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Flow of Genetics > DNA replication > Flashcards

DNA replication Flashcards

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1
Q

What polymerases exist within E.coli (bacterial cells) and its function?

A

Pol 1 - DNA repair + replication

Repair
Pol II, IV, V

Main Replicative Enzyme
Polymerase III

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2
Q

E.coli ( bacteria)
What’s the main replicative enzyme

A

Polymerase III (3)

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3
Q

What is the direction of synthesis of all polymerases?

A

5’ to 3’

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4
Q

How many genes does polymerase 1 code for?

A

1 gene

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5
Q

How many genes does polymerase 3 code for?

A

22 genes

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6
Q

How can we determine which genes (proteins) are important?

A

Simply knocking out a protein will be lethal.
1. Temperature sensitive mutants - specific proteins activated at certain temperatures, deactivates protein (wrong temperature) -> cell death
2. Cells begin to replicate, then deactivate one protein to see the effect.

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7
Q

What are the type of mutants?

A
  1. Quick stop
  2. Slow stop mutant
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8
Q

What is a quick stop mutant?

A

Stops DNA replication immediately

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9
Q

What is a slow stop mutant?

A

DNA replication cannot start after the first round of replication has finished

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10
Q

Problems that arise from DNA replication are due to what factors?

A
  1. Topology
    2.Polarity
    3.Fidelity
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11
Q

How does topology complicate DNA replication?

A
  1. Coiled strands
  2. Circular DNA molecules
  3. Antiparallal
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12
Q

How does polarity complicate DNA replication?

A
  1. Antiparallel strands - 5’ to 3’
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13
Q

How does fidelity complicate DNA replication?

A
  1. Mutations and errors - becomes unfunctional
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14
Q

How are DNA strands coiled?

A

Plectonemically - wound around each other
Interaction between 2 DNA strands
Therefore strands need to be separated for DNA replication

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15
Q

What enzyme separates DNA strands?

A

Helicases

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16
Q

What is the structure of helicase?

A

Hexamer Ring
6 Identical subunits
Opposing - ATP X2 , ADP + P x2 , EMPTY X2
6 potential ATP binding site

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17
Q

How does Helicase separate the DNA strand?

A

Helicase undergoes conformation between 3 states
ATP ,ADP + Pi , Empty as ATP is hydrolysed -Ripple Effect
Causes oscillation of helicase
Extends loops through is central hole
Where DNA binds to and is looped through the central
Separating the DNA strands
Towards 3’ end

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18
Q

How many nucleotides can Polymerase III synthesise?

A

1600nt/s
10nt/turn

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19
Q

What opens up due to helicase activity?

A

Replication bubble
1 replication fork on each strand on 3’ end

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20
Q

What problems arise from the unwinding the DNA

A
  • Mechanical strain in rest of the molecule
    -Resistance
    -Increased problem in circular DNA - molecule cannot rotate
    -Torsional strain -> SUPERCOILING
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21
Q

What is the linking number?

A

The number of times 2 strands cross each other

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22
Q

What is the equation of Lk ?

A

Lk = T (twist) + W (writhe)

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23
Q

What is the equation for twist?

A

T = number bp / helical repeats

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24
Q

what is twist and writhe?

A

T= number of duplex turns
W= number of duplex self crossings

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25
What is true about T equation?
Number of base pairs does not change, helical repeat can change
26
Lk0
Relaxed form W=0
27
When Lk < 0
Negative supercoiling
28
When Lk > 0
Positive supercoiling
29
What is true about W?
Releases tortional strain
30
Super helical Density given by 0~ , theta
change in Lk/ Lk0
31
When 0~ is 0 we say the DNA is ...
relaxed
32
What is the biological significance of negative supercoiling?
Easier to pull strands apart DNA replication Transcription Less twist Negative writhe
33
How is eukaryotic positive supercoiling relieved?
DNA wrapped around histone core Underwound - negative supercoiling Accessible for replication and transcription
34
What is the biological significance of positive supercoiling?
Helicase activity unwinds DNA strands Positive supercoiling ahead of replication forks on either side
35
What enzymes remove supercoiling?
Topoisomerase
36
How do you remove positive supercoiling?
Cleave both phosphodiester backbone
37
How to do you remove negative supercoiling?
Cleave one phosphodiester backbone Allow rotation More positive supercoiling
38
What are the types of topoisomerase?
IA IB IIA IIB
39
What is the mechanism of supercoiling removal by topoisomerase?
Phosphodiester bond is transferred to tyrosine residue on the enzyme Breaks the DNA strand Unbroken strand passed through gap Phosphodiester bond transferred back to DNA. Restores back other side. No ATP is used.
40
What type of topoisomerase cleave 1 backbone?
A types IA IIA
41
What type of topoisomerase cleave backbone of 2 strands?
B type IB IIB
42
What is the mechanism of type I topoisomerase?
Breaks backbone of one strand By transferring phosphodiester bond to tyrosine residue on enzyme 1 unbroken strand passed through gap PD bond transferred back to the DNA -> re-form the backbone on the other side Increases turn, reduces Lk by 1. Stops chromosome from being overly unwound
43
What do type 2 topoisomerase do?
Takes positive supercoil and reduces it back to RELAXED form.
44
What is a type 2 topoisomerase enzyme?
DNA Gyr(rendu)ase DNA gyrase
45
What is the mechanism of DNA gyrase in removing positive supercoiling?
Positive supercoiling DNA +1 DNA gyrase - A+B subunits Topoisomerase binds recognised by DNA binding protein ATP binds to A subunits PD broken by transfer to tyrosine residue on A subunit -> Phosphate DNA + Alpha subunits Horizontal section cut. 5'-P linked to Tyr on A subunit Vertical section passed through Reform backbone (ADP + P+H20) DNA now contains 1 negative supercoil = -1 Change of 2
46
Topoisomerase 1 reduces linking number by how much?
1
47
Topoisomerase 2 reduces linking number by how much?
2
48
When you run DNA on a agarose gel, what topology of DNA will run the fastest?
Supercoiled DNA
49
When you run DNA on a agarose gel, order the bands (slowest -> fastest)
Slowest - nicked/ relaxed DNA - incomplete digestion Then -> linear DNA Fastest- supercoiled
50
Bidirectional replication gives what type of replication (greek) ?
Theta Replication
51
Problems What is cantenes?
Replication of circular DNA 2 daughter rings are interlocked Forms catanene
52
Problems What enzyme separate cantenes?
Topoisomerase IV
53
What is process of removing catenes?
Decatenation
54
What are steps of decatenation?
1. Cleaves 2.Pass through 3.Re-seals
55
Problem- Antiparallel strands Type of replication
Semi-discontinuous
56
What is semi-discontinuous replication?
The lagging strand - 5' to 3' of template DNA loops back, primers add, synthesis from 5' to 3' The leading strand - 3' to 5' template strand Polymerase lays down 1 primer DNA replication 5' to 3'
57
What are the gaps called in the lagging strand?
Nicks Polymerase 3 cannot join nicks
58
Explain Okazaki experiment?
Grew E.coli injected w/T4 bacteriophage 3H-TTP added New DNA strands are radioactive Alkaline lysis ( break H-bonds) - > separates lagging and leading strand Radioactive ssDNA map out sizes
59
What did the Okazaki experiment prove?
Semi discontinuous replication How? Small fragments present (lagging strand) - short pieces always present- nicks in DNA Increasing time - increase size of DNA - lagging changing to leading 5'-3' (DNA ligase)
60
What are Okazaki fragments made up of?
RNA primer and DNA
61
What does DNA ligase mutants result to?
Remain as short fragments
62
What enzyme proves that Okazaki fragments are made up of RNA primer and DNA?
DNase Breaks down DNA not RNA
63
What mutations/errors can occur in DNA replication?
-incorrect nt added -RNA nt instead of DNA nt -nicks in backbone (fragments)
64
How to pb base pair correctly?
Induced Fit
65
Incorrect base pairing can result to?
Binding + shape discrimination
66
Even if H-bonds are possible, what leads to shape discrimination?
Steric Collision
67
Name purine bases?
Adenine Guanine
68
What are purines?
2 ring
69
Name pyrimidines?
Cytosine Thymine Uracil
70
What are pyrimidines?
1 ring
71
How does proofreading work?
Incorrect bp Polymerase stalls Substrate to exonuclease A.S Cleaves terminal phosphodiester bond Releases dNMP - A,G,T,C
72
The activity of DNA polymerase 1
DNA synthesis - 5' -3' Polymerase Proofreading- 3' - 5' exonuclease Nick translation (DNA repair) - exonuclease
73
Protease treatment gives rise to what 2 fragments?
1. Sm(n)all N-terminal fragment - 5' to 3' exonuclease 2. Large(c) C- terminal fragment (Klenow fragment) contains polymerase and 3' to 5' exonuclease activity
74
Why can't RNA be added to DNA strand?
Steric Clash Extra OH - Ribose
75
How are RNA in okazaki fragments removed?
Pol I - nick translation, binds to nicks 5' to 3' exonuclease Detaches after 100 bp - still fragmented Pol III - no nick translation
76
Why is U a problem?
Deamination of C -> U (mutation)
77
How is U removed?
Uracil-N-glycosylase enzyme Baseless nt recognised and cleaved by AP( apyrimidnic) endonuclease Nicked DNA, incorrect nt
78
Uracil-N- glycosylase mutants result?
No removal of Uracil No nicks/fragments
79
What seals DNA nicks?
DNA ligase
80
In circular chromosomes and plasmids initiation starts at the?
Origin of Replication ORI
81
The ORI is a region rich of which bases?
A-T Why - weaker H bonds - easier to separate
82
What is the initation point in e.coli?
OriC
83
Number of bp repeats on oric?
13bp 9bp

Flow of Genetics (3 decks)

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