DNA Replication 2 Flashcards
Does DNA replication only occur in one direction?
No - DNA replication is bi-directional and occurs at the replication forks.
Why do replication forks permits genomic stability when DNA replication is occurring?
Because ssDNA is kept to a minimum.
Why should single strand DNA be kept to a minimum?
ssDNA can be damaged by enzymes & mechanical shear (become fragmented).
Why is the simplest model for DNA replication wrong?
It shows both new strands could be synthesised at the same time as the duplex is opened.However, it requires that one new chain is synthesised in the 3’ – 5’ direction & DNA polymerase can NOT do this.
How is the leading strand of DNA synthesised?
The leading strand is continuously synthesised 5’-3’, in the direction of the replication fork movement.
How is the lagging strand of DNA synthesised?
The lagging strand is also synthesised 5’-3’ but discontinuously, as a series of short DNA pieces called Okazaki fragments. These are then joined to make long nascent DNA chains.
How many RNA primers does the leading strand require?
One RNA primer.
How many RNA primers does the lagging strand require?
Multiple RNA primers - one for each segment.
Does elongation of the lagging strand go in the same direction as the replication fork?
No - Elongation of the lagging strand is in the opposite direction to the direction of replication fork advance
What kind of DNA replication occurs with the lagging strand?
Discontinuous Replication.
How many replication forks are there in DNA replication and what does this mean for the direction of replication?
There are two replication forks - moving in opposite directions, therefore DNA replication is BI-DIRECTIONAL..
Since replication is bi-directional what does this mean each replication fork will have?
Each will have a leading and a lagging strand. (see diagram on phone for more help).
How many origins of replication do bacterial chromosomes have?
Normally only one origin of DNA replication.
How many origins of replication do eukaryotic chromosomes have?
They have multiple origins of replication. (Thousands)
Why do eukaryotes need multiple origins of replication?
There genomes are much larger - would take too long to replicate the DNA and the rate of replication is lower in eukaryotes, replication forks only travel at 50 nucleotides per second. (bacteria - 500-1000 nucleotides per second).
Why could the speed of the replication forks be slower in eukaryotes?
Increased difficult to replicate DNA that is packaged tightly in chromatin.
What is the origin of replication called in E.coli?
OriC - it is AT rich.
Describe how replication is initiated in E.coli?
OriC is bound by an initiator protein DnaA. Opens up a ~45 bp segment into single strands. DnaC binds and permits helicase, DnaB, binding.
Where does replication terminate in E.coli?
At specific ter sites.
At the end of replication how are the two double-stranded daughter chromosomes separated?
By DNA topoisomerase II
Who discovered DNA polymerase I and when?
Found in 1957 by A. Kornberg
What does the 5’-3’ exonuclease activity allow the DNA polymerase I to do?
Allows degradation of a strand ahead of the advancing polymerase. Useful in DNA repair and removal of RNA primers.
How larger is E.coli DNA polymerase I? (kDa)
102 kDa protein
What is the klenow fragment?
The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin.
Which domains of the DNA polymerase I enzyme does the klenow fragment contain?
3’-5’ exonuclease + polymerase. (klenow fragment - 68kDa)
Why is the klenow fragment helpful when carrying out experiments?
It retains the ability to proof read and polymerise DNA but cannot degrade any DNA - when you require DNA synthesis but no destruction of strand. It is often used for end “filling”.
Who discovered polymerase II and III in 1971?
Thomas Kornberg & Malcolm Gefter
Do polymerase II and III have the 5’-3’ exonuclease activity?
No - they are only capable of 3’-5’ exonuclease and 5’-3’ polymerase.
Which is the largest enzyme (Mr) and has the quickest rate of polymerisation?
DNA polymerase III - it’s Mr = 830,000 and it can polymerise at a rate of 250-1000 nucleotides per second.
What happens if there is an absence of DNA polymerase III?
It is lethal - the organism will die.
What type of enzyme is DNA polymerase III?
A holoenzyme.
Describe the structure of DNA polymerase III.
There are two core domains made up of the subunits - α, ε, and θ.These are linked to the are linked by a five-subunit clamp-loading complex (also known as the γ complex) with the composition τ2γδδ′.Two β clamps interact with the two-core subassembly, each clamp a dimer of the β subunit. The complex interacts with the DnaB helicase through the τ subunits.
What does an enzyme is distributive mean?
If an enzyme carries out a single reaction and then dissociates from substrates.
What does an enzyme is processive mean?
If an enzyme performs multiple actions before dissociating
Does DNA polymerase III dissociate easily from the template?
Yes it does - allowing it to recycle and begin synthesis of the next Okazaki fragment.
DNA polymerase III needs to remain on the template strand to synthesis the new strand how is this done?
The β-sliding clamp keeps the DNA polymerase on the DNA while it is moving – and increases the enzyme’s processivity.
When does the β-sliding clamp release the DNA polymerase III?
When it encounters dsDNA.
Why do DNA polymerase enzymes need a proof reading ability?
Reliance on complementary base pairing is not sufficient to ensure accuracy (non-Watson-Crick base-pairing is possible and does occur).
What do proof reading mechanisms ensure?
Proof-reading mechanisms ensure that fidelity is very high – only 1 error for every 10^9 nucleotides copied.
What is the first mechanism of quality control (proof reading) that the DNA polymerase has?
Correct nucleotide has higher affinity for polymerase as it correctly base-pair with template. Phosphodiester bond formation involves conformational change in DNA polymerase so incorrectly-bound nucleotides do not fit active site.
What is the second mechanism of quality control that the DNA polymerase has?
3’-5’ exonuclease activity of DNA polymerasesTakes place immediately after incorrect addition to growing chain.Polymerase can’t extend such a strand- requires a base-paired 3’-OH end of the primer strand.
Name all the main proteins involved in DNA replication.
DNA polymerase III DNA polymerase I DNA helicase DNA gyrase (Type II topoisomerase) DNA primase Single-stranded DNA binding protein DNA ligase
What is DNA primase called in bacteria?
DnaG
What is the replisome?
It is the combination of the the proteins involved in DNA replication - contains two DNA polymerases.
Describe the trombone model.
The lagging strand template DNA loops round, bringing the two DNA polymerases into a complex. This brings the 3’ end of a completed Okazaki fragment close to the start site for the next fragment.
What is the rate-limiting step in DNA replication?
Unwinding of the DNA.
Why does the lagging strand synthesis start later than the leading strand synthesis.
because it requires a high amount of primers.
What does the phrase - ‘DNA unwinding and synthesis are concomitant’ mean?
There are closely associated.
When can lagging strand synthesis start?
When sufficient ssDNA has been produced.
Describe the synthesis of the lagging strand.
After completing a fragment the lagging strand holoenzyme relocates to a new primer.Then locked in with β-clamp (about 1 a second).When the polymerase encounters the previously synthesised fragment, the Pol III core releases the DNA and loses its affinity for the β-clamp. However, it is held in place by connections to the Pol III core involved in leading strand synthesis.
How is synthesis of the lagging strand completed?
DNA polymerase III elongates chain from primer, then falls off.DNA polymerase I binds and uses its 5’-3’ exonuclease activity to remove the RNA from the Okazaki fragment, replacing it with DNA using its 5’-3’ polymerase activity.DNA ligase links the Okazaki fragments.
Which enzyme seals the Okazaki fragments together (the nick)?
The 3’-OH and the 5’ phosphate groups are joined together, catalysed by DNA ligase, in a reaction requiring ATP (in euakryotes, above), or NAD (nicotinamide adenine dinucleotide) in E. coli.
Overall, what type of replication occurs in DNA replication?
Semi-discontinuous, as the leading strand is synthesised continuously and the lagging strand is synthesised discontinuously.
What is the role of the DNA polymerase III α complex?
Synthesises RNA/DNA primers for Okazaki fragments in lagging strand synthesis- has both a primase and a polymerase activity.
How does DNA ligase ligate the Okazaki fragments between a 5’ phosphate and a 3’ hydroxyl?
DNA ligase must activate the 5’ phosphate by transferring AMP to it. After activation of the 5’ phosphate, displacement of AMP produces a phosphodiester bond to seal the nick.
