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BMS242 Cell and Molecular S1 > L5 DNA Replication > Flashcards

L5 DNA Replication Flashcards

1
Q

What does the semi conservative nature of DNA replication mean?

A

Each of the parental strands acts as a template for the synthesis of the daughter strands

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2
Q

What direction can DNA rep occur in

A

5’ –> 3’

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3
Q

What is found at the 3’ end

A

Hydroxyl

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4
Q

What is found at the 5’ end

A

Phosphate

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5
Q

What can be said about the orientation of the template strands

A

They are antiparallel

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6
Q

Why is replication essentially irreversible

A

Because it is coupled to the breakdown of pyrophosphate making is highly unfavourable

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7
Q

What is the structure of pyrophosphate

A

2 inorganic phosphate molecules

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8
Q

What enzyme catalyses the breakdown of pyrophosphate

A

Pyrophosphatase

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9
Q

What is the equilibrium constant for the replication … what does this mean

A

10^5

Highly favoured in the forward direction

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10
Q

What is a dNTP

A

Dinucelotide tripohsophate

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11
Q

What is a dNMP

A

Dinucleotide monophosphate

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12
Q

What is the formation for replication

A

dNTP + (dNMP)n –> (dNMP)n+1 + 2Pi ``

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13
Q

What enzyme seperates the paired DNA strands

A

DNA helicase

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14
Q

Why can the leading strand be continuously synthesied

A

5’ –> 3’ direction

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15
Q

What must the lagging strand be synthesied as

A

Okazaki framgments

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16
Q

All synthesis of DNA occurs as a result of extension of an

A

RNA primer

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17
Q

What enzyme lays the RNA primer

A

DNA primase

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18
Q

What does DNA primase require for it to work

A

DNA template

NTPs

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19
Q

What 4 enzymes are required for the synthesis of the lagging strand

A

DNA primase
DNA polymerase
Ribonuclease H
DNA ligase

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20
Q

Function of DNA primase

A

Makes and lays down the RNA primer

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21
Q

Function of DNA polymerase

A

Extension of the RNA primer with dNTP

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22
Q

Function of Ribonuclease H

A

Removes the RNA primer

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23
Q

What can be said about the specificity of ribonuclease H

A

Absolutely specific for RNA/DNA hybrid molecules

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24
Q

Function of DNA ligase

A

Seals the nick joining the Okazaki fragments

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25
Q

Does DNA ligase require ATP

A

Yes requires the energy of ATP hydrolysis

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26
Q

What is used in the first stage of ligation and released in the second

A

ATP used

AMP released

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27
Q

Describe the equations (two coupled) for ligation

A

ATP + 5’-P –> P-P + 5’-P-AMP

P-P –> 2 Pi + free energy

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28
Q

Describe the energy requirements of DNA helicase

A

Cont. using ATP

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29
Q

How does DNA used the energy released from ATP

A

Wraps around the parental strands when it spins the rotational force is converted to forward motion
ATP GIVES THE FORWARD MOMENTUM

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30
Q

Werners Syndrome

A

Progeria (premature ageing)
Autosomal recessive
In RECQ helicase gene WRU

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31
Q

Bloom syndrome

A

Rare cancer syndrome caused by loss of function in RecQ family DNA helicases which maintains the integrity of the genome

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32
Q

Describe how a sliding clamp is used to aid the processivity of DNA polymerase

A

Sliding clamp and clamp loader (w/ ATP) position close to primer;template interface
Clamp loader removed and replaced with DNA polymerase

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33
Q

The fact that DNA polymerase is unlikely to drop off can be summarised by what word

A

Processivity

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34
Q

What is the sliding clamp known as in humans

A

Proliferating cell nuclear antigen

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35
Q

What is the role of single strand binding proteins

A

Bind to the ssDNA of the replication fork making it available for synthesis and easing progression of the replication fork
Prevents any base pairing causing hair pins

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36
Q

SSBs bind cooperatively, what does this mean?

A

Once bound the probability of another one binding is high

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37
Q

What is the role of DNA topoisomerases - why are they needed

A

Prevent the DNA from becomming tangled
When the DNA is being unwound at the replication origin this introduces superhelical tension into the helix
role is to release the tension by nicking and resealing the backbone

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38
Q

TYPE 1 DNA TOPOISOMERASES
HOW DOES IT WORK
ATP?

A

Nick and reseal ONE of the two DNA strands

No atp demand

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39
Q

TYPE 2 DNA TOPOISOMERASES
HOW DOES IT WORK
ATP?

A

Nick and reseal both of the DNA strands

Requires ATP

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40
Q

Yeast replication origins

A

Autonomous replicating sequences (ARS) elements

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41
Q

Human replication origin

A

DNA sequence near to LMNB2 MYC HBB, but can also be defined by chromatin strucutre (lacking the nucelosomes)

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42
Q

Describe the importance of biphasic replication of the DNA

A

Temporal separation to ensure that each chromosome is replicated once per cycle

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43
Q

What is phase 1 of the biphasic replication

What cell cycle phase does it occur in

A

Replicator selection

G1

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44
Q

What is phase 2 of the biphasic replication

What cell cycle phase does it occur in

A

Origin activation

S

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45
Q

What occurs during replicator selection

A

Formation of the prereplicative complex

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46
Q

What occurs duing origin activation

A

Unwinding of the DNA and recruitment of DNA polymerases

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47
Q

Describe the phases of eukaryotic replicator selection

A

Origin recognition complex (ORC) binds
Helicase loading proteins, Cdc6 and Cdt1 bind to the ORC
Helicase Mcm2-7 binds to complete formation of the pre-RC

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48
Q

In G1 what can be said of the CDK levels

A

Low CDK

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49
Q

What is the effect of low CDK in G1

A

Allows formation of pre-RC complexes

Prevents activation of the pre-RC complex

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50
Q

In S phase what can be said of the CDK levels

A

High CDK

51
Q

What is the effect of high CDK levels in S

A

Prevents formation of any pre-RC complexes

Allows activation of existing pre-RC complexes

52
Q

What is the problem with finishing replication

A

Requirement of the RNA primer creates problems
Once ribonuclease H has removed all RNA primers, polymerase and ligase close all but one gap
Gives a natural tendancy of replicating chromosomes to become shorter

53
Q

What is the effect of telomerase

A

Extends the 3’ end (overhang) to compensate for the natural shortening

54
Q

What is the telomeric repeat

A

TTAGGG

55
Q

Describe the strucutre of telomerase

A

Ribonucleoprotein

With an intrinsic RNA component which acts as template for the synthesis

56
Q

What is the step wise telomere synthesis known as

A

The telomere shuffel

57
Q

How many nucelotides does telomerase translocated

A

6 nucleotides

58
Q

What is the 9 nucelotide RNA sequence on telomerase

A

AAUCCCAUU

59
Q

What is meant by DNA replication being semi-conservative

A

Each new daughter strand produced in DNA replication consists of one parental helix and one newly synthesised DNA strand

60
Q

What bonds are formed during the process of DNA replication

A

Phosphodiester bonds

61
Q

An incoming nucleotide can only be added to the free 5’ hydroxyl group on the terminal deoxyribose sugar of an existing polynucleotide chain, T or F

A

F - incoming nucleotides can only be added to the free 3’ hydroxyl group

62
Q

Recall the carbon nomenclature for a deoxyribose sugar

A

Carbon 1 (1’) is the carbon to the right of the oxygen atom in the deoxyribose ring. Move round in a clockwise direction so that carbon 5 (5’) is the CH2OH group attached to the deoxyribose ring

63
Q

Describe the chemical mechanism for the addition of a new nucleotide to the growing polynucleotide chain

A

The 3’ deoxyribose sugar undergoes nucleophilic attack on the first phosphate bond of an incoming nucleotide trisphosphate

64
Q

What is the other product formed from the addition of a nucleotide to the growing polynucleotide chain

A

Pyrophosphate

65
Q

What attributes of DNA replication make it irreversible

A

The formation of a new phosphodiester bond and the production of pyrophosphate is coupled to a second reaction catalysed by pyrophosphatase that converts pyrophosphate to two molecules of inorganic phosphate (2Pi)

66
Q

What reaction supplies the energy required for DNA synthesis

A

Breakage of 2 high energy phosphate bonds

67
Q

State the general equation for the addition of a nucleotide to the grown polynucleotide chain

A

dNTP + (dNMP)n –> (dNMP)n+1 + 2Pi

68
Q

What chemical variables explain the irreversible nature of DNA synthesis

A

Equilibrium constant, Keq of the magnitude of 105 and the Gibb’s free energy, ?G = -7kcal mol-1

69
Q

The irreversible nature of DNA synthesis accounts for the massive stability that is an inherent property of DNA molecules, T or F

A

T

70
Q

What can be said about the orientation of the two polynucleotide chains in a dsDNA molecule

A

They are orientated antiparallel to each other

71
Q

The primer sequence of DNA synthesis can only grow in a 3’ to 5’ direction, why is this

A

The addition of a dNTP can only occur by the nucleophilic attack of the 3’ carbon and hydroxyl on the 5’ phosphate of the incoming nucleotide

72
Q

Due to the fact that DNA synthesis can only occur in a 5’–>3’ direction, how many polymerase enzymes are required per replication fork

A

2

73
Q

Which enzyme is responsible for breaking the hydrogen bonds between complimentary base pairs

A

DNA helicase

74
Q

What localised structure is formed by the breaking of hydrogen bonds between complimentary base pairs in the parent strand

A

Replication fork

75
Q

Which enzyme is responsible for the synthesis of new DNA daughter strands

A

DNA polymerase

76
Q

Which proteins maintain the unwound parental DNA strands in a single stranded conformation and hence ease replication fork progression

A

SSBs – single stranded binding proteins

77
Q

DNA polymerase can only synthesis in a 3’ –> 5’ direction, T or F

A

F – vice versa

78
Q

What is different between the synthesis of the leading and lagging strand during DNA replication

A

The leading strand is synthesised continuously and precedes the synthesis of the lagging strand. The lagging strand however, is synthesised discontinuously i.e. with breaks in the polynucleotide chain

79
Q

What is the significance of DNA polymerases inability to work de novo

A

RNA primers required at the start of the replication of the leading strand and at the start of each Okazaki fragment of the lagging strand

80
Q

What enzyme is responsible for the synthesis of RNA primers

A

DNA primase

81
Q

RNA primers are required throughout both the leading and lagging strand synthesis, T or F

A

F – they are only required at the start of lagging strand synthesis but throughout leading strand synthesis

82
Q

What two features are required for synthesis of RNA primers

A

DNA template strand and nucleotide trisphosphates

83
Q

Explain the role of RNA primers in the synthesis of DNA

A

DNA polymerase requires an RNA primer in order to synthesise a new DNA strand. RNA primers supply DNA polymerase with base-paired chain ends to add new nucleotides to.

84
Q

Explain how DNA polymerase synthesises the lagging strand

A

Lagging strand DNA polymerase completes Okazaki fragments in the 5’ to 3’ direction and then starts synthesising a completely new fragment further along towards the 5’ end of the parental template strand

85
Q

What is the approximate size Eukaryotic primers and how often do they occur in the lagging strand

A

10 nucleotides long, occurring every 100-200 nucleotides in the lagging strand

86
Q

What causes the synthesis of each Okazaki fragment in the lagging strand to halt

A

When DNA polymerase reaches the RNA primer attached to the 5’ end of another fragment

87
Q

What is the initial product of DNA replication

A

A DNA-RNA hybrid molecule

88
Q

Explain the role of ribonuclease H in DNA replication

A

Ribonuclease H removes the RNA primers in the initial DNA-RNA hybrid molecule

89
Q

Which enzyme works with ribonuclease H to repair gaps in the DNA sequence

A

DNA polymerase – extends across the gaps created by RNA primer removal by ribonuclease H

90
Q

Which enzyme joins 3’ and 5’ ends of the Okazaki fragments together

A

DNA ligase

91
Q

Ligation of newly synthesised adjacent DNA fragments is a two-step reaction, requiring ATP hydrolysis, T or F

A

T

92
Q

What attribute of the ligation of DNA fragments makes it another example of an irreversible reaction

A

Like DNA synthesis ligation results in the formation of another molecule of pyrophosphate. This reaction is then coupled to a reaction the converts PPi to 2Pi

93
Q

Recall the general and word equation for the first reaction in DNA ligase activity

A

ATP + 5’P –> PPi + 5’P-AMP. Adenosine trisphosphate + 5’ phosphate –> pyrophosphate + 5’adenosine diphosphate

94
Q

In the reaction catalysed by DNA ligase, two phosphates are removed from ATP to form AMP which then binds to the 5’ phosphate in the polynucleotide chain, the two phosphates are referred to as pyrophosphate, T or F

A

T

95
Q

Recall the general and word equation for the reaction coupled to DNA ligase activity that accounts for its irreversible nature

A

PPi –> 2Pi + Free energy. Pyrophosphate is converted to two molecules of inorganic phosphate by pyrophosphatase, which also releases energy

96
Q

The ligation process is rendered energetically highly favourable by the conversion of pyrophosphate (PPi) to 2Pi by pyrophosphatase, T or F

A

T

97
Q

How does DNA helicase separate parental DNA strands at the replication fork

A

DNA Helicase sits like a nut on a bolt and uses the energy released by ATP hydrolysis to drive a rotational energy which is translated to a force that opens up the replication fork

98
Q

Werner’s syndrome is an example of a disease caused by mutations in a helicase. Describe the aetiology and symptoms of this condition

A

Werner’s syndrome is a progeria (premature ageing) caused by a autosomal recessive mutation (loss of function) in the RECQ helicase encoded by the WRN gene

99
Q

Give an example of another helicase mutation that causes disease, other than Werner’s syndrome

A

Bloom syndrome is another loss of function mutation that occurs in another Rec-Q family DNA helicase. The role of this helicase is to maintain genome integrity. This mutation causes a rare cancer phenotype with tumours in multiple tissues

100
Q

What is meant by the processivity of DNA polymerases

A

Processivity refers the tendency of polymerases to continue to synthesise as long as there is sufficient substrate available

101
Q

What protein is said to enhance the processivity of polymerases

A

Sliding clamp proteins

102
Q

How do proteins that increase polymerase processivity act

A

Sliding clamp proteins keep the DNA polymerase enzyme at the primer template junction by fixing itself to the primer template junction through association with a protein called a clamp loader.

103
Q

Describe the ternary structure formed by proteins that increase processivity and how this complex acts

A

Sliding clamp positioning is ATP-dependant. A ternary structure is formed by the sliding clamp and clamp loader proteins and the associated ATP. This complex sits behind the DNA polymerase and provides an extra impetus to drive it forward

104
Q

What is significant about sliding clamp proteins across Eukaryotes

A

Extremely highly conserved

105
Q

The human sliding clamp protein, proliferating cell nuclear antigen (PCNA) has a near identical structure to the homologue in yeast. What is PCNA significance in cancer

A

PCNA is a useful marker for hyperproliferative cells found in tumours

106
Q

How do SSBs ease replication fork progression

A

They act to prevent hydrogen bond formation between complimentary base pairs within the same ssDNA strand by binding to the sugar-phosphate backbone and allowing easy progress of polymerase

107
Q

What is the role of topoisomerases

A

Prevent the DNA from becoming tangled and supercoiled during DNA replication

108
Q

How do topoisomerases act

A

Topoisomerases release the tension created in the polynucleotide chains created by unwinding of the two ssDNA strands. This is achieved by the selective nicking and resealing of regions in the DNA molecule

109
Q

Explain the major differences between the two types of topoisomerases

A

Type I Topoisomerases nick and release one of the 2DNA strands and effective remove one turn in the molecule. This is an ATP-independent reaction. Type II Topoisomerases nick a reseal both DNA strands by causing dsDNA breaks effectively removing two turns of the supercoiled helix. This reaction is ATP-dependent

110
Q

What is the name given to specific DNA sequences that direct the initiation of DNA replication by recruiting replication initiation proteins

A

Replicator sequences

111
Q

Whilst in yeast, autonomously replicating sequences (ARS) direct DNA replication initiation, similar structures in humans have proven elusive but seem to be defined by chromatin structure rather than DNA sequence, T or F

A

T

112
Q

Near to which genes have human DNA replication initiation sequences been found

A

LMNB2 (laminin B), MYC and HBB (Haemoglobin B)

113
Q

Eukaryotic DNA replication is monophasic, T or F

A

F – its biphasic

114
Q

Where in the cell cycle does replicator selection and formation of the pre-replicative complex occur

A

G1

115
Q

What DNA replication event occurs in S phase

A

Origin activation – the unwinding of DNA and recruitment of DNA polymerase

116
Q

Temporal separation of replicator selection and origin activation ensures what

A

Each origin is used and each chromosome is only replicated once per cell cycle

117
Q

How does Eukaryotic replicator selection occur

A

Origin replication complex (ORC) binds to the replicator sequence. Helicase loading proteins then bind to ORC to convert the single stranded replicator sequence into a pair of replication forks. Mcm2-7 then also binds to complete formation of the pre-RC

118
Q

Give example of helicase loading proteins that bind to ORC

A

Cdc6 and Cdt1

119
Q

Cyclin dependent kinases are important in the temporal control of DNA replication. During which stage of the cell cycle are cdk levels particularly high and particularly low

A

G1 phase – low cdk activity. S phase – high cdk activity

120
Q

How is cyclin-dependant kinase (cdk) activity important in limiting pre-RC formation and activation to specific points in the cell cycle

A

Cdk levels are high during S phase of the cell cycle. High cdk levels leads to the phosphorylation of already formed pre-RC thus activating them and leading to formation of the replication origin. Cdk also acts to phosphorylate the individual components of the pre-RC, particularly the Cdc6, Cdt1 and ORC elements. Phosphorylation of these constituent elements leads to their inactivation and hence inhibition of new pre-RC formation during S phase. During G1 cdk levels are low and thus there is little phosphorylation of Cdc6 and Cdt1 and hence more pre-RC formation

121
Q

What accounts for the end replication problem during DNA replication

A

The need for an RNA primer for initiation of DNA synthesis. Ribonuclease H removes the last RNA primer in the linear chromosome sequence however DNA polymerase can no longer extend the DNA sequence. This would result in a gradual reduction in the length of replicated DNA by the length of one primer with each subsequent replication

122
Q

How is the end replication problem overcome

A

Addition of non-coding telomeric repeat sequences to the 3’ end of the DNA sequence. These are long enough to enable DNA primase to bind and initiate new RNA primer synthesis and prevent chromosome shortening

123
Q

Describe the composition of telomeric repeat sequences

A

Telomeres consist of a hexanucleotide repeat sequence (TTAGGG)

124
Q

How does the telomerase enzyme act to create and maintain these telomeric repeats at the end of linear chromosomes

A

Telomerase is a ribonucleoprotein with an intrinsic RNA component containing the complimentary RNA sequence to the telomeric repeat sequence (AAUCCC). This RNA component acts as a template on which telomere repeat sequences are synthesised in a step-wise process known as the telomere shuffle. This allows addition of multiple TTAGGG repeats to the 3’-OH at each telomere.

BMS242 Cell and Molecular S1 (13 decks)

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