L5 DNA replication Flashcards
Which one of the following conclusions is supported by Chargaff’s chromatography data on the base composition of DNA in different species?
a) DNA is helical in structure
b) DNA contains pairings between one purine and one pyrimidine
c) AT base-pairs are joined by two hydrogen bonds
d) DNA strands are anti-parallel
e) Eukaryotic chromosomes are linear
b) DNA contains pairings between one purine and one pyrimidine
What are the three types of DNA replication?
- Conservative
- Semi - Conservative
- Dispersive
What is conservative replication of DNA?
- Original DNA strand is not replicated
- A completely new, identical DNA molecule is synthesised from scratch
What is semi - conservative DNA replication?
Two new DNA double helices are formed. Each new helix contains one original strand (one from parent DNA) and one newly synthesised strand
What is dispersive DNA replication ?
Here the parental DNA molecule is broken down into small fragments. These fragments then serve as templates for the synthesis of new DNA segments, resulting in two daughter DNA molecules that are a mixture of parental and newly synthesized DNA.
Which of the three types of replication is proven to be appropriate in human beings?
Semi - Conservative.
Who was responsible for the proven type of replication?
Matthew Meselson and Franklin Stahl (1958)
What did Meselson and Stahl use to do their experiment?
Used differences between isotopes to do their experiment
In this case, Nitrogen: DNA contains lots of Nitrogen atoms
How did they use isotopes of nitrogen to differentiate DNA?
They took advantage of their weight difference between 14N and 15N using it to seperate them.
Give a short summary of the Meselson and Stahl experiment.
- Bacteria grown in 15N will be heavy DNA.
- They transferred them to media containing 14N, the new DNA will be light.
- The heavy and light molecules are then seperated by ultracentrifugation.
What was added into the experiment for creating a density gradient to seperate the DNA molecules?
Caesium chloride gradients.
How was DNA with different densities seperated?
- DNA is isolated from cells which is then mixed with CsCl solution and placed in a centrifuge.
- It was then centrifuged for days at a very high speed.
What was observed after centrifugation?
- DNA molecules moved to positions where their density was equal to CsCl solution.
- There were three segments of DNA seperated.
- The most heaviest DNA will be at the bottom.
- The hybrid DNA is at the centre.
- The light DNA is at the top.
Why was the conservative replication ruled out of the experiment?
- This model would have predicted two distinct bands in the first generation: one heavy and one light but this was not observed
Why was the dispersive replication ruled out of the experiment?
This model would have predicted a single band of intermediate density in all generations, as DNA would be a mixture of N14 and N15 fragments. This was not observed
Describe the replication fork
1) Unwinding: Enzymes like helicase unwind the double-stranded DNA helix, breaking hydrogen bond between pairs
2) Separation: Two single strands of DNA are separated, creating a Y-shaped structure
3) Template for replication: Each single strand serves as a template for the synthesis of a new complementaty DNA strand
4) Enzyme activity: DNA polymerase helps add new nucleotides to growing DNA strands
What are all the enzymes involved in DNA replication? (6)
- Primase
- DNA polymerase
- DNA ligase
- Topoisomerase
- Helicase
- Single strand binding protein.
What are the functions of DNA polymerase?
- They add nucleotides one at a time in the 5’ - 3’ direction.
- They use template strands to form H-Bonds.
- They add tens or hundreds of nucleotides per second.
What is the function of Primase?
Primase is responsible for generating a primer and they are made up of usually RNA.
What does ligase do?
Ligase joins the loose ends together into a single strand of DNA by base pairing nucleobases.
What does topoisomerase do?
Topoisomerase relieves pressure from
overwinding around the replication bubble by
making and resealing breaks in the DNA
What does helicase do?
Helicase breaks hydrogen bonds
between the two DNA strands,
separating them
What does SSB (Single strand binding) protein do?
SSB binds to the separated strands,
preventing them from reannealing
What are leading strands?
They are the strands which get replicated continuously in the 5’ to 3’ direction.
What are lagging strands?
Strands that are discontinuous and are replicated in pieces.
What are the discontinous pieces of the lagging strand called?
Okazaki fragments
How does the erosion of genetic material at ends of linear chromosomes happen?
- Primer removal at the end of the chromosomes leaves a gap that cannot be filled as there is no DNA polymerase following up.
- So, every round of replication a little piece is lost.
- This is a problem for the lagging strand at each end of the linear DNA.
How is this erosion of end chromosomes solved?
Telomeres
What are telomeres?
Short DNA sequences that are repeated over and over at the ends of the chromosomes.
Which enzyme is responsible for the replinishing of telomeres at the end of each replication?
Telomerase.
If DNA replication were conservative, how many DNA bands would
Meselson and Stahl observe after two rounds of replication in the
presence of the “heavy” nitrogen isotope?
a) 0
b) 1
c) 2
d) 3
e) 4
C. 2
