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DNA Replication. Flashcards

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1
Q

Define a cofactor?

A

An inorganic molecule that is used to help some enzymes to produce their product.

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2
Q

What are many cofactors made out if?

A

Many cofactors are made of metallic substances.

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3
Q

Define DNA replication?

A

The process by which each strand DNA is copied to produce 2 identical daughter strands.

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4
Q

What does the letter N mean when it appears in a strand of DNA or RNA?

A

I stands for any nucleotide.

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5
Q

When does DNA replication take place?

A

During cell division.

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6
Q

What does DNA replication allow DNA to do?

A

It allows for DNA to be copied and passed on to future generations.

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7
Q

What occurs during DNA replication?

A

DNA strands are copied to produce 2 identical daughter strands.

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8
Q

How long does DNA replication take in prokaryotes?

A

Around 20 minutes.

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9
Q

How long does DNA replication take in eukaryotes?

A

Up to 24 hours (in yeast it takes around 4 hours).

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10
Q

What is the first step of DNA replication?

A

The unwinding of the DNA molecule and the separation of the complimentary base pairs.

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11
Q

What enzyme will unwind and separate the base pairs within the DNA molecule?

A

A helicase.

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12
Q

What happens in DNA replication when the strands have been separated?

A

An enzyme called DNA polymerase III will read a strand of DNA.

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13
Q

What is the name of the strand that is read by DNA polymerase?

A

The template strand.

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14
Q

What is the name of the strand that is synthesised by DNA polymerase?

A

A daughter strand.

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15
Q

What direction is DNA synthesised in?

A

In the 5 prime to 3 prime direction.

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16
Q

In what direction will DNA polymerase read a DNA strand?

A

In the 3 prime to 5 prime direction.

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17
Q

What enzyme will DNA polymerase III follow up the DNA molecule?

A

The helicase.

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18
Q

What are the 2 synthesised DNA strands known as?

A

The leading strand.

The lagging strand.

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19
Q

Do the DNA polymerases that form the leading and lagging strands move in the same direction?

A

They both move in the 3 to 5 direction.

But, due to the antiparallel nature of DNA they will move in opposite directions relative to each other.

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20
Q

Will the same enzyme synthesise the leading and lagging strands?

A

An individual DNA polymerase 3 will synthesise each strand.

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21
Q

Which daughter strand of DNA will contain a fragmented copy of the DNA?

A

The lagging strand will create a fragmented copy of DNA.

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22
Q

What does DNA polymerase need to bind to, to be able to synthesise DNA?

A

DNA polymerase III cannot synthesise any DNA unless it finds a free 3 prime hydroxyl group.

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23
Q

What molecules will help to add free 3 prime hydroxyl groups to DNA so that RNA polymerase can read the DNA strand?

A

A primer.

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24
Q

What is a primer molecule composed of?

A

They are small sequences of nucleotides that have a free 3 prime hydroxyl group.

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25
Q

What enzyme is responsible for synthesising primers?

A

RNA primase.

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26
Q

What happens to the DNA fragments on the lagging strand once the DNA has been copied?

A

DNA polymerase I will remove the primers and fill in the gaps between the DNA fragments.

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27
Q

What is created when DNA polymerase I removes the primers on the lagging strand?

A

Okazaki fragments.

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28
Q

What elements of the primers will DNA polymerase I leave behind when it forms Okazaki fragments?

A

A phosphate and hydroxyl group at the beginning and end of each new sequence of DNA.

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29
Q

How are the Okazaki fragments removed from the lagging strand?

A

B an enzyme called DNA ligase.

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30
Q

Which DNA strand is synthesised into a continuous strand of DNA?

A

The leading strand.

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31
Q

Who suggested that DNA must have a copy system?

A

Watson and Crick.

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32
Q

What are the 3 theories of DNA replication that scientists came up with?

A

The dispersive DNA replication.

The semi-conservative model.

The conservative model.

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33
Q

What is the dispersive model of DNA replication?

A

That a DNA strand is broken into many small pieces.

These small pieces are copied and re-assembled to create a new strand that is made up of old and new DNA.

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34
Q

What is the semi conservative model of DNA replication?

A

That the DNA strand opens up and each individual strand acts as a template to produce 2 new daughter strands.

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35
Q

What is the conservative model of DNA replication?

A

That nothing happens to the double stranded DNA molecule as a new strand is copied from it.

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36
Q

What is the correct model of DNA replication?

A

The semi-conservative model.

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37
Q

Which scientists proved that the semi conservative model was the correct model of replication and when?

A

Meselson and Stahl. In 1958.

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38
Q

What did Meselson and Stahl use in their experiment to prove the semi conservative model of replication was true?

A

E.Coli bacteria that had been cultured in a medium containing ammonium chloride.

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39
Q

What form of ammonium chloride did Meselson and Stahl use when culturing their E.coli for the 1st time?

A

One with a heavy isotope of nitrogen.

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40
Q

Why did Meselson and Stahl use a heavy isotope of nitrogen in their ammonia when culturing their E.coli?

A

So they could identify it in the new DNA strands of newly formed E.coli.

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41
Q

What happened to the E.coli once they had been cultured in the ammonia containing the heavy isotope of nitrogen during Meselson and Stahls experiment?

A

They were transferred to a medium containing a lighter isotope of nitrogen and left in for 20 minutes so that 1 cell division could take place.

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42
Q

What happened to the E.coli in Meselson and Stahls experiment after it had been cultured in the medium containing the lighter isotope of nitrogen?

A

Samples were collected and centrifuged so that the density of the DNA could be measured.

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43
Q

What did Meselson and Stahls find after they centrifuged the E.coli that had performed 1 cell division?

A

That the DNA had formed 1 density band, showing that the light and heavy DNA had mixed together to form a hybrid.

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44
Q

Which model was disproved after Meselson and Stahls centrifuged the E.coli that had performed 1 cell division?

A

The conservative model.

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45
Q

Why was the conservative model disproved after centrifugation of the E.coli that had performed 1 cell division Meselson and Stahls experiment?

A

Because 2 density bands should have been visible after 1 division.

1 density band representing the heavier parent strands and 1 strand representing the lighter daughter strands.

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46
Q

Why would 2 bands be visible after 1 division of E.Coli in Meselson and Stahls experiment?

A

Because the heavy parent strands would have to use the lighter nitrogen to make the daughter strands, resulting in 2 bands.

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47
Q

Why would the daughter strands in Meselson and Stahls experiment be made of lighter nitrogen?

A

Because they could only be made from lighter nitrogen as they were produced in the lighter solution.

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48
Q

What models could be true after Meselson and Stahl allowed their E.coli to perform 1 cell division?

A

The semi-conservative and dispersive models.

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49
Q

Why would the conservative and dispersive models only produce 1 density band after 1 cell division had been performed?

A

As they would use both the light and heavy nitrogen to create 2 different hybrid DNA molecules.

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50
Q

What experiment did Meselson and Stahl perform to determine whether the dispersive model or semi-conservative model was true once the conservative model had been ruled out?

A

They left the E.Coli in the lighter medium for 40 minutes so they could perform 2 cell divisions.

After centrifugation, 2 distinct density bands had been formed.

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51
Q

What isotopes were represented in the 2 density bands that were formed in the 2nd step of Meselson and Stahls experiment?

A

One band was coated with both isotopes.

The other was only coated with the lighter isotope.

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52
Q

What model did the 2 density bands formed in the 2nd step of Meselson and Stahls experiment disprove?

A

The dispersive model.

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53
Q

Why was the dispersive model disproved in the 2nd step of Meselson and Stahls experiment?

A

If it were true then both density bands would contain both isotopes.

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54
Q

Why would each of the 2 bands formed in the 2nd step of Meselson and Stahls experiment contain both isotopes if the dispersive model were true?

A

As the older heavier DNA would have been broken up and re-used creating a DNA strand with both heavy and light isotopes in it.

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55
Q

What happened to the DNA during the first step of Meselson and Stahls experiment?

A

The heavy strands were copied and each produced a daughter strand made of light nitrogen.

This created 2 DNA molecules each consisting of 1 heavy strand and 1 light strand.

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56
Q

Which of the 2 DNA molecules had been coated with which isotope in the 2nd step of Meselson and Stahls experiment?

A

The 2 original strands were coated with the heavy isotopes.

The copied strands were coated with lighter isotopes.

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57
Q

What happened to the DNA during the second step of Meselson and Stahls experiment?

A

In the 2nd division light nitrogen was used to create new DNA molecules.

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58
Q

How does the 2 bands formed in the 2nd step of Meselson and Stahls experiment explain that the semi conservative model was true?

A

Because 1 band is represented by the 2 DNA molecules formed in the 1st division and contained heavy and light strands.

And the other band was created by the 2 DNA molecules formed in the 2nd division and contained only light strands.

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59
Q

Who was responsible for identifying and researching the enzymes responsible for DNA replication?

A

Arthur Kornberg.

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60
Q

What is the most important enzyme in DNA replication?

A

DNA polymerase. DNA is always synthesised from the 5 prime end, ending with the 3 prime end, meaning that DNA polymerase must read the parent strand in the 3 prime to 5 prime direction.

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61
Q

Is there more than 1 type of DNA polymerase?

A

Yes, but they all synthesise DNA in the same way.

Most organisms have more than one type of polymerase.

62
Q

What does DNA polymerase use to build a DNA molecule?

A

DNTPs (deoxynucleoside triphosphate’s) which contain the 4 bases;

Adenine.
Thymine.

Cytoosine.
Guanine.

63
Q

What direction does DNA polymerase read the template strand in?

A

In the 3 to 5 direction.

64
Q

What does DNA polymerase add to each template strand?

A

Complimentary base pairs to each base.

This creates a daughter strand on each parent strand.

65
Q

What do many enzymes require to help them do their job?

A

Cofactors.

66
Q

What co-factor does DNA polymerase require?

A

Magnesium (Mg2+).

67
Q

What location on DNA will DNA polymerase start reading the strand in?

A

DNA polymerase must start at a 3 prime position.

68
Q

What is the location where DNA synthesis begins known as?

A

The origin of replication (ORI).

69
Q

How many ORI’s do prokaryotic genomes have?

A

A single ORI.

70
Q

How many ORI’s do eukaryotic genomes have?

A

Multiple ORI’s.

71
Q

Will prokaryotic replication ever stop?

A

No.

Once replication has begun it will not stop until the whole circular genome is copied.

72
Q

What does the DNA replication process create in prokaryotes?

A

An entire new genome.

73
Q

How does DNA replication know when to stop?

A

There is a short sequence of DNA called a termination sequence which signals for replication to stop.

74
Q

Do eukaryotic chromosomes have any replication termini or termination sequences?

A

No.

75
Q

DNA is synthesised from what end?

A

From the 5 end.

This means that nucleotides are added to the 3 end.

76
Q

Why do eukaryotic organisms need multiple ORI’s of replication?

A

As they have many more base pairs than prokaryotic organisms.

77
Q

The human genome consists of around how many base pairs?

A

3.4 billion base pairs.

78
Q

How many base pairs are found on the average human chromosome?

A

Around 100 million base pairs.

79
Q

What will define how many ORI’s a eukaryotic organism has?

A

The more complex the eukaryotic organism, the more ORI’s are present.

80
Q

What is the origin of replication in E.Coli is known as?

A

The ORI-C.

81
Q

How many base pairs are found in the ORI-C in E.coli?

A

Around 245 base pairs with 2 important sequences.

82
Q

What are the 2 important sequences found in the ORI-C of E.coli?

A

A series of three 13 nucleotide sequences in a tandem array.

A series of four 9 nucleotide sequences.

83
Q

Why are the series of three 13 nucleotide sequences important in the ORI-C of E.coli?

A

They contain many As and Ts allowing the DNA to open much more easily at this location.

84
Q

What are the four 9 nucleotide sequences in the ORI-C of E.coli used for?

A

The are spread throughout the ORI and are where the DNA-A proteins bind to.

85
Q

What is the job of the DNA-A binding proteins in the ORI-C of E.coli?

A

They are initiator proteins that are used to begin replication.

86
Q

How do the DNA-A binding proteins in the ORI-C of E.coli work?

A

They recognise and bind to the 9 nucleotide sequences causing the ORI-C to condense moving the 9 nucleotide regions closer to the 13 nucleotide sequences.

87
Q

What happens in the DNA replication of E.coli once the ORI.C has condensed?

A

DNA-A proteins separate the strands in the A-T region and DNA helicase can then continue the separation.

88
Q

What is the DNA helicase that is used in DNA replication in E.coli known as?

A

DNA-B.

89
Q

Why do 2 separated DNA strands want to recombine?

A

As the base pairs are complimentary.

90
Q

How do E.coli stop the 2 separated DNA strands from re-combining in DNA replication?

A

Single strand binding proteins (SSB proteins) bind to the single DNA strands and stop the re-combination.

91
Q

What enzyme will tell DNA-B to start and stop working in DNA replication in E.coli?

A

DNA C.

92
Q

What enzyme is responsible for delivering DNA-B to the ORI in DNA replication of E.coli?

A

DNA-C.

93
Q

What happens once DNA-B is delivered to the ORI in DNA replication of E.coli?

A

It clamps around each individual strand and unwinds the DNA in opposite directions.

94
Q

How many helicases work on 1 DNA molecule?

A

One on each strand.

95
Q

What needs to be open for DNA replication to occur?

A

The ORI.

96
Q

What enzyme will synthesise a primer molecule allowing DNA polymerase to bind to the DNA strand?

A

DNA primase.

97
Q

What enzyme will remove the primers from the DNA strands?

A

DNA polymerase I removes the primers leaving Okazaki fragments.

98
Q

Which enzyme will stitch together the Okazaki fragments on the lagging strand?

A

DNA ligase

99
Q

What strand will Okazaki fragments be formed on?

A

On the lagging strand.

100
Q

Why is it difficult for DNA polymerase to copy the lagging strand?

A

It only works in a 3 to 5 direction, making it hard to synthesise both strands from the same origin as they are antiparallel.

101
Q

What is the phase of replication known as when nucleotides are being added to the daughter strands?

A

Elongation.

102
Q

How does DNA primase work?

A

It reads the template strand and forms a small piece of RNA of around 15 nucleotides and has a 3 prime hydroxyl group on it.

103
Q

The primer on what strand only needs to be synthesised once?

A

The leading strand.

104
Q

The primer on what strand only needs to be synthesised more than once?

A

The lagging strand.

105
Q

DNA helicase opens up a DNA strand in what direction?

A

In the 5 to 3 direction from the leading strand.

106
Q

What direction can the leading strand be read in?

A

The 5 to 3 direction.

107
Q

How does DNA polymerase synthesise its daughter strand from the leading strand?

A

It will synthesise 1 continuous piece of DNA.

108
Q

What direction is the lagging strand in relative to the leading strand?

A

It is in the 3 to 5 direction relative to the leading strand.

109
Q

The polymerase that works on the lagging strand must go in the what direction relative to the helicase?

A

The opposite direction.

110
Q

The polymerase that works on the leading strand must go in the what direction relative to the helicase?

A

The same direction.

111
Q

What must be laid down on the lagging strand to allow DNA polymerase to copy it?

A

Multiple primers.

112
Q

What are Okazaki fragments?

A

Fragments of DNA that are separated by primers.

113
Q

How many base pairs are found in Okazaki fragments in bacteria?

A

Between 1000 and 2000 base pairs.

114
Q

What enzyme will remove the primers from the Okazaki fragments?

A

DNA polymerase I in prokaryotes.

RNAseh, FEN-1 protein and DNA polymerase I in eukaryotes.

115
Q

What enzyme stitches the fragments of DNA together once DNA polymerase I has removed the primers?

A

DNA ligase creates phosphodiester bonds between the nucleotides of different fragments.

116
Q

Which polymerase cannot synthesise a daughter strand without a primer?

A

DNA polymerase III.

117
Q

What helps DNA polymerase III attach to the DNA strand?

A

A clamp protein.

118
Q

What does DNA polymerase III do once it is attached to the DNA strand?

A

It moves down the strand identifying each base and creating its corresponding base.

119
Q

If DNA polymerase finds a sequence of CAT on a DNA strand, what will it synthesise?

A

GTA.

120
Q

How does DNA polymerase bring in the bases that are used to make the nucleotides in the daughter strand?

A

As deoxynucloeside triphosphates.

121
Q

How does DNA polymerase alter the deoxynucloeside triphosphates so they can be added to the daughter strand?

A

It will cleave 2 of the 3 phosphates from the DNTP.

The resulting molecule is added to the daughter strand, creating a longer chain.

122
Q

How does DNA polymerase III read the parent strand?

A

In the 5 to 3 direction.

123
Q

How does DNA polymerase III synthesise the daughter strand?

A

In the 3 to 5 direction.

124
Q

What is a tautomeric shift in cytosine?

A

When an isomer of C is created.

125
Q

What does the isomer of C represent when a tautomeric shift occurs?

A

It will represent a T molecule which the polymerase will place next to an A.

126
Q

Can the isomer of C from a tautomeric shift pair with an A?

A

No.

127
Q

What happens when an isomer of C from a tautomeric shift is placed next to an A in a DNA strand?

A

A bulge is created.

128
Q

What will DNA polymerase do if a bulge in the strand is created by the isomer of C?

A

It will correct its mistake and go back to remove the isomer of C and replace it with a T.

129
Q

Why does DNA polymerase III have proof-reading ability in the 3 to 5 direction?

A

Because it can correct mistakes it has made.

E.g. The removal of C isomers.

130
Q

What proof-reading ability is DNA polymerase III said to have?

A

3 prime to 5 prime exonuclease activity.

131
Q

What does the 3 prime to 5 prime exonuclease activity of DNA polymerase III prevent from happening?

A

DNA mutations.

132
Q

What are the 3 prokaryotic DNA polymerases?

A

DNA Polymerase I.

DNA Polymerase II.

DNA Polymerase III.

133
Q

What is DNA polymerase I involved in?

A

DNA repair.

134
Q

What is DNA polymerase II involved in?

A

Repairing damaged DNA.

135
Q

What is DNA polymerase III involved in?

A

Synthesising new strands.

136
Q

What are the 3 characteristics of DNA polymerase I?

A

5-3 exonuclease ability.

5-3 polymerase activity.

3-5 exonuclease activity.

137
Q

What is 5-3 exonuclease ability?

A

The ability to remove RNA primers.

138
Q

What is 5-3 polymerase activity?

A

The ability to add nucleotides to DNA.

139
Q

What is 5-3 exonuclease activity?

A

Proofreading ability.

140
Q

What is the main characteristic of DNA polymerase II?

A

3-5 exonuclease activity.

141
Q

What are the 2 characteristics of DNA polymerase III?

A

5-3 polymerase activity.

3-5 exonuclease activity.

142
Q

What about the structure of eukaryotic chromosomes makes it difficult for DNA replication to occur?

A

The ends of the linear chromosomes.

143
Q

What about the ends of linear chromosomes makes it difficult for replication to occur?

A

When the primer sequence is removed from the 5 prime end as it is impossible to replace that section of DNA.

144
Q

Why is it impossible to replace the primer at the 5 prime end of a DNA strand?

A

As DNA polymerase would have to move against the 5 to 3 direction and there is no 3 prime hydroxyl group for the polymerase to attach to.

145
Q

How does the removal of the primer at the 5 prime end cause the daughter strand to differ from the parent strand?

A

The daughter strand will be shorter than the parent strand as the 5 prime end begins further up the strand.

146
Q

What happens if the primer at the 5 prime end isn’t replaced?

A

The daughter strand will get shorter and shorter meaning that DNA would be lost each time replication occurs.

147
Q

What happens on the 3 prime end to help replace the primer from the 5 prime end on linear eukaryotic DNA?

A

The 3 prime end of the parent strand is extended to form a structure known as a telomere.

148
Q

What enzyme will extend the 3 prime end of the parent strand during the replacement of the primer on the 5 prime end?

A

Telomerase.

149
Q

What are telomeres?

A

Sections of non coding DNA consisting of repetitive DNA sequences made up of nucleotides that spell TTAGGG.

150
Q

How does telomerase work?

A

It has an RNA primer that corresponds to the repetitive sequences on telomeres.

It binds to the 3 prime overhang and adds nucleotides to the 3 prime end.

This process extends the 3 prime overhang by around 500 bases.

151
Q

What ability does telomerase have?

A

Reverse transcriptase ability.

This allows it to use its RNA template to form DNA

152
Q

What happens when the 3 prime end of a DNA strand has been extended by telomerase?

A

A primase comes and adds a primer to the 3 prime end.

DNA polymerase uses the primer to add complimentary base pairs to the lagging strand before the primer is removed.

Molecular Biology. (22 decks)

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