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MIMS > DNA > Flashcards

DNA Flashcards

1
Q

How do you prove that DNA is the genetic material

A

Take a pathogenic bacterium (S strain) which undergoes a random mutation forming R strain (non-pathogenic).
Grow R cells in presence of heat killed S cells
R strain cells are transformed to S strain whose daughters are pathogenic
Test each molecule for transformation: only DNA results in S strain

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2
Q

What do S strain cells cause

A

Pneumonia

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3
Q

3 things DNA needs to do

A

Be stored

Be propagated

Be read

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4
Q

What are nucleic acids made of

A

Sugar
Phosphate
Aromatic Bases

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5
Q

How remember purine from Pyrimidine

A

Purine: 2 rings
Pyrimidine: 1 rings

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6
Q

Which bases are purines and Pyrimidines

A

Purine:
A
G

Pyrimidine
T
C
U

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7
Q

Is DNA branched

A

No it is a linear chain of nucleotides

5’ to 3’ polarity

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8
Q

Which is the 5’ end

A

Where phosphate bonds to CH2

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9
Q

Which is the 3’ end

A

Where phosphate group binds directly to C in ring

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10
Q

Give 6 types of nucleic acid polymers

A 
DNA
mRNA
tRNA
rRNA
miRNA
IncRNA
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11
Q

What is chargaff’s rule

A

%A=%T

%C=%G

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12
Q

What does it mean to say DNA is an anti parallel double helix

A

One strand is 3’ to 5’ and the other is 5’ to 3’

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13
Q

How many base pairs per turn

Why is it not an integer

A

10.5

It is an average

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14
Q

How are the planes of bases related to the helix axis

A

Perpendicular

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15
Q

What basic fact about sequence specific recognition by transcription factors is important

A

The edges of bases are exposed to solvent

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16
Q

Give and briefly describe the 3 types of DNA

Which is most common

A

A-DNA - squashed, right handed
B-DNA - most common
Z- DNA - left handed, less regular, less stable and therefore transient

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17
Q

How much DNA must be packed into a cell?

What is the average cell size?

How is this done

A

2m

10μm

Supercoiling

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18
Q

What is supercoiling

A

The coiling of a coil

In DNA it’s when the axis of the double helix is wound upon itself

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19
Q

Is the supercoiled state relaxed

A

No it is high energy and generates structural strain

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20
Q

How is DNA usually wound

Why

A

Underwound

Facilitates compaction and strand separation

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21
Q

How can supercoiling be measured

A

Topology

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22
Q

How can supercoiling be controlled

A

Enzymes can change the degree of cooling. These are called topoisomerases

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23
Q

How do topoisomerases work briefly

A

Cleaning and rejoining DNA strands

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24
Q

What is a nucleoid

A

A rosette model of DNA organisation

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25
Q

What is the basic unit of eukaryote chromosome structure

A

The nucleosome

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26
Q

What is a nucleosome

A

where DNA is wrapped around a protein barrel made of a Histone octamer

DNA wraps 2x around the barrel

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27
Q

Are histones charged

A

Yes they are positive

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28
Q

What histones do nucleosomes contain

How much DNA is there

A

2 copies of H2A, H2B, H3 and H4 and one copy of H1

200 nucleotides worth

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29
Q

What does H1 do

A

It is a linker histone, linking the entry and exit points of DNA as well as binding DNA between nucleosomes

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30
Q

What is a chromatin

A

Chromosomal DNA packaged with histones. It’s simplest form is the 10nm fibre

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31
Q

What is a euchromatin

A

An open chromatin which is more accessible (thus transcriptionally active)
Like beads on a string

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32
Q

What is a heterochromatin

A 
Condensed chromatin
Less accessible (therefore transcriptionally inactive)

Known as a solenoid

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33
Q

What is SMC

A

Structural. Maintenance of Chromosome proteins

Multi-domain ATPases

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34
Q

Give overview of SMC dimer

A

2 molecules of anti parallel coil with a head and hinge at either end and an arm in between
Hinge of each join the two molecules and the head is where the ATP binds

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35
Q

Give the 2 important SMC proteins

What do they do

A

Cohesin
Condensin

Encircle DNA

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36
Q

What are cohesinopathies

A

Developmental disorders caused by mutations in Cohesin leading to defects such as limb deformities and craniofacial anomalies

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37
Q

What is the loop extrusion model

A

Condensin extrude loops of DNA
Condensin molecules approach each other and arrange around the longitudinal axis to form threadlike structures
These structures pack in layers to form an X shaped chromosome

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38
Q

Give 4 implications of DNA packing

A

Chromatin status
Accessibility for gene transcription
Local chromatin status in dynamic
Alterations in chromatin status are important in disease

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39
Q

What is chromatin status

A

The degree of DNA compaction that regulates its accessibility

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40
Q

3 things which affect chromatin status

A

H1: higher H1 levels favour chromatin condensation (less accessible)

Post translational modifications if histones that reduce positive charge (lysine acetylation)

Chromatin remodelling

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41
Q

How are histones modified

A

Covalently by post translational modifications on the unstructured tails that project out eg methylation or phosphorylation

These can be inherited (epigenetics)

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42
Q

What does lysine acetylation do to histones

A

Reduces compaction by removing positive charge having a direct effect on chromatin condensation

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43
Q

Are histone modifications static?

A

No they are dynamic: chromatin writers and erasers add and remove chemical signals

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44
Q

What do chromatin readers do

A

Recognise each unique set of modifications (Histone Code) and trigger a transcriptional response

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45
Q

What are Bromodomain proteins

A

A family of chromatin readers that are used to treat cancer as they recognise acetylated Lys

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46
Q

Why is the nucleosome position important?

Are they randomly placed and even?

A

It affects availability of DNA binding sites for transcription factors

No: there are regions of high nucleosome density and nucleosome free regions

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47
Q

What is nucleosome remodelling

A

The assembly, movement and editing of nucleosomes, which requires ATP hydrolysis

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48
Q

What are nucleosome remodellers

A

Protein complexes with ATPase activity

They all have a DNA translocation motor and reader subunits for targeting the remodeller to specific chromatin sites

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49
Q

What does nucleosome assembly involve

A

A strand of DNA undergoes deposition of H3-H4 tetramer to give random spacing.
Then the nucleosomes are matured and spaced regularly by enzymes

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50
Q

How may a nucleosome remodeller change chromatin access

A

Repositioning to give irregular spacing
Nucleosome ejection
Histone dimer eviction

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51
Q

What does INO80 do

A

Nucleosome editing By exchanging histones

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52
Q

Are interphase chromosomes arranged randomly in the nucleus

A

No they occupy spatially distinct regions called chromosome territories

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53
Q

WHat is Burkitt’s lymphoma

A

Translocation between MYC Gene and one of 3 immunoglobulin Gene variants located on different chromosomes

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54
Q

What is the most common translocation in Burkitt’s lymphoma

What is the order of likelihood and why

A

MYC:IGH

IGH>IGL>IGK

IGH is spatially the closest to MYC

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55
Q

What is a TAD

A

Topologically Associating Domains
A contiguous region along the chromosome which contains the majority of interaction sites

TADs are made of many chromatin fibres

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56
Q

Order TAD, compartments, territories and chromatin fibres according to size

A

Territory> compartment> TAD> chromatin fibre

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57
Q

What are the 3 theories of DNA replication

A

Semi conservative
Conservative
Dispersive

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58
Q

What is semi conservative replication

A

When DNA splits into 2 strands which are transcribe another strand and are incorporated into it

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59
Q

Describe conservative replication

A

2 newly synthesised strands form an identical double helix while the original DNA remains as it is

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60
Q

Who identified the mode of DNA replication

A

Meselson and Stahl, 1958

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61
Q

How did Meselson and Stahl prove DNA replication

A

E. Coli culture was put into a radioactive NH4Cl medium where the N is N-15. This is transferred to a N-14 NH4Cl medium.
They took a sample after 20 mins (1 cell division) and 40 mins (2 cell divisions)
DNA extracted using density gradient centrifugation

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62
Q

What would have been Meselson and Stahl’s possible outcomes for their experiments

A

Conservative: 2 bands after centrifuge (1 parent and 1 daughter)
Dispersive: 1 band (both a mix of parent and daughter), 1 band after 2nd division
Semi conservative: 1 band (both half parent half daughter) then 2 bands after 2nd division (1 for just new DNA and 1 for original and new mix)

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63
Q

Describe the initiation of replication in prokaryotes

A

E. Coli has 1 origin which is defined by initiator proteins

Bacterial initiator is DnaA which binds to oriC

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64
Q

What is the OriC locus made of?

A

5 DnaA boxes and an A/T rich region

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65
Q

Why is it important that the OriC region is A/T rich?

A

They have fewer Hydrogen bonds so are weaker and can be untwisted more easily

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66
Q

What does the DnaA do

A

Occupies DnaA boxes and forms a large protein- DNA complex

DnaA binding facilitates unwinding of AT-rich region

DnaA polymerises onto ssDNA of unwound origin

DnaB helicase is recruited to the unwound origin with the help of loafer protein, DnaC

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67
Q

Which enzyme type unwinds DNA

A

Helicases

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68
Q

What is the structure of a replicative helicase

A

Hexameric ring with ATP at the interface between subunits
One DNA strand it threaded through the ring as the other is peeled off
Require ATP hydrolysis

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69
Q

What stops 2 strands sticking back together

A

SSB: binds to both strands to stop rebonding but is easily hydrolysed otherwise it would interfere and damage signalling

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70
Q

What does DNA ligase do

A

“Glues” together Okazaki fragments

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71
Q

Which direction does DNA polymerase work

A

5’ to 3’ direction

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72
Q

What does topoisomerase do

A

Unravel double helix

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73
Q

What is Chargaff’s rule

A

%A=%T

%G=%C

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74
Q

What is it called when the 2 strands of DNA come apart

A

Melting

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75
Q

What did Max Delbrück say

A

The DNA strands would have to be pulled apart to replicated/ be copied

This is the untwiddling problem

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76
Q

What does DnaA do

A

Creates a small melted region which acts as a loading site for DnaB

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77
Q

What is DnaB

A

A helicase that uses ATP hydrolysis to direct unwinding of double stranded DNA

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78
Q

How do replication forks arise

A

Replication initiates at the origin and spreads bidirectionally giving 2 y-shaped structures

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79
Q

What enzyme is responsible for synthesising DNA

Give 2 difficulties of this enzyme

A

DNA polymerase

Only works in 5’ to 3’ direction
Can only extend synthesis from a pre-existing 3’ OH group of DNA

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80
Q

How to get over the fact that DNA polymerase cannot begin chain synthesis itself

A

Primer

Primase, a specialised RNA polymerase that acts to make a short RNA molecule that is then extended by DNA polymerase

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81
Q

Which strand is the leading strand

A

3’ to 5’

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82
Q

How is the lagging strand replicated

A

In short pieces called Okazaki fragments which are then stitched together to make a continuous strand

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83
Q

How long are Okazaki fragments in

a) humans?
b) bacteria?

A

a) 100-200 bases long

b) 1000 bases long

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84
Q

Why must RNA primers be removed

A

The genome is made of DNA only

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85
Q

What enzyme carries out DNA replication in bacteria

What happens when it encounters an RNA primer

A

DNA polymerase III

it stalls when it encounters RNA ahead of it and the enzyme DNA polymerase I
Is recruited to the 3’ OH left by DNA polymerase III

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86
Q

How does DNA polymerase I deal with RNA primers

A

The 5’ to 3’ exonuclease activity chews this up, allowing the polymerase activity to make DNA in its place

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87
Q

What is 5’ to 3’ exonuclease activity

A

Hydrolyses RNA and DNA from 5’ end

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88
Q

Describe the difference in leading and lagging strand activity in E. coli

A

Leading: goes half way round the chromosome where it meets the fork coming in the opposite direction (keeps going for 2,500,000) bases

Lagging: only goes for 1000 bases

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89
Q

Discuss the processivity of DNA polymerase

A

Not great
It can’t hold onto the DNA v well so relies on a sliding clamp (the β clamp) to ensure it remains attached to the strand and is capable of processive synthesis

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90
Q

How many times does the β clamp have to attached to a single strand

A

Once on leading strand

Many times on lagging strand

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91
Q

What is Nick translation

A

5’ to 3’ activity by DNA polymerase I

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92
Q

What is the sliding clamp?

A

It is the β subunit of DNA polymerase III

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93
Q

What is the holoenzyme complex

A

The interaction of several proteins to form one replication machine

It acts to couple leading and lagging strand synthesis with replication form progression

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94
Q

Why was the trombone model proposed

A

Leading and lagging strands head off in different directions and must be brought together by holoenzyme complex

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95
Q

How does the trombone model work

A

The lagging strand is primed then bent back around to engage with DNA polymerase III forming a loop of increasing size that is periodically released allowing a novel priming event to occur

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96
Q

What is the DNA replication sequence

A 
Origin recognition 
Helicase recruitment 
DNA melting
Priming
Elongation
Association of polymerase with a sliding clamp
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97
Q

Why do eukaryotic cells have multiple origins of replication

A

To all DNA replication in a reasonable timeframe

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98
Q

What recognises origins in eukaryotic chromosomes

A

A 6 protein complex called ORC

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99
Q

What does ORC stand for and what does it do

A

Origin Recognition Complex

Recruits the DNA melting helicase (MCM, which also has 6 subunits)

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100
Q

What does ORC require to recruit MCM

A

Cdc6 and Cdt1

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101
Q

What is the most significant mechanistic distinction between prokaryotic and eukaryotic DNA replication

A

The way in which primers are removed

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102
Q

How are RNA primers removed in eukaryotes

What kind of enzyme is used

A

By RNaseH which digests RNA that is base paired to DNA. This structure is then cut off by Fen1 and the resulting nick is fixed by DNA ligase
This processes are coordinated by the eukaryotic sliding clamp, PCNA

A specialised ribonuclease

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103
Q

Why is DNA in eukaryotes controlled by the cell cycle

A

So DNA is replicated only once per cycle

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104
Q

What worries Watson and Crick about their model and replication

A

Since it is 2 chains intertwined it was difficult to foresee how they would separate and replicate without tangling

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105
Q

What stops replicating DNA tangling

A

Topoisomerases which release topological tension in DNA molecules

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106
Q

How does each type of topoisomerase work

A

Type I: nick a single strand and swivel it around another before resealing the nick

Type II: cut both strands and allow another duplex to pass through the gap, which is then resealed

107
Q

Does DNA topoisomerase 1 require ATP

Why (2)

A

No

Formation of covalent bond is isoenergetic with the phospho-ester bond

DNA swivels due to the torque caused by supercoiling

108
Q

Which drugs target the action of topoisomerases

A

Etoposide and doxorubicin as inhibitors for cancer chemotherapy

109
Q

What are often exploited in eukaryotes for Drug therapy (DNA replication)

A

MCMs as markers of cellular proliferation

110
Q

What is a genome

A

The complete set of genetic material for that organism (usually DNA expect for some viruses that use RNA)

111
Q

In eukaryotes what DNA is included in the genome

A

The DNA found in nuclear chromosomes

Not mitochondrial DNA

112
Q

Is a chromosome all equally condensed

A

No there are some less dense regions (euchromatin) and more condensed regions (heterochromatin)

113
Q

Which regions of the genome are not transcribed

A

The heterochromatin

114
Q

What are the 2 major regions of heterochromatin in mammalian chromosomes

A

Centromeres and telomeres

115
Q

What are centromeres

Where are they found

A

Regions of the chromosome that contain repetitive sequences and are essential structures for chromosome pairing and mitosis

In the middle of the chromosome in humans but at one end of mice chromosomes

116
Q

What are telocentric chromosomes

A

Centromeres in mice found at the end of the chromosome

117
Q

Does centromere position matter

A

Yes as it determines the length of the two arms of the chromosome

118
Q

Which arm is shorter in a chromosome

A

p (like petit)

q is the long one

119
Q

What happens in extreme forms of DNA condensation into heterochromatin

A

Inactivation of the X chromosome in female (XX) cells to form Barr bodies

120
Q

What is Lyonisation

A

Formation of Barr bodies by extreme DNA condensation into heterochromatin

Named after Mary Lyons

It results in cells only having one active copy of the X chromosome

121
Q

How and why are X chromosomes in females silenced

A

Women have XX so one is silenced so you don’t have excess copies of all the genes in the X

It is silenced by compacting it into heterochromatin resulting in dosage compensation

122
Q

Which X is silenced in females

A

Random

This leads to the patchwork of colours on a calico cat coat

123
Q

Can male calico cats have different colour patches

A

Usually no, in theory only female
But there are some rare examples of Male cats who have extra copies of the X chromosome
This is the feline equivalent of Klinefelter’s syndrome

124
Q

What is Klinefelter’s syndrome

Give symptoms

A

When males are born with an extra X chromosome

May lead to slower development, dyslexia, difficulty socialising, and in adult life leads to more feminine features as well as infertility and reduced sex drive

125
Q

Why does the lagging strand replication mechanism make telomeres a requirement

What would happen without telomeres

A

The end of the chromosome either isn’t replicated or is left with an RNA-DNA duplex

There would be a progressive shortening of chromosome and potential loss of genetic information as the RNA would not be replicated

126
Q

What is the telomere sequence in humans

What does it do

A

TTAGGG

Produces a protective cap for the chromosome end

127
Q

How is a telomere added

A

Via the action of telomerase

128
Q

Discuss the structure of telomerase

A

Contains both a protein and RNA component

The RNA component serves as a template for DNA synthesis making telomerase a self templating reverse transcriptase

129
Q

Why are reverse transcriptases important

Where are they often found

A

They use RNA to make DNA (over turning the central dogma proposed by Crick)

In many RNA viruses eg HIV

130
Q

How does telomere action counteract the lagging strand problem

A

Adds long repetitive stretches of single stranded DNA to the end of the chromosome
This DNA can adopt some unusual structures such as a loop that closes off the end of the chromosome (the T loop) and quadruplex DNA structures

131
Q

What is the purpose of quadruplex DNA structures

A

Prevent the exposed chromosome ends from being mistakenly recognised by the cell’s DNA damage response systems as damaged DNA

132
Q

Why does telomerase add such a long stretch of DNA to the end of each chromosome

A

Normal adult differentiated cells have low levels of telomerase so each time they divide, their telomeres get shorter. Once telomeres reach a critical length the cells read this as getting old and stop dividing. Reintroduction of telomeres increase lifespan

133
Q

What contributes to immortality in cancer

A

Cancer cells have elevated telomerase levels

134
Q

How can knowledge of telomeres help cancer treatment

A

Telomerase inhibitors may work as possible anti cancer drugs

135
Q

Which DNA sequencing method did Sanger invent

A

Chain terminator method

136
Q

What does the Sanger method of DNA sequencing rely on

A

The fact DNA polymerase can incorporate 2’3’- dideoxynucleotides (ddNTPs) into growing DNA chains
As the ddNTP does not have a 3’ OH, it terminates the chain

137
Q

What was the original technique used by Sanger

A

4 reactions set up where DNA synthesis is primed by synthetic oligonucleotides. The dNTPs had a radio label and were spiked with a ddNTP
This meant the new DNA chains randomly terminate at either A G C or T residues
Then perform gel electrophoresis
The gel is exposed to X Ray film to visualise the DNA bands and the sequence is read from bottom to top

138
Q

How is the Sanger method done now

A

Uses ddNTPs conjugated with fluorescent markers, enabling a sequencing reaction

Fine capillaries are now used for separating DNA fragments not gels

139
Q

What is reannealing

A

The study of DNA composition by studying the rate at which denatured DNA reformed into double stranded DNA in solution

More complex DNA structures take longer to reform

140
Q

Why does more complex DNA take longer to form

A

The probability of finding a complementary stretch of DNA in a complex population is lower

141
Q

How big is an E. coli genome

What about yeast

A

4.7 x10^6 bases

13x10^6

142
Q

How big is the human genome

A

> 3x10^9

143
Q

How big is the genome of A. Dubia (an amoeba) compared to human

A

200x larger in the amoeba

144
Q

Does bigger genome= bigger genes

A

No
The worm C. elegans has 10^8 bases but 19,000 genes
The human has a genome of 3x10^9 bases but has only 25000

145
Q

Human genome is 250x bigger than those orb of yeast but only encodes 5x more genes. Why is this

A

Human coding sequences are stuffed with junk DNA

95% is thought to be non-coding

146
Q

What are the 4 classes of repetitive sequence found in the human genome

Give their % of the genome

A

Simple sequence repeat (CACACA) (3% of. Genome)
Segmental duplications (5%)
Transposon derives repeats (45%)
Inactive copies of partially retrotransposed cellular genes

147
Q

Is the genome static

A

No

148
Q

What are mobile genetic elements

Give 2 broad types

A

Regions of DNA that can move around and insert in other parts of the genome

This can cause mutations depending where they insert

Transposons and retrotransposons

149
Q

What moves DNA transposons

A

Transposase

150
Q

What are retrotransposons

A

Where DNA is transcribed to RNA and then reverse transcribed to DNA which is Inserted back into a different part of the genome

151
Q

What are “jumping genes”?

A

Mobile genetic elements

152
Q

Why are transposons bad for antibiotics

A

Many transposons carry antibiotic resistance genes so can propagate resistance within and even between strains and species

153
Q

Give 2 examples of diseases resulting from the mutagenic quality of transposition

A

Muscular dystrophy

Haemophilia

154
Q

How could transposons be useful

A

Facilitate shuffling of coding sequences or moving genes so that they are under the control of a new promoter (changing when and where it is expressed)

They may help generate our immune system diversity system

155
Q

What do recombinases do

A

Mediate diversity generation are related to transposon-encoded enzymes and may therefore be derived from an ancient transposon

156
Q

Give uses of the genome sequencing in medicine

A

Genetic diagnosis

Mutation detection

157
Q

How does genome sequencing help with CF

A

Can diagnose the type of CF and therefore it’s severity and the patient’s long term survival

158
Q

What does reannealing of DNA depend on

A

Temperature
Length of DNA
base composition
Iconic composition of the solvent

159
Q

What are DNA endonucleases

A

Enzymes in bacteria that digest DNA to destroy incoming viral DNA

160
Q

What is the restriction modification system

A

The anti-viral defence in bacteria that uses DNA endonuclease to destroy incoming viral DNA

161
Q

How do DNA endonucleases distinguish between bacterial and viral DNA

A

They recognise specific sequences which may be absent from the bacterial genome
Also specific for methylated or non methylated DNA

162
Q

Where can DNA be methylated

A

A or C residues

163
Q

Why is it called the restriction modification system

A

Bacteria modify their DNA so they can selectively attack viral DNA, restricting the viral infection

164
Q

Which type of restriction enzyme are most widely used

A

Type II

These recognise sequences that are commonly palindromic and can range from a couple of bases to tens of bases

165
Q

What is a sticky end

A

When a restriction enzyme cuts each strand in different positions, generating overhanging stretches of DNA

alternatively, the enzyme could simply cut both DNA strands to generate 2 blunt ended DNA molecules

166
Q

What can DNA ligase and restriction enzymes do together

A

Pasting together bits of DNA that have compatible sticky ends (making DNA constructs)

167
Q

Is DNA charged

What other feature is useful

A

Yes it is negative (due to phosphate backbone)

Chemically stable in solution

168
Q

As DNA is negative and stable in solution. What does this property allow

A

DNA can be separated by size when put in an electrical field in a porous material (usually an agarose or polyacrylamide gel)

After separation DNA can be visualised directly by dyes that bind double stranded DNA and fluorescence under ultra violet light

169
Q

What is a common dye used for DNA

A

Ethidium bromide which intercalates into DNA

However it is a mutagen and probs a carcinogen

170
Q

What is the southern blot

A

Separate DNA in an electrical field with fluorescent dye
Transfer DNA from the gel to a filter
Analyse using hybridisation to check for specific sequences

171
Q

What is the most common way to copy your favourite piece of DNA

A

Cut and paste it into a plasmid And introduce it into the appropriate bacterium
Now your DNA will be copied and amplified and easily purified back from growing bacteria

172
Q

What is a plasmid

A

Circular pieces of extrachromosomal DNA in bacteria

173
Q

How do you copy and paste your favourite DNA

A

Use restriction enzymes to cut the plasmid open so you can ligase your piece of DNA into the circle

174
Q

What is an alternative to plasmids for DNA copying

A

Using bacteriophages

Use restriction enzymes and DNA ligase to cut along with the virus when the virus infects the bacteria

175
Q

What is a disadvantage of using plasmids or bacteriophages to copy DNA

A

There is a limit to the length of DNA you can propagate

176
Q

What is PCR

A

Polymerase chain reaction

177
Q

How does PCR work

A

You know the sequence at the end of the stretch of DNA so you design short primers which provide a free 3’ end of DNA that DNA polymerase can extend from

It proceeds in a series of cycles, increasing the amount of DNA exponentially

178
Q

Give the 3 steps in each cycle of PCR

A

Heat to >90 degrees to separated double stranded DNA
Use lower temperature to allow primers to hybridise to their target sequence
Allow DNA polymerase to extend the DNA

(Melting, reannealing, extension)

179
Q

What is required for PCR

A 
Short primers of 20 bases that hybridise to DNA flanking the region to be amplified. 
Template DNA 
dNTPs
Buffer
DNA polymerase
180
Q

Where does the DNA polymerase come from in PCR

A

The DNA polymerase is Taq polymerase from Thermus aquaticus that grows in hot springs so can survive temperature changes

181
Q

What is 3C

A

Chromosome Conformation Capture

DNA is digested in situ, crosslinked, and ligated together
Cross links are then reversed and ligated fragments are sequences

182
Q

What is CRISPR/Cas9 based on

A

Immunity in archaea and some bacteria

183
Q

What is CRISPR

A

Clustered Regularly Interspersed Short Palindromic Repeats

Short viral DNA sequences present in microbial genome that are transcribed into RNA and used as guides to direct the Cas9 nuclease to cleave the DNA of an invading virus

184
Q

How is CRISPR/Cas9 used in experiments

A

Can do gene knockouts and allele replacements by preparing guide RNAs to target the desired genomic locus

185
Q

What is better: mutated DNA or cell death?

A

Cell death: higher eukaryotes May induce apoptosis rather than live with potentially mutated DNA

186
Q

Give 2 types of point mutation

A

Transition

Trans version

187
Q

What is a transition mutation

A

When a purine is replaced by a different purine

188
Q

What is transversion mutation

A

When a purine is replaced by a Pyrimidine (or vice versa)

189
Q

What kind of damage can the DNA back bone undergo

A

It can be chronically modified or even broken

These breaks can be on 1 strand or both strands resulting in a double strand break (DSB)

190
Q

What is the Ames test

A

Screening for mutagenic chemicals using salmonella

191
Q

How does the Ames test work

A

A salmonella strain that cannot synthesis His is grown in the absence of His
Mutations that enable His synthesis result in Colony growth
Number of colonies is proportional to how mutagenic the chemical is

192
Q

What is the safe threshold for a mutagenic agent

A

None

It is either mutagenic or not

193
Q

Give 3 endogenous and exogenous causes of DNA lesions

A

Endogenous: ROS
Reactive chemicals
Chemical instability

Exogenous: UV light
Ionising radiation
Genotoxic chemicals

194
Q

Give 4 possible errors that can occur during replication

A

Base mismatch
Deletion/ insertion
Low fidelity copying by trans lesion synthesis DNA polymerases
Incomplete replication

195
Q

What gives DNA polymerase high fidelity

A

The incoming nucleotide must have properly paired with its Partner on the opposite strand before DNA polymerase adds it to the 3’ end of the previous base

196
Q

If the polymerase incorporates the wrong nucleotide, how is the error fixed

A

Detected by polymerase itself
And corrected by 3’ to 5’ exonuclease activity associated with the enzyme, allowing the enzyme to back up. It removes the bad nucleotide and the. Polymerase drives the enzyme forwArds to redo

197
Q

Discuss Huntington’s Disease

A

Autosomal dominant

Caused by insertion of a repeated sequence of CAG in Huntington gene

198
Q

How many repeats are needed for symptoms to appear in Huntington’s

A

Less than 30 CAG repeats and they don’t have the disease

>40 repeats and they develop the cognitive and motor symptoms of the disease

199
Q

How is anticipation exhibited in Huntington’s disease

Why might this be

A

Affected offspring often develop the disease at a younger age than affected parents as they have more copies of the repeats

One possibility: hairpin forms in repeat region chasing expansion here
Or
DNA repair systems may recognise misaligned bases in the hair pin and in the process of fixing it, expand the repeat sequence

200
Q

What are the 3 major repair mechanisms for single stranded damage

A

Base excision
Nucleotide excision
Mismatch repair

201
Q

How does each case of single stranded repair work overall

A

Recognition of incorrect/ damaged base
Removal/ repair of base or nucleotide
Filling gap with DNA polymerase and ligase

202
Q

What are the 2 major classes of repair mechanism for double stranded break

A

Non homologous end joining

Homologous end joining

203
Q

How can UV light affect DNA

Will the base have to be removed

A

Causes adjacent T residues to form diners through a cyclobutyl ring, deforming the double helix and obstructing DNA replication and RNA transcription

No it can be directly reversed

204
Q

How can a mutated thymidine from UV radiation be corrected?

A

Photoreactivation of the T dimer, catalysed by DNA photolyase enzymes, converts the T dimer back to 2 single T residues

205
Q

What happens if C deaminates spontaneously or by oxidising agents?

A

Forms U

U pairs with A so could result in a conversion if a CG base pair to a TA in later generations

206
Q

When a C is mutated to a U, how does the body cope?

A

Uracil DNA glycosylase (UDG) recognises U in DNA.

It flips it out of the double helix and cleaves the glycosidic linkage to the deoxyribose

207
Q

what is the result of UDG action

A

Creation of an “abasic site” (no bases are present)

208
Q

What happens to an abasic site after the action of UDG

A

Recognised by a nuclease that cleaves the backbone producing a 3’OH upstream of the site.
This acts as a priming site for a DNA polymerase

209
Q

What does the DNA polymerase do to the 3’OH during removal of an abasic site

A

DNA polymerase removes the abasic site and incorporates a C opposite the templated G

210
Q

In
a) eukaryotes
b) prokaryotes
Which DNA polymerase is involved in removal of an abasic site

A

a) DNA polymerase β

b) DNA polymerase 1

211
Q

Does UDG work for all mutated bases?

A

No it can only remove U but there are other DNA glycosylases that are specialised for other altered bases

212
Q

Give an example of oxidative DNA damage

A

8-oxoGuanine

213
Q

What happens if 8-oxoGuanine is left unrepaired

Where is the often found

What happens in the case of cytosine deamination?

A

Causes a GC to TA transversion

In human cancer cells

A specific glycosylase removes the oxidised base

214
Q

Which method of DNA modification is not mutagenic

What is it used for?

A

Methylation on a C or A

To control gene expression

215
Q

Discuss DNA methylation in bacteria

A

The Dam methyltransferase adds a methyl group to the A residue in the sequence GATC
This protects the DNA from digestion by the bacterium’s own restriction endonucleases

216
Q

Discuss DNA methylation in eukaryotes

A 
Only C (not A) can be methylated 
Occurs in CpG islands 
This switches off expression of nearby genes
It is the basis of genomic imprinting
217
Q

What is a CpG island

A

Regions containing repeats of CG

218
Q

What is genomic imprinting

Eg?

A

When some genes show parent of origin

Eg: only the copy you inherit from your father is expressed, the maternal copy is methylated and silent

219
Q

True or false:

Photo lyases repair the covalent linkage made between T’s (Pyrimidine dimers) when exposed to UV light in humans

A

False

These enzymes are not found in humans

220
Q

What is NER

A

Nucleotide excision repair

The complex system for repairing Pyrimidine dimers and other base/nucleotide changes in DNA

221
Q

How is the UV induced T dimer fixed in humans

Name 2 other enzyme types that are involved

A

The NER pathway detects the dimer and unwinds the DNA around the lesion.
The strand is cut up- and down-stream of the lesion, giving a short single stranded gap
The gap is filled by DNA polymerase and sealed by DNA ligase

Helicases and nucleases

222
Q

What is xeroderma pigmentosum

A

An autosomal recessive syndrome

It is caused by the inherited mutation of genes that encode for enzymes in the NER

223
Q

Give 3 symptoms of xeroderma pigmentosum

A

Extreme sensitivity to sunlight
Premature skin aging
Predisposition to skin cancers

224
Q

What is the issue with a mutation causing an A to be swapped with a G

A

A new strand of DNA generated during replication is not methylated until some time after replication
Thus the newly generated DNA is hemimethylated with the parental template strand marked by methyl groups but not the new strand

225
Q

How does mismatch repair machinery work

A

Preferentially repairs non methylated new DNA by excising a single strand of the new DNA that flanks the error (similar to the action of NER)

226
Q

What are mutations in the mismatch repair system associated with in humans?

What is the name of the syndrome?

A

Inherited predisposition to cancer
Especially colorectal cancer

Hereditary nonpolyposis colorectal cancer syndrome

227
Q

What are the 2 main mechanisms to ensure DSB repair

A

Non homologous end joining (NHEJ)

Homologous recombination

228
Q

How does NHEJ work

A

Sticks broken ends of DNA back together which can restore original sequence but ends require some cleaning up.
May result in loss of DNA sequences at the joining site

229
Q

What are the resting phases of the eukaryotic cell cycle

A

G1 and G0

230
Q

When is NHEJ particularly important

A

In G1 and G0 (rearing phase)

231
Q

What can loss of function mutations in NHEJ cause

Eg

A

Human Cancers

Eg mutations in DNA ligase IV are found in certain leukaemias

232
Q

What does HR take advantage of

A

The fact that most organisms have more than a single copy of a given chromosome and the extra copy can act as a template for repair of a damaged chromosome

233
Q

How does HR work in human DNA repair

A

DSB is processed to trim back the 5’ end, leaving an extended 3’ end which is recognised by a highly conserved protein.
This protein scans the genome for identical double stranded sequences and directs “strand invasion”, replacing one duplex strand with the single strand from the damaged DNA.
This allows prime replication from invaded end, copying the intact strand

234
Q

Which proteins recognise the single stranded 3’ end in HR in

a) bacteria
b) eukaryotes

A

a) RecA

b) RAD51

235
Q

What is a Holliday junction

A

Mobile 4 way junctions in DNA resulting from HR repair

236
Q

What do Holliday junctions do

What allows this

A

Slide along DNA

The junctions are between homologous sequences

237
Q

What enzyme facilitates branch migration of Holliday junctions in bacteria?

How does this work?

A

The RuvAB complex

It is a molecular motor that uses ATP to push the junction

238
Q

How can a Holliday junction be resolved to 2 linear duplexes

A

In a way to restore parental configuration in both duplexes

Or

Crossed over configuration where DNA is swapped between the 2 original molecules

239
Q

When would it be desirable to have a Holliday junction have a cross over configuration with the DNA swapped between the 2 molecules after HR?

A

During gene shuffling in meiosis to increase genetic diversity

240
Q

Other than repair of DSBs, what else is HR useful for

A

Restarting stalled or broken DNA replication forks

241
Q

What is the average of failed replication forks per cell cycle

A

10/ one eukaryotic cell cycle

242
Q

Name 6 proteins involved in HR that will likely cause cancer if mutated

A 
BRCA2
ATM
Chk2
p53
Nbs1
Mre 11
243
Q

Give an example of DSB processes that can have deleterious consequences

A

A translocation between parts of chromosomes 9 and 22

This is associated with Chronic Myelogenous Leukaemia

244
Q

Give an example of the cell harnessing DSB repair for positive purposes

Elaborate

A

Antibody diversity generation

Number of different antibodies produced depends on number of combinations possible. Recombination events used to do this are error prone, thus increasing diversity

245
Q

How is even more diversity added for antibody production

What mediates this process and when can its importance be seen

A

Terminal transferase adds extra nucleotides at the junction point, increasing diversity by being extra sloppy in how many bases they add

The NHEJ pathway
Mutations in NHEJ proteins result in immunodeficiency and failure to perform antibody diversity generation

246
Q

How might targeting cellular mechanisms that maintain molecular stability be used in clinical medicine?
What is this technique called

A

Cancer cells often inactivate some of their own DNA repair mechanisms to acquire the mutator phenotype and become overly reliant on a smaller set of repair pathways. If these pathways are targeted, you can induce cancer cell death, with only minor harm to healthy cells

Synthetic lethality

247
Q

Has synthetic lethality been effective?

A

Yes: Olaparib inhibits PARP.

PARP is highly effective in cancer patients with defective BRCA2 genes so PARP inhibition results in cancer cell death

248
Q

Name a non hydrolysable analogue or lactose

A

IPTG

249
Q

Name 2 small molecule inhibitors of RNA synthesis

A

Actinomycin D

Rifamycin

250
Q

Describe the action of actinomysin D

A

Blocks all RNA synthesis immediately by binding tightly to dsDNA, intercalating between neighbouring GC base pairs

251
Q

Does actinomysin D have any clinical relevance

A

Generally too toxic for clinical use but it’s inhibitor of the growth of rapidly dividing cells makes it an effective therapeutic agent against some cancers

252
Q

Describe the action of rifamycin

Give its other name

A

An antibiotic that blocks all bacterial RNA synthesis by binding to β subunit of RNA polymerase

DOES NOT affect eukaryotic transcription, generally used to treat TB

AKA rifampicin

253
Q

Name an RNA dependent DNA polymerase

A

Telomerase

254
Q

What are restriction enzymes

A

Bacterial enzymes that cut DNA at/ near specific recognition sequences

255
Q

Is DNA right or left handed?

A

Right handed!!!

256
Q

Describe Z DNA

A

LEFT handed double helix (THE ONLY RIGHT HANDED ONE - Z is far from A and B)

257
Q

Difference between A and B DNA

A

A is Shorter, more compact helical structure whose base pairs are not perpendicular to the helix axis

258
Q

Which cancer can bevacizumab treat

A

Colon - blocks VEGF

259
Q

What is CHK1 activated by

A

Double strand breaks and then will phosphorylate CDC25 (resulting in CDC25’s degradation)

CHK1 also phosphorylates WEE1, activating WEE1

260
Q

What is the core complex of RNAi

A

RISC

RNA induced silencing complex

261
Q

What is the protein domain present in Fos and Jun transcription factors

A

Leu zipper

262
Q

What technique is used for measuring the on and off rate of binding between macro molecules

A

Surface plasmon resonance

263
Q

What allosterically activates glycogen synthase

A

G-6-P

264
Q

What enzyme is required to synthesise ketone bodies

A

Acetyl CoA acetyltransferase

MIMS (12 decks)

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