DNA Replication. Flashcards
1
Q
Define a cofactor?
A
An inorganic molecule that is used to help some enzymes to produce their product.
2
Q
What are many cofactors made out if?
A
Many cofactors are made of metallic substances.
3
Q
Define DNA replication?
A
The process by which each strand DNA is copied to produce 2 identical daughter strands.
4
Q
What does the letter N mean when it appears in a strand of DNA or RNA?
A
I stands for any nucleotide.
5
Q
When does DNA replication take place?
A
During cell division.
6
Q
What does DNA replication allow DNA to do?
A
It allows for DNA to be copied and passed on to future generations.
7
Q
What occurs during DNA replication?
A
DNA strands are copied to produce 2 identical daughter strands.
8
Q
How long does DNA replication take in prokaryotes?
A
Around 20 minutes.
9
Q
How long does DNA replication take in eukaryotes?
A
Up to 24 hours (in yeast it takes around 4 hours).
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.
11
Q
What enzyme will unwind and separate the base pairs within the DNA molecule?
A
A helicase.
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.
13
Q
What is the name of the strand that is read by DNA polymerase?
A
The template strand.
14
Q
What is the name of the strand that is synthesised by DNA polymerase?
A
A daughter strand.
15
Q
What direction is DNA synthesised in?
A
In the 5 prime to 3 prime direction.
16
Q
In what direction will DNA polymerase read a DNA strand?
A
In the 3 prime to 5 prime direction.
17
Q
What enzyme will DNA polymerase III follow up the DNA molecule?
A
The helicase.
18
Q
What are the 2 synthesised DNA strands known as?
A
The leading strand.
The lagging strand.
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.
20
Q
Will the same enzyme synthesise the leading and lagging strands?
A
An individual DNA polymerase 3 will synthesise each strand.
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.
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.
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.
24
Q
What is a primer molecule composed of?
A
They are small sequences of nucleotides that have a free 3 prime hydroxyl group.
25
What enzyme is responsible for synthesising primers?
RNA primase.
26
What happens to the DNA fragments on the lagging strand once the DNA has been copied?
DNA polymerase I will remove the primers and fill in the gaps between the DNA fragments.
27
What is created when DNA polymerase I removes the primers on the lagging strand?
Okazaki fragments.
28
What elements of the primers will DNA polymerase I leave behind when it forms Okazaki fragments?
A phosphate and hydroxyl group at the beginning and end of each new sequence of DNA.
29
How are the Okazaki fragments removed from the lagging strand?
B an enzyme called DNA ligase.
30
Which DNA strand is synthesised into a continuous strand of DNA?
The leading strand.
31
Who suggested that DNA must have a copy system?
Watson and Crick.
32
What are the 3 theories of DNA replication that scientists came up with?
The dispersive DNA replication.
The semi-conservative model.
The conservative model.
33
What is the dispersive model of DNA replication?
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.
34
What is the semi conservative model of DNA replication?
That the DNA strand opens up and each individual strand acts as a template to produce 2 new daughter strands.
35
What is the conservative model of DNA replication?
That nothing happens to the double stranded DNA molecule as a new strand is copied from it.
36
What is the correct model of DNA replication?
The semi-conservative model.
37
Which scientists proved that the semi conservative model was the correct model of replication and when?
Meselson and Stahl. In 1958.
38
What did Meselson and Stahl use in their experiment to prove the semi conservative model of replication was true?
E.Coli bacteria that had been cultured in a medium containing ammonium chloride.
39
What form of ammonium chloride did Meselson and Stahl use when culturing their E.coli for the 1st time?
One with a heavy isotope of nitrogen.
40
Why did Meselson and Stahl use a heavy isotope of nitrogen in their ammonia when culturing their E.coli?
So they could identify it in the new DNA strands of newly formed E.coli.
41
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?
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.
42
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?
Samples were collected and centrifuged so that the density of the DNA could be measured.
43
What did Meselson and Stahls find after they centrifuged the E.coli that had performed 1 cell division?
That the DNA had formed 1 density band, showing that the light and heavy DNA had mixed together to form a hybrid.
44
Which model was disproved after Meselson and Stahls centrifuged the E.coli that had performed 1 cell division?
The conservative model.
45
Why was the conservative model disproved after centrifugation of the E.coli that had performed 1 cell division Meselson and Stahls experiment?
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.
46
Why would 2 bands be visible after 1 division of E.Coli in Meselson and Stahls experiment?
Because the heavy parent strands would have to use the lighter nitrogen to make the daughter strands, resulting in 2 bands.
47
Why would the daughter strands in Meselson and Stahls experiment be made of lighter nitrogen?
Because they could only be made from lighter nitrogen as they were produced in the lighter solution.
48
What models could be true after Meselson and Stahl allowed their E.coli to perform 1 cell division?
The semi-conservative and dispersive models.
49
Why would the conservative and dispersive models only produce 1 density band after 1 cell division had been performed?
As they would use both the light and heavy nitrogen to create 2 different hybrid DNA molecules.
50
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?
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.
51
What isotopes were represented in the 2 density bands that were formed in the 2nd step of Meselson and Stahls experiment?
One band was coated with both isotopes.
The other was only coated with the lighter isotope.
52
What model did the 2 density bands formed in the 2nd step of Meselson and Stahls experiment disprove?
The dispersive model.
53
Why was the dispersive model disproved in the 2nd step of Meselson and Stahls experiment?
If it were true then both density bands would contain both isotopes.
54
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?
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.
55
What happened to the DNA during the first step of Meselson and Stahls experiment?
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.
56
Which of the 2 DNA molecules had been coated with which isotope in the 2nd step of Meselson and Stahls experiment?
The 2 original strands were coated with the heavy isotopes.
The copied strands were coated with lighter isotopes.
57
What happened to the DNA during the second step of Meselson and Stahls experiment?
In the 2nd division light nitrogen was used to create new DNA molecules.
58
How does the 2 bands formed in the 2nd step of Meselson and Stahls experiment explain that the semi conservative model was true?
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.
59
Who was responsible for identifying and researching the enzymes responsible for DNA replication?
Arthur Kornberg.
60
What is the most important enzyme in DNA replication?
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.
61
Is there more than 1 type of DNA polymerase?
Yes, but they all synthesise DNA in the same way.
Most organisms have more than one type of polymerase.
62
What does DNA polymerase use to build a DNA molecule?
DNTPs (deoxynucleoside triphosphate’s) which contain the 4 bases;
Adenine.
Thymine.
Cytoosine.
Guanine.
63
What direction does DNA polymerase read the template strand in?
In the 3 to 5 direction.
64
What does DNA polymerase add to each template strand?
Complimentary base pairs to each base.
This creates a daughter strand on each parent strand.
65
What do many enzymes require to help them do their job?
Cofactors.
66
What co-factor does DNA polymerase require?
Magnesium (Mg2+).
67
What location on DNA will DNA polymerase start reading the strand in?
DNA polymerase must start at a 3 prime position.
68
What is the location where DNA synthesis begins known as?
The origin of replication (ORI).
69
How many ORI's do prokaryotic genomes have?
A single ORI.
70
How many ORI's do eukaryotic genomes have?
Multiple ORI’s.
71
Will prokaryotic replication ever stop?
No.
Once replication has begun it will not stop until the whole circular genome is copied.
72
What does the DNA replication process create in prokaryotes?
An entire new genome.
73
How does DNA replication know when to stop?
There is a short sequence of DNA called a termination sequence which signals for replication to stop.
74
Do eukaryotic chromosomes have any replication termini or termination sequences?
No.
75
DNA is synthesised from what end?
From the 5 end.
This means that nucleotides are added to the 3 end.
76
Why do eukaryotic organisms need multiple ORI's of replication?
As they have many more base pairs than prokaryotic organisms.
77
The human genome consists of around how many base pairs?
3.4 billion base pairs.
78
How many base pairs are found on the average human chromosome?
Around 100 million base pairs.
79
What will define how many ORI's a eukaryotic organism has?
The more complex the eukaryotic organism, the more ORI’s are present.
80
What is the origin of replication in E.Coli is known as?
The ORI-C.
81
How many base pairs are found in the ORI-C in E.coli?
Around 245 base pairs with 2 important sequences.
82
What are the 2 important sequences found in the ORI-C of E.coli?
A series of three 13 nucleotide sequences in a tandem array.
A series of four 9 nucleotide sequences.
83
Why are the series of three 13 nucleotide sequences important in the ORI-C of E.coli?
They contain many As and Ts allowing the DNA to open much more easily at this location.
84
What are the four 9 nucleotide sequences in the ORI-C of E.coli used for?
The are spread throughout the ORI and are where the DNA-A proteins bind to.
85
What is the job of the DNA-A binding proteins in the ORI-C of E.coli?
They are initiator proteins that are used to begin replication.
86
How do the DNA-A binding proteins in the ORI-C of E.coli work?
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
What happens in the DNA replication of E.coli once the ORI.C has condensed?
DNA-A proteins separate the strands in the A-T region and DNA helicase can then continue the separation.
88
What is the DNA helicase that is used in DNA replication in E.coli known as?
DNA-B.
89
Why do 2 separated DNA strands want to recombine?
As the base pairs are complimentary.
90
How do E.coli stop the 2 separated DNA strands from re-combining in DNA replication?
Single strand binding proteins (SSB proteins) bind to the single DNA strands and stop the re-combination.
91
What enzyme will tell DNA-B to start and stop working in DNA replication in E.coli?
DNA C.
92
What enzyme is responsible for delivering DNA-B to the ORI in DNA replication of E.coli?
DNA-C.
93
What happens once DNA-B is delivered to the ORI in DNA replication of E.coli?
It clamps around each individual strand and unwinds the DNA in opposite directions.
94
How many helicases work on 1 DNA molecule?
2.
One on each strand.
95
What needs to be open for DNA replication to occur?
The ORI.
96
What enzyme will synthesise a primer molecule allowing DNA polymerase to bind to the DNA strand?
DNA primase.
97
What enzyme will remove the primers from the DNA strands?
DNA polymerase I removes the primers leaving Okazaki fragments.
98
Which enzyme will stitch together the Okazaki fragments on the lagging strand?
DNA ligase
99
What strand will Okazaki fragments be formed on?
On the lagging strand.
100
Why is it difficult for DNA polymerase to copy the lagging strand?
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
What is the phase of replication known as when nucleotides are being added to the daughter strands?
Elongation.
102
How does DNA primase work?
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
The primer on what strand only needs to be synthesised once?
The leading strand.
104
The primer on what strand only needs to be synthesised more than once?
The lagging strand.
105
DNA helicase opens up a DNA strand in what direction?
In the 5 to 3 direction from the leading strand.
106
What direction can the leading strand be read in?
The 5 to 3 direction.
107
How does DNA polymerase synthesise its daughter strand from the leading strand?
It will synthesise 1 continuous piece of DNA.
108
What direction is the lagging strand in relative to the leading strand?
It is in the 3 to 5 direction relative to the leading strand.
109
The polymerase that works on the lagging strand must go in the what direction relative to the helicase?
The opposite direction.
110
The polymerase that works on the leading strand must go in the what direction relative to the helicase?
The same direction.
111
What must be laid down on the lagging strand to allow DNA polymerase to copy it?
Multiple primers.
112
What are Okazaki fragments?
Fragments of DNA that are separated by primers.
113
How many base pairs are found in Okazaki fragments in bacteria?
Between 1000 and 2000 base pairs.
114
What enzyme will remove the primers from the Okazaki fragments?
DNA polymerase I in prokaryotes.
RNAseh, FEN-1 protein and DNA polymerase I in eukaryotes.
115
What enzyme stitches the fragments of DNA together once DNA polymerase I has removed the primers?
DNA ligase creates phosphodiester bonds between the nucleotides of different fragments.
116
Which polymerase cannot synthesise a daughter strand without a primer?
DNA polymerase III.
117
What helps DNA polymerase III attach to the DNA strand?
A clamp protein.
118
What does DNA polymerase III do once it is attached to the DNA strand?
It moves down the strand identifying each base and creating its corresponding base.
119
If DNA polymerase finds a sequence of CAT on a DNA strand, what will it synthesise?
GTA.
120
How does DNA polymerase bring in the bases that are used to make the nucleotides in the daughter strand?
As deoxynucloeside triphosphates.
121
How does DNA polymerase alter the deoxynucloeside triphosphates so they can be added to the daughter strand?
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
How does DNA polymerase III read the parent strand?
In the 5 to 3 direction.
123
How does DNA polymerase III synthesise the daughter strand?
In the 3 to 5 direction.
124
What is a tautomeric shift in cytosine?
When an isomer of C is created.
125
What does the isomer of C represent when a tautomeric shift occurs?
It will represent a T molecule which the polymerase will place next to an A.
126
Can the isomer of C from a tautomeric shift pair with an A?
No.
127
What happens when an isomer of C from a tautomeric shift is placed next to an A in a DNA strand?
A bulge is created.
128
What will DNA polymerase do if a bulge in the strand is created by the isomer of C?
It will correct its mistake and go back to remove the isomer of C and replace it with a T.
129
Why does DNA polymerase III have proof-reading ability in the 3 to 5 direction?
Because it can correct mistakes it has made.
E.g. The removal of C isomers.
130
What proof-reading ability is DNA polymerase III said to have?
3 prime to 5 prime exonuclease activity.
131
What does the 3 prime to 5 prime exonuclease activity of DNA polymerase III prevent from happening?
DNA mutations.
132
What are the 3 prokaryotic DNA polymerases?
DNA Polymerase I.
DNA Polymerase II.
DNA Polymerase III.
133
What is DNA polymerase I involved in?
DNA repair.
134
What is DNA polymerase II involved in?
Repairing damaged DNA.
135
What is DNA polymerase III involved in?
Synthesising new strands.
136
What are the 3 characteristics of DNA polymerase I?
5-3 exonuclease ability.
5-3 polymerase activity.
3-5 exonuclease activity.
137
What is 5-3 exonuclease ability?
The ability to remove RNA primers.
138
What is 5-3 polymerase activity?
The ability to add nucleotides to DNA.
139
What is 5-3 exonuclease activity?
Proofreading ability.
140
What is the main characteristic of DNA polymerase II?
3-5 exonuclease activity.
141
What are the 2 characteristics of DNA polymerase III?
5-3 polymerase activity.
3-5 exonuclease activity.
142
What about the structure of eukaryotic chromosomes makes it difficult for DNA replication to occur?
The ends of the linear chromosomes.
143
What about the ends of linear chromosomes makes it difficult for replication to occur?
When the primer sequence is removed from the 5 prime end as it is impossible to replace that section of DNA.
144
Why is it impossible to replace the primer at the 5 prime end of a DNA strand?
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
How does the removal of the primer at the 5 prime end cause the daughter strand to differ from the parent strand?
The daughter strand will be shorter than the parent strand as the 5 prime end begins further up the strand.
146
What happens if the primer at the 5 prime end isn't replaced?
The daughter strand will get shorter and shorter meaning that DNA would be lost each time replication occurs.
147
What happens on the 3 prime end to help replace the primer from the 5 prime end on linear eukaryotic DNA?
The 3 prime end of the parent strand is extended to form a structure known as a telomere.
148
What enzyme will extend the 3 prime end of the parent strand during the replacement of the primer on the 5 prime end?
Telomerase.
149
What are telomeres?
Sections of non coding DNA consisting of repetitive DNA sequences made up of nucleotides that spell TTAGGG.
150
How does telomerase work?
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
What ability does telomerase have?
Reverse transcriptase ability.
This allows it to use its RNA template to form DNA
152
What happens when the 3 prime end of a DNA strand has been extended by telomerase?
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.
