DNA Replication 2 Flashcards Preview

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Flashcards in DNA Replication 2 Deck (55)
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1
Q

Does DNA replication only occur in one direction?

A

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

2
Q

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

A

Because ssDNA is kept to a minimum.

3
Q

Why should single strand DNA be kept to a minimum?

A

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

4
Q

Why is the simplest model for DNA replication wrong?

A

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.

5
Q

How is the leading strand of DNA synthesised?

A

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

6
Q

How is the lagging strand of DNA synthesised?

A

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.

7
Q

How many RNA primers does the leading strand require?

A

One RNA primer.

8
Q

How many RNA primers does the lagging strand require?

A

Multiple RNA primers - one for each segment.

9
Q

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

A

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

10
Q

What kind of DNA replication occurs with the lagging strand?

A

Discontinuous Replication.

11
Q

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

A

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

12
Q

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

A

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

13
Q

How many origins of replication do bacterial chromosomes have?

A

Normally only one origin of DNA replication.

14
Q

How many origins of replication do eukaryotic chromosomes have?

A

They have multiple origins of replication. (Thousands)

15
Q

Why do eukaryotes need multiple origins of replication?

A

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).

16
Q

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

A

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

17
Q

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

A

OriC - it is AT rich.

18
Q

Describe how replication is initiated in E.coli?

A

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

19
Q

Where does replication terminate in E.coli?

A

At specific ter sites.

20
Q

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

A

By DNA topoisomerase II

21
Q

Who discovered DNA polymerase I and when?

A

Found in 1957 by A. Kornberg

22
Q

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

A

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

23
Q

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

A

102 kDa protein

24
Q

What is the klenow fragment?

A

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

25
Q

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

A

3’-5’ exonuclease + polymerase. (klenow fragment - 68kDa)

26
Q

Why is the klenow fragment helpful when carrying out experiments?

A

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”.

27
Q

Who discovered polymerase II and III in 1971?

A

Thomas Kornberg & Malcolm Gefter

28
Q

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

A

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

29
Q

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

A

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

30
Q

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

A

It is lethal - the organism will die.

31
Q

What type of enzyme is DNA polymerase III?

A

A holoenzyme.

32
Q

Describe the structure of DNA polymerase III.

A

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.

33
Q

What does an enzyme is distributive mean?

A

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

34
Q

What does an enzyme is processive mean?

A

If an enzyme performs multiple actions before dissociating

35
Q

Does DNA polymerase III dissociate easily from the template?

A

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

36
Q

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

A

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

37
Q

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

A

When it encounters dsDNA.

38
Q

Why do DNA polymerase enzymes need a proof reading ability?

A

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

39
Q

What do proof reading mechanisms ensure?

A

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

40
Q

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

A

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.

41
Q

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

A

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.

42
Q

Name all the main proteins involved in DNA replication.

A

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

43
Q

What is DNA primase called in bacteria?

A

DnaG

44
Q

What is the replisome?

A

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

45
Q

Describe the trombone model.

A

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.

46
Q

What is the rate-limiting step in DNA replication?

A

Unwinding of the DNA.

47
Q

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

A

because it requires a high amount of primers.

48
Q

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

A

There are closely associated.

49
Q

When can lagging strand synthesis start?

A

When sufficient ssDNA has been produced.

50
Q

Describe the synthesis of the lagging strand.

A

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.

51
Q

How is synthesis of the lagging strand completed?

A

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.

52
Q

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

A

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.

53
Q

Overall, what type of replication occurs in DNA replication?

A

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

54
Q

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

A

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

55
Q

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

A

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.