DNA replication 2

Question 1

Does DNA replication only occur in one direction?

Answer 1

No - DNA replication is bi-directional and occurs at the replication forks.

Question 2

Why do replication forks permits genomic stability when DNA replication is occurring?

Answer 2

Because ssDNA is kept to a minimum.

Question 3

Why should single strand DNA be kept to a minimum?

Answer 3

ssDNA can be damaged by enzymes & mechanical shear (become fragmented).

Question 4

Why is the simplest model for DNA replication wrong?

Answer 4

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.

Question 5

How is the leading strand of DNA synthesised?

Answer 5

The leading strand is continuously synthesised 5’-3’, in the direction of the replication fork movement.

Question 6

How is the lagging strand of DNA synthesised?

Answer 6

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.

Question 7

How many RNA primers does the leading strand require?

Answer 7

One RNA primer.

Question 8

How many RNA primers does the lagging strand require?

Answer 8

Multiple RNA primers - one for each segment.

Question 9

Does elongation of the lagging strand go in the same direction as the replication fork?

Answer 9

No - Elongation of the lagging strand is in the opposite direction to the direction of replication fork advance

Question 10

What kind of DNA replication occurs with the lagging strand?

Answer 10

Discontinuous Replication.

Question 11

How many replication forks are there in DNA replication and what does this mean for the direction of replication?

Answer 11

There are two replication forks - moving in opposite directions, therefore DNA replication is BI-DIRECTIONAL..

Question 12

Since replication is bi-directional what does this mean each replication fork will have?

Answer 12

Each will have a leading and a lagging strand. (see diagram on phone for more help).

Question 13

How many origins of replication do bacterial chromosomes have?

Answer 13

Normally only one origin of DNA replication.

Question 14

How many origins of replication do eukaryotic chromosomes have?

Answer 14

They have multiple origins of replication. (Thousands)

Question 15

Why do eukaryotes need multiple origins of replication?

Answer 15

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).

Question 16

Why could the speed of the replication forks be slower in eukaryotes?

Answer 16

Increased difficult to replicate DNA that is packaged tightly in chromatin.

Question 17

What is the origin of replication called in E.coli?

Answer 17

OriC - it is AT rich.

Question 18

Describe how replication is initiated in E.coli?

Answer 18

OriC is bound by an initiator protein DnaA. Opens up a ~45 bp segment into single strands. DnaC binds and permits helicase, DnaB, binding.

Question 19

Where does replication terminate in E.coli?

Answer 19

At specific ter sites.

Question 20

At the end of replication how are the two double-stranded daughter chromosomes separated?

Answer 20

By DNA topoisomerase II

Question 21

Who discovered DNA polymerase I and when?

Answer 21

Found in 1957 by A. Kornberg

Question 22

What does the 5’-3’ exonuclease activity allow the DNA polymerase I to do?

Answer 22

Allows degradation of a strand ahead of the advancing polymerase.
Useful in DNA repair and removal of RNA primers.

Question 23

How larger is E.coli DNA polymerase I? (kDa)

Answer 23

102 kDa protein

Question 24

What is the klenow fragment?

Answer 24

The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin.

Question 25

Which domains of the DNA polymerase I enzyme does the klenow fragment contain?

Answer 25

3’-5’ exonuclease + polymerase.

klenow fragment - 68kDa

Question 26

Why is the klenow fragment helpful when carrying out experiments?

Answer 26

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”.

Question 27

Who discovered polymerase II and III in 1971?

Answer 27

Thomas Kornberg & Malcolm Gefter

Question 28

Do polymerase II and III have the 5’-3’ exonuclease activity?

Answer 28

No - they are only capable of 3’-5’ exonuclease and 5’-3’ polymerase.

Question 29

Which is the largest enzyme (Mr) and has the quickest rate of polymerisation?

Answer 29

DNA polymerase III - it’s Mr = 830,000 and it can polymerise at a rate of 250-1000 nucleotides per second.

Question 30

What happens if there is an absence of DNA polymerase III?

Answer 30

It is lethal - the organism will die.

Question 31

What type of enzyme is DNA polymerase III?

Answer 31

A holoenzyme.

Question 32

Describe the structure of DNA polymerase III.

Answer 32

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.

Question 33

What does an enzyme is distributive mean?

Answer 33

If an enzyme carries out a single reaction and then dissociates from substrates.

Question 34

What does an enzyme is processive mean?

Answer 34

If an enzyme performs multiple actions before dissociating

Question 35

Does DNA polymerase III dissociate easily from the template?

Answer 35

Yes it does - allowing it to recycle and begin synthesis of the next Okazaki fragment.

Question 36

DNA polymerase III needs to remain on the template strand to synthesis the new strand how is this done?

Answer 36

The β-sliding clamp keeps the DNA polymerase on the DNA while it is moving – and increases the enzyme’s processivity.

Question 37

When does the β-sliding clamp release the DNA polymerase III?

Answer 37

When it encounters dsDNA.

Question 38

Why do DNA polymerase enzymes need a proof reading ability?

Answer 38

Reliance on complementary base pairing is not sufficient to ensure accuracy (non-Watson-Crick base-pairing is possible and does occur).

Question 39

What do proof reading mechanisms ensure?

Answer 39

Proof-reading mechanisms ensure that fidelity is very high – only 1 error for every 10^9 nucleotides copied.

Question 40

What is the first mechanism of quality control (proof reading) that the DNA polymerase has?

Answer 40

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.

Question 41

What is the second mechanism of quality control that the DNA polymerase has?

Answer 41

3’-5’ exonuclease activity of DNA polymerases
Takes 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.

Question 42

Name all the main proteins involved in DNA replication.

Answer 42

DNA polymerase III	
DNA polymerase I 
DNA helicase	
DNA gyrase (Type II topoisomerase)	
DNA primase		
Single-stranded DNA binding protein	
DNA ligase

Question 43

What is DNA primase called in bacteria?

Answer 43

DnaG

Question 44

What is the replisome?

Answer 44

It is the combination of the the proteins involved in DNA replication - contains two DNA polymerases.

Question 45

Describe the trombone model.

Answer 45

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.

Question 46

What is the rate-limiting step in DNA replication?

Answer 46

Unwinding of the DNA.

Question 47

Why does the lagging strand synthesis start later than the leading strand synthesis.

Answer 47

because it requires a high amount of primers.

Question 48

What does the phrase - ‘DNA unwinding and synthesis are concomitant’ mean?

Answer 48

There are closely associated.

Question 49

When can lagging strand synthesis start?

Answer 49

When sufficient ssDNA has been produced.

Question 50

Describe the synthesis of the lagging strand.

Answer 50

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.

Question 51

How is synthesis of the lagging strand completed?

Answer 51

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.

Question 52

Which enzyme seals the Okazaki fragments together (the nick)?

Answer 52

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.

Question 53

Overall, what type of replication occurs in DNA replication?

Answer 53

Semi-discontinuous, as the leading strand is synthesised continuously and the lagging strand is synthesised discontinuously.

Question 54

What is the role of the DNA polymerase III α complex?

Answer 54

Synthesises RNA/DNA primers for Okazaki fragments in lagging strand synthesis- has both a primase and a polymerase activity.

Question 55

How does DNA ligase ligate the Okazaki fragments between a 5’ phosphate and a 3’ hydroxyl?

Answer 55

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.