
DNA - C is a DNA helicase
ATP hydrolysis allows the helicase to untwist the DNA by breaking H bonds of the base pairs.
What are replication bubbles? What keeps them bubbles?
They are regions within the DNA that are “opened” by action of specific proteins in the ORC
Single stranded binding proteins bind to each opened strand to keep the strands independent.
What happens after formation of the replication bubble?
Once the double stranded DNAs are unzipped into single stranded DNAs, Primase, which is an RNA polymerase, will stick to the single stranded DNA and put down an RNA primer providing a 3’-OH group.
Now the DNA polymerase can latch on and put new DNA down.
What does DNA polymerase do?
- It synthesizes new strands starting from the RNA primer in the 5’ to 3’ direction.
- It can proofread in the 3’ to 5’ direction using its exonuclease activity.
- It can remove primers in the 5’ to 3’ direction using its exonuclease activity. (different polymerase)
What does a primer provide for DNA Polymerase?
How do primers get knocked off the DNA strand?
RNA polymerase provides a 3’-OH for DNA polymerase to attach and start adding nucleotides.
They first lose some stability when DNA polymerase starts working and synthesizes around 8-10 nucleotides.
How can there be leading and lagging strands on both new strands that are being synthesized?
Because there is two replication forks. At the opening of each fork, the DNA can be added by DNA polymerase continuously. These are the leading strands in respect to the two forks.
But, behind each leading strand is open DNA that cannot simply go away from the fork in a continuous fashion. Instead, behind each leading strand there needs to multiple initiations (RNA primers) to keep things moving in the 5’ to 3’ direction. These segments constitute the lagging strand and are called Okazaki fragments.
What makes DNA polymerase able to remove RNA primers?
The hydroxyl on the second carbon of the ribose sugar of the RNA’s bases is large and makes it unstable and prone to nucleophilic attack.
