DNA and Replication. Flashcards
Describe and explain the type of DNa HYPOTHESIS
- SEMICONSERVATIVE
- CONSERVATIVE
- DISPERSIVE
Why was the semiconservative replication not accepted initially?
The semiconservative replication model was initially thought to be impossible, because DNA is plectonemic – strands can’t be separated without unwinding (As many DNA were circular, and how do you separate circular strands? It’s hard to separate circular strands)
A typical bacterial chromosome is a circular DNA molecule of about 4,000,000 bp (4Mb)
Imagine this is converted to a linear molecule
A bacterium can divide every 20 minutes
So, it must replicate its genome every 20 minutes
So, it must replicate 200,000 bp per minute
There are 10 bp per turn of the helix
The DNA molecule would have to rotate at
20,000 revs per minute! Too fast/Impossible. Therefore, scientists thought it was impossible to replicate using the semiconservative replication.
Explain the set-up of the Meselson- Stahl experiment?
· It was designed to distinguish between the three modes of DNA replication and work out which occurs in E. coli bacteria
· (Very simple and well-designed experiment!)
· A culture of bacteria was grown in a medium containing heavy nitrogen
· The commonest isotope of nitrogen has an atomic weight of 14 – 14N
· The heavy isotope of nitrogen has an atomic weight of 15 – 15N
· Nitrogen is a component of the DNA bases so it can get into DNA
· All the bacteria ‘s DNA was labeled with heavy 15N
· Different weight nitrogen can be distinguished by using density gradient centrifugation
· DNA + CsCL solution (6 M)
· Centrifugate
· Density gradient forms is the CsCl solution/ Buoyant density
· Band will form in the CsCl solution based on the N weight.
CONCLUISION:
Heavy DNA bacteria is mixed with light DNA bacteria
First division after 20 mins: 50% light- 50% heavy DNA. (Using the experiment method) the band is in the middle.
2 Replication after 40 mins: 2 bands = 1 band contains (14N-14N) so it’s at the top and the other (14N-15N) bear in mind all these are replicated in 14N culture, which means the free nucleotide that move in and complementary base pair with template strand are 14N isotopes.
As the second replication happens it’s result shows that it must be due to semi-conservative replication because with dispersive we would have the exact amount of isotope as each other in the buoyancy in the middle as we have equal amount of both isotopes in the second replication as well , whereas with conservative only there would be 2 different isotopes one heavy and the other light which is not the case as proved by Meselson’s experiment. So, the right one is semi-conservative replication which gives expected correct results.
What are TOPOismorase?
DNA topoisomerase separates strands without unwinding DNA double helix.
What does DNA topoisomerase I do?
DNA topoisomerase type I in action, makes single strand ‘cross over’ the other strand so relaxing DNA strands and creating space for the DNA replication for the new complementary strands.
- Type topoisomerase breaks strand and crosses it over the other DNA strand so it’s more relaxed therefore less likely to supercoil
What does DNA TOPOISMOERASE II do?
pe II DNA isomerase (major enzyme used) circular DNA
- DNA topoisomerase binds to first DOUBLE strand called G-segment, which gets completely cut so other DOUBLE strand will pass through therefore crossing over
- DNA T segment which is the other DOUBLE strand crosses over
DNA strands are released as space is created preventing supercoiling
What is DNA direction synthesis occured on?
DNA synthesis is always in the 5’ to 3’ direction.
What is required to start replication at fork?
A primer is required to start synthesis
How is DNA corrected?
Exonuclease activity.
During replication the separated single strands must be protected
The strands are protected by single-strand binding proteins or SSBs, prevent strands from attaching to each other/prevent attacks by nuclease as single strand DNA is more susceptible to DNases
What happens towards the end of DNA replication?
DNA tolomerase adds a lot of nucleotide sequences which will allow the strand to be completed as the primer will be able to bind to so DNA synthesis will occur
sequence added by TOLOMERASE TTAGGG
