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BMS 242 - Advanced Cell and Molecular > DNA replication > Flashcards

DNA replication Flashcards

1
Q

How is DNA ‘semi-conservative’?

A

Each parent strand is used as a template

Contains information which dictates the order of the nucleotides in the newly synthesised strand

Each daughter strand contains one strand from the parent strand

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2
Q

Which direction does DNA synthesis occur in?

How is this different to the movement of RNA pol II?

A

5’ –> 3’

RNA pol II moves along DNA in the 3’ –> 5’ direction

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3
Q

Which bonds are present in DNA?

A

Phosphodiester bonds

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4
Q

How does DNA replication occur?

A

Sequential polymerisation of of new nucleotide at the 3’ end:

Nucleophillic attack by the 3’ -OH on the sugar on the incoming DEOXYRIBONUCLEOSIDE TRIOSPHOSPHATE

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5
Q

Why is DNA synthesis effectively an ‘irreversible reaction’?

A

It is coupled to the breakdown of PPi (pyrophosphate) to 2Pi (2 x inorganic phosphate)

The breakdown of the 2 high energy phosphate bond provides the free energy for DNA synthesis

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6
Q

Why is DNA synthesis a high exothermic reaction?

A

Production of 2 inorganic phosphates from pyrophospahte as each deoxyribonucleoside phosphate is added to the growing DNA chain

Breaking a phosphate bond releases high energy required for the DNA synthesis

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7
Q

Why are molecules of DNA added as deoxyribonucleoside TRIphosphates?

A

COUPLED REACTION:

To able the 2 of phosphate molecules to be released as PRYOPHOSPHATE

And then, to allow pyrophosphate to be broken down into 2 individual phosphate molecules

Breakage of TWO high energy bonds

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8
Q

What enzyme break pyrophosphate into 2 inorganic phosphate molecules?

A

Pyrophosphatase

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9
Q

What is the ‘equation’ for DNA synthesis?

A

dNTP + (dNMP)n –> (dNMP)n+1 + 2Pi

Deoxyribonucleoside TRIphosphate + Deoxyribonucleoside MONOphosphate –> …

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10
Q

How is DNA synthesis initiated?

A

By creating a replication fork where DNA strands are separated

By the extension of an RNA primer

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11
Q

Which enzyme separates the paired DNA strands?

A

DNA helicase

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12
Q

How does DNA synthesis occur on the ‘lagging strand’?

What does this mean?

A

In the OPPOSITE direction to the replication fork

Still in the 5’ –> 3’ direction (DNA is anti-parallel)

Means that the lagging strand cannot be synthesised continuously

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13
Q

What are Okazaki fragments?

A

Short fragments formed on the lagging strand of DNA synthesis, due to the antiparallel and unidirectional orientation of DNA

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14
Q

What enzyme synthesises the RNA primer on DNA?

A

DNA primase (a type of RNA polymerase)

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15
Q

How long is the DNA primer?

A

20 nucleotides in length

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16
Q

How does DNA primase bind to DNA?

A

WITHOUT the need for a primer

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17
Q

What does DNA polymerase do?

On the lagging strand?

A

Extends the RNA primer laid down by DNA primase

On the lagging strand:
- Extends the primer to the 5’ end of the previous Okazaki fragment

  • THEN, extends the 3’ end of the UPSTREAM Okazaki fragments after ribonuclease H has removed the RNA template
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18
Q

What does ribonuclease H do?

A

Removes the RNA primer between the junction of Okazaki fragments

Creating a gap

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19
Q

What is ribonuclease H specific for?

A

The RNA component of the RNA-DNA hybrid molecule

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20
Q

What does DNA ligase do?

A

Seals the nick between 2 Okazaki fragments in a 2 step reaction to create one CONTINUOUS strand

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21
Q

Where does DNA ligase get the energy from to ligate Okazaki fragments together?

A

ATP hydrolysis

From the breakdown of pyrophosphate by pyrophosphatase

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22
Q

What is the first step of catalysation by DNA ligase ‘sealing the nick’?

A
  • Covalent attachment of AMP from ATP to the 5’ end of the nick, forming ADP (adenosine diphosphate)

Uses 1 molecule of ATP

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23
Q

What is the second step of DNA ligase ‘sealing the nick’?

A
  • NUCLEOPHILLIC attack (by the 3’ hydroxyl) on the phosphate bond between the 2 phosphates within the ADP
  • Releases AMP
  • Creates a new phosphodiester bond in the backbone of DNA
24
Q

Why is ‘sealing the nick’ energetically favourable and irreversible?

A

COUPLED REACTION of conversion of PPi to 2Pi

25
Q

What does DNA helicase use to separate parental strands of DNA at the replication fork?

How is it used?

A

ATP

Used to give DNA helicase its forwards momentum to unwind DNa

26
Q

How does DNA helicase move on DNA?

A

SPINS around and wraps itself around ONE of the strands parental DNA

27
Q

What is Werner syndrome?

What is is caused by?

A

Premature ageing

Caused by:
- Autosomal recessive mutations in the WRN gene encoding RecQ helicase (a DNA helicase)

  • Causes inefficient replication of the genome during GROWTH and REGENERATION of adult tissues
28
Q

What is bloom syndrome?

What is is caused by?

A

Cancer

Caused by:
- Loss-of-function mutation in RecQ-family DNA helicase which maintains GENOME INTEGRITY (in DNA repair)

29
Q

What is the processivity of DNA polymerase enhanced by?

A

Association with the SLIDING CLAMP

30
Q

Describe processivity

A

Once the first nucleotide has been polymerised, there is a HIGH PROBABILITY that the next and next will be

31
Q

How does the sliding clamp work?

A

2 additional proteins assemble onto the DNA PRIOR to the recruitment of DNA polymerase at the PRIMER-TEMPLATE JUNCTION

1) Sliding clamp - FIXES DNA pol to the primer-template junction
2) Clamp loader - Attaches the sliding clamp to the primer-template junction

Prevents DNA polymerase from FALLING off the DNA template

32
Q

How is DNA polymerase attached to the primer-template junction?

A

1) Clamp loader with ATP takes the clamp and fixes it around the primer-template junction

2) Once the ring of the clamp is complete:
- ATP –> ADP + Pi
- Clamp loader dissociates and DNA polymerase is recruited

33
Q

What do single-stranded binding proteins do?

A

Keep single-stranded DNA in the replication fork UNBOUND

Make the DNA nucelotides readily available to be read by DNA polymerase

34
Q

What would happen is SSBs were not present?

A

2 parental strands (between the point of DNA helicase and DNA polymerase) would rebind to eachother, once DNA helicase gets far enough ahead

35
Q

How do SSBs attach to DNA?

A

Rapid, cooperative way

Decorate the parental strands between DNA helicase and DNA polymerase

36
Q

What do DNA topoisomerases do?

What does this allow?

A

Prevent DNA becoming tangled during DNA replication:

  • Nick and reseal the backbone of DNA
  • To allow the strands to rotate around eachother and release the stress on the DNA strands
37
Q

Why are DNA topoisomerases needed?

A

Unwinding of DNA at the replication fork by DNA helicase induces SUPERCOILING into the DNA helix, ahead of DNA helicase

Prevents progression of the enzymes

38
Q

What do Type I topoisomerases do?

A

Nick and reseal ONE of the 2 DNA strands

39
Q

What do Type II topoisomerases do?

A

Nick and reseal BOTH of the 2 DNA strands

40
Q

Which type of topoisomerase uses ATP?

A

Type II (nick and reseal BOTH)

41
Q

What is highly conserved between E.coli and human replication forks?

A

KEY components:

RNA primers
DNA polymerase 
SSBPs
Sliding clamp
Ribonuclease H 
DNA ligase
DNA topoisomerases I and II
42
Q

Where is DNA replication initiated?

A

At the replication origin

43
Q

What does the replicator origin in DNA do?

A

Recruit INITIATOR proteins

Which direct the initiation of DNA replication

44
Q

Describe the initiation of DNA replication in eukaryotes

A

Biphasic:

1) Replicator Selection: formation of a pre-replicative complex
2) Origin Activation: unwinding of DNA and recruitment of DNA polymerase

45
Q

When does the replicator selection phase of DNA replication initiation occur?

A

In G1

46
Q

When does the origin activation phase of DNA replication initiation occur?

A

S

47
Q

What ensures that each replication origin is only used ONCE per cell cycle?

A

Temporal seperation of the 2 stages:

1) Replicator selection
2) Origin activation

48
Q

Describe the replicator selection phase of DNA replication initiation

A

Formation of a Pre-Replicative Complex (pre-RC):

1) Origin Recognition Complex (ORC) binds to replicator sequence in the replicator origin
2) ORC recruits helicase-loading proteins Cdc6 and Cdt1 (which bind to the ORC)
3) Helicase Mcm2-7 binds to the complete formation of the pre-RC
- Forming a STABLE complex at a SELECTED replicator origin

49
Q

How many molecules of helicase Mcm2-7 are needed in DNA replication and why?

A

2

As there are 2 replication forks which move away from eachother

50
Q

Describe the origin activation phase of DNA replication initiation

A

High levels of cyclin-dependant kinase (Cdk) activity:

  • BIND to and ACTIVATE the existing Pre-RC
  • PREVENT new Pre-RC from forming
51
Q

Why can the selected origins not be activated in G1?

A

There is no Cdks present to activate the Pre-RC

52
Q

How do Cdk levels ensure that chromosomes are replicated exactly ONCE per cell cycle?

A
  • Levels of Cdks remain HIGH in G2 - preventing the formation of any new pre-RC
  • Levels drop at the end of M phase - allowing new pre-RC to form at the stat of G1
53
Q

Describe the ‘End Replication Problem’

A
  • Created by the need for RNA primers for DNA replication
  • RNA primer removed by ribonuclease H
  • All the gaps which are created by the removal of the primer can be closed by DNA polymerase and DNA ligase
  • APART FROM ONE: at the far end, as there is no DNA sequence before it for DNA polymerase to elongate
  • If nothing done about this: progressive shortening of this DNA strand, which could result in the loss of important genes
54
Q

What prevents the loss of DNA sequence at the 5’ end of the lagging strand?

A

TTAGGG repeats added to the 3’ end of the template strand by telomerase

55
Q

What is the structure of telomerase?

A
  • Ribonucleoprotein: made of many proteins and ONE intrinsic RNA
  • Intrinsic RNA has a sequence of 9 nucleotides long: AAUCCCAAU
  • Which has 6 nucleotides complimentary to TTAGGG
56
Q

What is the ‘telomerase shuffle’?

A
  • The synthesis of the telomere repeat sequences, which is done as a step-wise process
  • Joins to the DNA sequence with the 6 complimentary nucleotides and then shuffles forwards 6 nucleotides so that the back end of the RNA is anneled to the front end of the newly synthesised DNA

BMS 242 - Advanced Cell and Molecular (24 decks)

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