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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
Q

What is true about T equation?

A

Number of base pairs does not change, helical repeat can change

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26
Q

Lk0

A

Relaxed form
W=0

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27
Q

When Lk < 0

A

Negative supercoiling

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28
Q

When Lk > 0

A

Positive supercoiling

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29
Q

What is true about W?

A

Releases tortional strain

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30
Q

Super helical Density given by 0~ , theta

A

change in Lk/ Lk0

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31
Q

When 0~ is 0 we say the DNA is …

A

relaxed

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32
Q

What is the biological significance of negative supercoiling?

A

Easier to pull strands apart
DNA replication
Transcription
Less twist
Negative writhe

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33
Q

How is eukaryotic positive supercoiling relieved?

A

DNA wrapped around histone core
Underwound - negative supercoiling
Accessible for replication and transcription

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34
Q

What is the biological significance of positive supercoiling?

A

Helicase activity unwinds DNA strands
Positive supercoiling ahead of replication forks on either side

35
Q

What enzymes remove supercoiling?

A

Topoisomerase

36
Q

How do you remove positive supercoiling?

A

Cleave both phosphodiester backbone

37
Q

How to do you remove negative supercoiling?

A

Cleave one phosphodiester backbone
Allow rotation
More positive supercoiling

38
Q

What are the types of topoisomerase?

A

IA IB
IIA IIB

39
Q

What is the mechanism of supercoiling removal by topoisomerase?

A

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
Q

What type of topoisomerase cleave 1 backbone?

A

A types
IA IIA

41
Q

What type of topoisomerase cleave backbone of 2 strands?

A

B type
IB IIB

42
Q

What is the mechanism of type I topoisomerase?

A

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
Q

What do type 2 topoisomerase do?

A

Takes positive supercoil and reduces it back to RELAXED form.

44
Q

What is a type 2 topoisomerase enzyme?

A

DNA Gyr(rendu)ase
DNA gyrase

45
Q

What is the mechanism of DNA gyrase in removing positive supercoiling?

A

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
Q

Topoisomerase 1 reduces linking number by how much?

A

1

47
Q

Topoisomerase 2 reduces linking number by how much?

A

2

48
Q

When you run DNA on a agarose gel, what topology of DNA will run the fastest?

A

Supercoiled DNA

49
Q

When you run DNA on a agarose gel, order the bands (slowest -> fastest)

A

Slowest - nicked/ relaxed DNA - incomplete digestion
Then -> linear DNA
Fastest- supercoiled

50
Q

Bidirectional replication gives what type of replication (greek) ?

A

Theta Replication

51
Q

Problems
What is cantenes?

A

Replication of circular DNA
2 daughter rings are interlocked
Forms catanene

52
Q

Problems
What enzyme separate cantenes?

A

Topoisomerase IV

53
Q

What is process of removing catenes?

A

Decatenation

54
Q

What are steps of decatenation?

A
  1. Cleaves
    2.Pass through
    3.Re-seals
55
Q

Problem- Antiparallel strands
Type of replication

A

Semi-discontinuous

56
Q

What is semi-discontinuous replication?

A

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
Q

What are the gaps called in the lagging strand?

A

Nicks
Polymerase 3 cannot join nicks

58
Q

Explain Okazaki experiment?

A

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
Q

What did the Okazaki experiment prove?

A

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
Q

What are Okazaki fragments made up of?

A

RNA primer and DNA

61
Q

What does DNA ligase mutants result to?

A

Remain as short fragments

62
Q

What enzyme proves that Okazaki fragments are made up of RNA primer and DNA?

A

DNase
Breaks down DNA not RNA

63
Q

What mutations/errors can occur in DNA replication?

A

-incorrect nt added
-RNA nt instead of DNA nt
-nicks in backbone (fragments)

64
Q

How to pb base pair correctly?

A

Induced Fit

65
Q

Incorrect base pairing can result to?

A

Binding + shape discrimination

66
Q

Even if H-bonds are possible, what leads to shape discrimination?

A

Steric Collision

67
Q

Name purine bases?

A

Adenine
Guanine

68
Q

What are purines?

A

2 ring

69
Q

Name pyrimidines?

A

Cytosine
Thymine
Uracil

70
Q

What are pyrimidines?

A

1 ring

71
Q

How does proofreading work?

A

Incorrect bp
Polymerase stalls
Substrate to exonuclease A.S
Cleaves terminal phosphodiester bond
Releases dNMP - A,G,T,C

72
Q

The activity of DNA polymerase 1

A

DNA synthesis - 5’ -3’ Polymerase
Proofreading- 3’ - 5’ exonuclease
Nick translation (DNA repair) - exonuclease

73
Q

Protease treatment gives rise to what 2 fragments?

A
  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
Q

Why can’t RNA be added to DNA strand?

A

Steric Clash
Extra OH - Ribose

75
Q

How are RNA in okazaki fragments removed?

A

Pol I - nick translation, binds to nicks
5’ to 3’ exonuclease
Detaches after 100 bp - still fragmented
Pol III - no nick translation

76
Q

Why is U a problem?

A

Deamination of C -> U (mutation)

77
Q

How is U removed?

A

Uracil-N-glycosylase enzyme
Baseless nt
recognised and cleaved by AP( apyrimidnic) endonuclease
Nicked DNA, incorrect nt

78
Q

Uracil-N- glycosylase mutants result?

A

No removal of Uracil
No nicks/fragments

79
Q

What seals DNA nicks?

A

DNA ligase

80
Q

In circular chromosomes and plasmids initiation starts at the?

A

Origin of Replication
ORI

81
Q

The ORI is a region rich of which bases?

A

A-T
Why - weaker H bonds - easier to separate

82
Q

What is the initation point in e.coli?

A

OriC

83
Q

Number of bp repeats on oric?

A

13bp
9bp

Flow of Genetics (3 decks)

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