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Human Molecular B > Lecture 4 - DNA Replication > Flashcards

Lecture 4 - DNA Replication Flashcards

1
Q

Pol A

A

DNA replication

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2
Q

Pol B

A

Base excision Repair

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3
Q

Pol G

A

Mitochondrial Replication

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4
Q

PolDi

A

DNA replication

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5
Q

Pol E

A

DNA replication

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6
Q

Pol Z

A

Bypass synthesis

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7
Q

Pol H

A

Bypass synthesis

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8
Q

Pol Q

A

DNA repair

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9
Q

Pol I

A

Bypass synthesis

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10
Q

Pol K

A

Bypass synthesis

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11
Q

Pol L

A

Base excision Repair

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12
Q

Pol M

A

Non homologous end joining

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13
Q

Pol S

A

Sister chromatid cohesion

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14
Q

REVIL

A

Bypass synthesis

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15
Q

TDT

A

Antigen receptor diversity

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16
Q

In the human genome, how many DNA polymerase enzymes are involved in DNA replication of genome?

A

3

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17
Q

What is the size of eukaryotic replicon?

A

40-100kb in length

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18
Q

Individual replicons are activated at characteristic times during which phase?

A

S Phase

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19
Q

What does regional activation pattern suggest?

A

Replicons near one another are activated at the same time

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20
Q

What is key to initiation of replication?

A

Creating the replication fork at the origin oriC

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21
Q

What are key elements in initiation?

A

Initiation requires several enzymatic activities:
Helicases
Single-strand binding protein
Synthesis of primers

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22
Q

What does initiation at oriC require?

A

Sequential assembly of large protein complex on the membrane

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23
Q

Define licensing factor

A

A factor located in the nucleus and necessary for replication that is inactivated or destroyed after one round of replication
New factors must be provided for further rounds of replication to occur

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24
Q

Prior to replication

A

Nucleus contains active licensing factor

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25
Q

After replication

A

Licensing factor is in the nucleus (inactive)

Licensing factor in the cytoplasm cannot enter nucleus

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26
Q

What does dissolution of nuclear membrane during mitosis allow?

A

Licensing factor to associate with nuclear material

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27
Q

What does cell division generate?

A

Daughter nuclei competent go support replication

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28
Q

Name the proteins that are licensing factors

A

MCM proteins
Cdc6
Cdt1

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29
Q

What is ORC?

A

A protein complex that is associated with origins throughout the cell cycle

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30
Q

What is Cdc6?

A

Unstable protein
Synthesised only in G1
Binds to ORC and allows MCM proteins to bind

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31
Q

What is Cdt1?

A

Facilitates MCM loading on origins

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32
Q

What happens to Cdt1 and Cdc6 once replication starts?

A

They leave ORC

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33
Q

How does negative regulation of replication occur?

A

G2 nuclei synthesise Geminin
Geminin sequester Cdt1
MCM proteins are prevented from assembling on freshly synthesised DNA
Geminin is degraded on completion of mitosis
DNA can recommence at the next S Phase

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34
Q

Which enzymes are involved in DNA assembly?

A

DNA polymerases

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35
Q

When DNA is synthesised which end are nucleotides added to?

A

3’-OH end

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36
Q

What direction will DNA grow in?

A

5’ to 3’

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37
Q

Nucleophilic attack by the ribose 3’-OH group on alpha phosphate of the incoming nucleotide will have a triphosphate which will release what?

A

Pyrophosphate (PP) - A diphosphate

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38
Q

What is the construction of DNA process?

A

Primer has 3’-OH end
The incoming nucleotide will have 5’ triphosphate
This will cause the release of diphosphate upon addition

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39
Q

What do bacterium or eukaryotic cell have?

A

Several different DNA polymerase enzymes

40
Q

What are the 2 processes that bacterial DNA polymerase undertakes?

A

a) semiconservative replication

b) Repair reactions

41
Q

Which gene codes for enzyme I?

42
Q

What is the function of enzyme I?

A

Major repair enzyme

43
Q

Which gene codes for enzyme II?

44
Q

What is the function of enzyme II?

A

Replication restart

45
Q

Which gene codes for enzyme III?

46
Q

What is the function of enzyme III?

47
Q

Which gene codes for enzyme IV?

48
Q

What is the function of enzyme IV?

A

Translation replication

49
Q

Which gene codes for enzyme V?

A

umuD’ 2 C

50
Q

What is the function of enzyme V?

A

Translation replication

51
Q

What is an example of nuclease activity of DNA polymerase?

A

Bacterial DNA polymerase I had an exonuclease effect

Combined with DNA synthesis to perform nick translation

52
Q

What is the role of nick translation?

A

Replaces part of preexisting strand of duplex DNA with newly synthesised material

53
Q

What is the process of Nick translation?

A

Nick generated 3’-OH
5’p groups DNA synthesis extends 3’end
Old strand is degraded by 5’ to 3’ exonuclease

54
Q

What is high-fidelity DNA polymerases involved with?

A

Involved in replication

Have a precisely constrained active site that favours binding of Watson-Crick base pairs

55
Q

Define processivity

A

The ability of an enzyme to perform multiple catalytic cycles win a single template instead of dissociating after each cycle
DNA polymerases generally require additional factors for processivity

56
Q

What is the common structure of DNA polymerase?

A

Large cleft composed of 3 domains that resemble a hand

DNA lies across the palm in a groove created by the fingers and thumb

57
Q

Leading strand

A

5’ to 3’

58
Q

Lagging strand

A

3’ to 5’

59
Q

What are the discontinuous short fragments of the lagging strand called?

A

Okazaki fragments

60
Q

What is semi-discontinuous replication?

A

The mode of replication in which one new strand is synthesised continuously while the other is synthesised discontinuously

61
Q

What are helicase?

A

Separate the strands of DNA using energy provided by hydrolysis of ATP
A hexameric helicase moves along one strand of DNA
Encircling it and binding to duplex DNA
Base pairs are separated
The helicase releases duplex

62
Q

What is DnaB?

A

Helicase that unwinds DNA for replication in E.coli

63
Q

What is MCM?

A

Helicase that unwinds DNA for replication in prokaryotes

64
Q

What is a single stranded binding protein (SSB) required for?

A

Maintain separated strands

65
Q

What does all DNA polymerases require?

A

Free 3’-OH priming end to initiate DNA synthesis

66
Q

What can provide the 3’-OH priming end?

A

RNA primer
Nick in DNA
Priming protein

67
Q

What is RNAa primer synthesised by?

68
Q

What is a primase?

A

A type of RNA polymerase that synthesises short segment of RNA that will be used as primers for DNA replication

69
Q

Where does primase exist?

A

Complex with DNA polymerase alpha

70
Q

How do leading and lagging catalytic units behave?

A

Differently

71
Q

In E.coli which enzyme units are required to synthesis the leading and lagging strand?

72
Q

In E.coli which enzyme is required to synthesise the leading and lagging strands?

A

DnaE - DNA pol III alpha

73
Q

Which three DNA polymerases make up the eukaryotic replication dork?

A

Alpha
Delta
Epsilon

74
Q

What is DnaB?

A

Helicase that unwinds DNA for replication in E.coli

75
Q

What is MCM?

A

Helicase that unwinds DNA for replication in prokaryotes

76
Q

What is a single stranded binding protein (SSB) required for?

A

Maintain separated strands

77
Q

What does all DNA polymerases require?

A

Free 3’-OH priming end to initiate DNA synthesis

78
Q

What can provide the 3’-OH priming end?

A

RNA primer
Nick in DNA
Priming protein

79
Q

What is RNAa primer synthesised by?

80
Q

What is a primase?

A

A type of RNA polymerase that synthesises short segment of RNA that will be used as primers for DNA replication

81
Q

Where does primase exist?

A

Complex with DNA polymerase alpha

82
Q

How do leading and lagging catalytic units behave?

A

Differently

83
Q

In E.coli which enzyme units are required to synthesis the leading and lagging strand?

84
Q

In E.coli which enzyme is required to synthesise the leading and lagging strands?

A

DnaE - DNA pol III alpha

85
Q

Which three DNA polymerases make up the eukaryotic replication dork?

A

Alpha
Delta
Epsilon

86
Q

What is the role of polymerase alpha l/primase complex?

A

Complex initiates and then primes the synthesis of both DNA strands (approx 10-30 bases)
Synthesises RNA primer and then extends the DNA by 20 bases
Not processive and remains associated with lagging strand

87
Q

What is the role of processive DNA polymerase Epsilon?

A

Elongates the leading strand

88
Q

What is the role of DNA polymerase delta?

A

Elongates the lagging strand

89
Q

What is PCNA?

A

A sliding clamp that helps processivity if polymerase Epsilon and delta

90
Q

What is the clamp loader?

A

Places the sliding clamp (PCNA) on DNA where they form a circular clamp around the DNA, increases processivity

91
Q

What is an example of clamp loader in eukaryotes?

92
Q

What is the process of polymerase switching?

A

DNA synthesis starts with polymerase alpha
Then replaced by the more processive delta and Epsilon
Pol alpha must contact RPA for stable attachment to the primed site
The RFC-PCNA complex competes with pol alpha for RPA
RFC loads PCNA onto DNA but remains associated with PCNA
Pol delta competes with RFC for both RPA and PCNA thereby recruiting PCNA and the 3 primer terminus for chain extension

93
Q

Why is the core on the leading strand processive?

A

It’s clamp (PCNA) keeps it on the DNA

94
Q

What happens with regards to clamping in Okazaki fragments?

A 
The clamp associated with the core on the lagging strand dissociates at the end of each Okazaki fragments and reassembles for the next fragment 
Core polymerase (pol delta) and the clamp (PCNA eukaryotes, clamp Beta in bacteria) dissociate at completion of Okazaki fragments synthesis and reassociate at the beginning
95
Q

What are Okazaki fragment linked by?

96
Q

What does synthesis of Okazaki fragment require?

A 
Priming
Extension 
Removal of RNA primer 
Gap filling 
Nick ligation
97
Q

How does DNA Ligase seals nicks between adjacent nucleotides?

A

Employing an enzyme-AMP intermediate

Human Molecular B (13 decks)

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