DNA Replication and Chromosomes: DNA replication Flashcards
Where does DNA replication occur?
In a cell.
Of what is DNA replication a part?
The cell cycle.
Which are critical aspects of cell biology?
The enzymes required for DNA replication, DNA mutation, and repair.
Cell cycle control.
What do the critical aspects of cell biology allow?
The development of modern molecular biology techniques.
For what is the replication of each chromosome required?
For each round of cell growth and division.
With what does the replication of chromosomes be tuned?
The replication of all other cell components and structures.
When does DNA synthesis occur?
During ‘S-phase; of Interphase of cell cycle.
Where does DNA synthesis result?
In duplicate copies of each chromosome before cell division in M-phase.
What did Watson and Crick suggested with their base-pairing model of DNA, in 1953?
A replication mechanism.
How was the replication mechanism termed later on?
Semi-conservative replication.
What was happening in the semi-conservative replication?
Parental DNA strands were separated and served as templates.
Why were the parental DNA strands separated and served as templates?
For the replication of new daughter strands.
Who did question the semi-conservative replication and suggested other mechanisms for DNA replication?
Delbruck and others.
How many theories about DNA replication were in 1957?
3.
Which were the 3 theories about DNA replication in 1957?
- Semi-conservative.
- Conservative.
- Dispersive replication.
Who did address the 3 theories of DNA replication, and how?
Meselson and Stahl, with experiments.
How were the experiments of Meselson and Stahl characterised?
The most beautiful experiment in biology.
What did Meselson and Stahl do in their experiment in 1958?
They grew Escherichia coli in 15N media for one generation –> transferred cells into 14N media –|> continued incubation.
What happened in the Meselson-Stahl experiment?
DNA isolated from samples.
What did Meselson and Stahl do once they managed to isolate DNA from E. coli samples with 15N and 14N with media?
Took DNA from cultures –> ultra-centrifuged DNA –> photographed –> scanned –> determined DNA concentrations in tubes.
What did 14N and 15N controls confirm?
Light and heavy DNA positions.
What did the Meselson-Stahl experiment confirm?
DNA was replicated in a semi-conservative manner.
Exactly what Watson and Crick said in 1953.
What is responsible for DNA replication in vivo?
A replication complex/’machine’.
What does a replication machine generate?
Copies of whole chromosomes in each cell cycle.
Where are some elements also involved in DNA synthesis?
In DNA repair.
With what is DNA repair associated?
With replication.
As what does DNA repair result?
As the DNA damage result.
What does DNA Topoisomerase do?
It cuts DNA.
Why does DNA Topoisomerase cut DNA?
To release supercoils and allow it to relax.
What does DNA Helicase do?
It unwinds and opens out the DNA duplex.
Why does DNA Helicase unwind and open out the DNA duplex?
To provide single-stranded regions.
What are the single-stranded regions?
The start-points for DNA replication.
How is single DNA strand replication characterised?
Simple.
How is replication of both strands of DNA at the same time, characterised?
Difficult.
Why is replication of both DNA strands at the same time difficult?
Because the DNA Polymerases will get in each other’s way.
What does each strand replication need to avoid DNA Polymerases getting in each other’s way?
They need to tune with the other strand’s replication.
What does Topoisomerase cut exactly of DNA?
The phosphodiester backbone of DNA of the double helix.
Why does topoisomerase cut the phosphodiester backbone of the double helix of DNA?
To allow it be opened up without untwisting.
What do type 1 topoisomerases relax?
Single-stranded DNA.
What do type 2 topoisomerases relax?
Double-stranded DNA.
How is E. coli Topoisomerase 2 known?
DNA Gyrase.
What does Helicase do?
It melts the hydrogen bonds between base pairs in the double helix.
Open the DNA structure.
Forms the replication fork.
What do Helicase monomers form around the leading strand?
A doughnut shape hexamer.
What does the formation of a doughnut shaped hexamer around the leading strand pull through?
New sequences.
Unwinding parental double-stranded ahead of hexamer.
How is the lagging strand during DNA replication?
Free.
Not bound by Helicase.
Where do single-strand DNA-binding (SSB) proteins load onto?
On exposed single-strand sections.
Why do single-strand DNA-binding proteins load on exposed single-strand sections?
To straighten them out.
When do single-strand DNA-binding proteins fall off the exposed single-strand sections?
When RNA/DNA Polymerase comes along.
What does DNA Primase produce at the exposed single-stranded regions?
An RNA primer.
Why does DNA Primer produce an RNA primer at the exposed single-stranded regions?
To allow DNA Polymerase begin synthesis.
What does DNA Polymerase do to DNA once it produces RNA primer?
It extends primer with new DNA.
How is the extension of DA on the leading strand?
Continuous.
How is the extension of DNA on the lagging strand?
Broken into lots of small sections.
What does the extension of DNA on the lagging strand produce?
Okazaki fragments.
What can DNA Polymerase redo?
Base pairs mistakes.
What does DNA Polymerase manage to do when it can redo base pair mistakes?
Reduce level of random mutation.
How many exonuclease activities does DNA Polymerase have?
2.
Which are the 2 exonuclease activities DNA Polymerase have?
- 3’ –> 5’ exonuclease activity.
2. 5’ –> 3’ exonuclease activity.
What does the 3’ –> 5’ exonuclease activity allow DNA Polymerase to do?
Remove a mistake.
Replace the wrong nucleotide with the correct one.
What does the 5’ –> 3’ exonuclease activity allow DNA Polymerase to do?
Remove the next RNA primer when the replication machine has move far enough along the template strand.
Where must DNA Polymerase move?
Along the template strand in 5’ –> 3’ direction.
Why must DNA Polymerase move along the template strand in 5’ –> 3’ direction?
To add nucleotides to the growing 3’ -OH end of the new leading strand.
In what orientation is the other parental strand?
In the opposite orientation.
In what direction must a DNA Polymerase synthesizing a new lagging strand for the template (opposite strand) go?
In the opposite direction of the template strand.
What does DNA Ligase use?
ATP.
Why does DNA Ligase use ATP?
To catalyse the formation of phosphodiester bonds between Okazaki fragments.
What does ATP donate?
Pyrophosphate.
Why does ATP donate pyrophosphate?
To form a new 5’-triphosphate tail.
Where is the new 5’-triphopshate tail joined?
To the adjacent 3’-OH group.
Where is DNA Ligase essential?
In cloning.
What does DNA Ligase allow to happen?
2 pieces of DNA –> be linked together.
What does looping the DNA around allow?
DNA Polymerase synthesizing leading strand and DNA Polymerase synthesizing lagging strand –> movie in same direction.
By what is the replication machine lead?
By DNA Topoisomerase.
What does the replication machine include?
DNA Helicase.
RNA Primase.
DNA Ligase.
What is different in prokaryotic and eukaryotic enzymes?
Different names of enzymes included in the replication machine.
Where does DNA replication start?
At specific ‘origins’ of replication.
Where does DNA replication move?
In both directions.
What do large chromosomes have?
Multiple origins.
Why do large chromosomes have multiple origins?
To reduce time of replication of DNA before cell division.
How many times must each origin fire during DNA synthesis?
Only once.
How is the fire of origin organised?
By timing the synthesis of proteins which control the Origin Recognition. Complex (ORC).
Why is the synthesis of the proteins which control the ORC timed?
To trigger the S-Phase.
What does ORC recruit?
Helicase.
Why does ORC recruit Helicase?
To form the replication fork.
What does Phosphorylation of ORC send?
A signal that synthesis has begun.
What does ORC phosphorylation prevent?
Further initiation from the origin site.
