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Biomed 2 > Genetic information > Flashcards

Genetic information Flashcards

1
Q

what excludes incorrect dNTP

A

steric collisions

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2
Q

at what rate does Pol II add an incorrect dNTP

A

1 per 100,000 bp

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3
Q

by how much does proof reading in the cell drop the error rate of mutations

A

by 100x

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4
Q

features of Pol III

A

3’-5’ exonuclease activity
can remove the last nucleotide if it was incorrect
exonuclease will cleave the nt at the phosphodiester terminal, releasing a dNMP

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5
Q

functions of Pol I

A

5’-3’ polymerase
3’-5’ exonuclease - proof reading
5’-3’ exonuclease - nick translation

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6
Q

function of 5’-3’ exonuclease in Pol I

A

can remove the nucleotide in front of it

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7
Q

why can’t rNTPs be directly incorporated onto growing DNA strands

A

extra OH in ribose causes a steric clash

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8
Q

structure of okazaki fragments

A

RNA at the 5’ end
Nick at the 3’ end

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9
Q

which Pol binds nicks

A

Pol I

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10
Q

what removes RNA primers

A

Pol I 5’-3’ exonuclease

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11
Q

when does Pol I detaches

A

after 1000bp
leaves behind a nick

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12
Q

what degrades RNA

A

RNAse H

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13
Q

what are psuedo-okazaki fragments

A

leading strand also consists of fragments that need to be joined together

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14
Q

what does the synthetic pathway for synthesising dTTP include

A

dUTP

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15
Q

what does Pol III do that needs to be corrected and what does it cause

A

incorporates a U instead of a T every 300 times (every 1200 bp)
needs to be corrected, leaving nicks
fragments every 1200bp of DNA

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16
Q

what addition of U is not offensive

A

U added to the opposite of A is not a problem

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17
Q

what addition of is a problem

A

U formed by the deamination of C
leads to a mutation

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18
Q

what removes every U, offensive or not and what does it produce

A

Uracil-N-glycosylase
baseless nucleotide

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19
Q

what is the function of apyrimidinic endonuclease

A

cleaves phosphodiester backbones of baseless nt

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20
Q

what enzyme removes and replaces the baseless nt and fills the nick

A

Pol I

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21
Q

what fills the DNA nicks left behind by the Pol’s and what can’t it do

A

DNA ligase
cant do RNA-DNA

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22
Q

what is an origin of replication (ori) and where is it located

A

circular chromosomes and plasmids
region of repetitive ds DNA rich in A-T

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23
Q

what binds to 9bp repeats and why

A

DnaA
causes the DNA to super coil at 9bp repeats
opens up the 3-13bp repeats

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24
Q

function of DnaC

A

binds to ssDNA and loads a DnaB helicase onto 3’ strand
Dna C detaches

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25
Q

function of DnaG primase

A

after 65 nt have been unwound by helicase
DnaG bind them to form a primasome

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26
Q

features of primase

A

RNA polymerase
self priming, adds 5’ RNA to 3’ DNA
around 10 nt in length
no proof reading
activity is increased in the presence of helicase - co-operativity

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27
Q

function of single stranded binding protein

A

binds to exposed ssDNA and prevents re-annealing

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28
Q

features of single stranded binding protein (SSB)

A

encoded by ssb gene
forms a tetramer
not sequence specific
leaves base exposed when bound
binds to co-operatively to ssDNA

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29
Q

what do the first primer and SSB trigger the arrival of

A

Pol III holoenzyme at the 3’ end

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30
Q

structure of Pol III holoenzyme

A

3x Pol III core
3x Tau proteins
Clamp loader: accessory proteins - binds SSB

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31
Q

function of the clamp loader and Pol III core

A

The clamp loader loads a β clamp onto the
DNA. Pol III core binds to the β clamp.

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32
Q

function of the clamp loader

A

Binds β clamp proteins
* Transfers the β clamp
onto DNA at primer 3’ end

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33
Q

what detaches the loader clamp loader

A

ATP hydrolysis

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34
Q

features of the β-clamp

A

Encoded by dnaN gene
* Forms a ring dimer
* Not sequence specific
* Binds Pol III core and imparts processivity
→ Goes from 10s of bp to >50,000

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35
Q

function of Pol III in replisome assembly

A

travels to replication fork
synthesises the leading strand and displaces the SSB
as it catches the helicase, a replisome forms

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36
Q

Pol III holoenzyme and primasome occupy how much space

A

around 50nm around the replication fork

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37
Q

function of DNA gyrase

A

binds to remove positive supercoiling

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38
Q

what happens as helicase unwinds the duplex

A

primase re-binds and synthesises a new primer

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39
Q

step 2 of the lagging strand synthesis

A

A β clamp is added to the primer by the clamp loader.

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40
Q

step 3 of lagging strand synthesis

A

As more ssDNA is created, the lagging strand starts to loop back, reversing the primer polarity

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41
Q

step 4 of lagging strand synthesis

A

A Pol III core binds once enough ssDNA has emerged for the β clamp to reach it

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42
Q

step 5 of lagging strand synthesis

A

First Okazaki fragment starts. DNA is pulled by helicase and Pol III. The lagging strand loops out, picking up SSB.

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43
Q

step 6 of lagging strand synthesis

A

Okazaki fragment lengthens.
Lagging strand loop gets longer.

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44
Q

step 7 of lagging strand synthesis

A

First Okazaki fragment finished.
Primase re-binds helicase, then adds a second primer.

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45
Q

step 8 of lagging strand synthesis

A

Pol III core and β clamp detach from DNA, releasing completed fragment.

46
Q

what happens after lagging strand synthesis is completed

A

Primer 2 gets a β
clamp. Looping process
repeats for primer 2

47
Q

how is the lagging strand edited

A

Pol I binds the end of the first okazaki fragment and replaces the RNA with DNA - leaves a nick
DNA ligase seals the nick

48
Q

what is Ter and Tus

A

To prevent the forks overshooting there are 23 bp sequences called Ter, which bind the Tus protein

49
Q

why can Tus only be displaced by the replication fork in one direction

A

the helicase would stall

50
Q

when can DNA gyrase no longer bind

A

when the forks are within 200bp long

51
Q

what relieves positive supercoiling when DNA gyrase can’t bind, which decatenates the molecules

A

toposisomerase IV

52
Q

what does OriC contain

A

GATC sequnces
substrates for DNA adenosine methylase (dam)

53
Q

what does hemimethylated GATC bind to

A

SeqA protein

54
Q

why does initiation only occur once per cycle

A

prevents DnaA from binding to OriC

55
Q

why are GATC sites methylated by dam very slowly (13 mins)

A

newly synthesized dsDNA remains hemimethylated and new initiation is prevented

56
Q

what are the requirements for PCR

A

Taq enzyme
primers
template DNA
dNTP’s

57
Q

what are the conditions required for denaturing annealing and extending

A

denaturing - 95C
annealing - 55C
extending - 72C

58
Q

function of Pol 1/2/3/4/5

A

I - DNA repair and replication
II - DNA repair
III - principal DNA replication enzyme
IV - DNA repair
V - DNA repair

59
Q

features of Pol I

A

one gene
109kDa
~400 copies per cell
10 nts/s - 20-100 nts at a time
too slow - >100 hours per genome

60
Q

features of Pol III

A

22 genes
10E6kDa
~ 10 copies per cell
~1600 nts/s - >50,000 nts at a time
40 minutes per genome

61
Q

difference between fast and slow stop mutants

A

fast - stops replication immediately
slow - allows current round of replication to finish but new one can’t start

62
Q

what is a temperature sensitive mutant

A

allow proteins to be switched on and off by changing the temperature

63
Q

how are DNA strands coiled

A

plectonomically

64
Q

how does helicase function

A

uses ATP hydrolysis
3bp/ATP
hexamer ring surrounds a ssDNA
both helicases move towards the 3’ end of the strand they’re clamped to

65
Q

what does unwinding at one part of the duplex cause

A

torsional strain elsewhere
supercoiling

66
Q

what equation describes DNA topology

A

Lk = T + W

67
Q

what does Lk, T, W mean

A

Lk - linking number (fixed value in circular DNA)
T - twists, number of duplex turns
W - writhe, number of duplex self crossings

68
Q

what does a Lk > Lk° and Lk < Lk° indicate

A

Lk > Lk° - there is positive supercoiling
Lk < Lk° - there is negative supercoiling

69
Q

in relaxed DNA what does σ equal

A

σ = 0

70
Q

what type of coiled is purified cellular DNA and give the σ value

A

negatively supercoiled
σ = -0.06

71
Q

when is eukaryotic DNA negatively supercoiled

A

around histones when forming a nucleosome

72
Q

how does positive supercoiling affect replication

A

needs to be removed for replication to continue

73
Q

what type of enzymes cause a change in Lk

A

topoisomerases

74
Q

difference between type I/II topoisomerases

A

type I - cleaves backbone of one strand, allowing duplex rotations and loss of negative supercoils
type II - cleaves backbone of both strands, using ATP and introduces a negative supercoil

75
Q

outline the mechanism of topoisomerase I of supercoil removal and how does it affect the Lk

A

phosphodiester bond is transferred to tyrosine residue on enzyme - breaks one DNA strand
unbroken strand passes through the gap
phosphodiester bond is transferred back to DNA - reforming the backbone on the other side
Lk - +1

76
Q

outline the mechanism of topoisomerase II of positive supercoil removal

A

horizontal section is cut
5’-P is linked to tyrosine
vertical section passed through
backbone reformed
Lk - -2

77
Q

how would you distinguish between linear and supercoiled DNA in agarose gel

A

supercoiled DNA travels faster than linear DNA

78
Q

what is a catanene

A

when circular DNA molecules are replicated and the two daughter rings interlock

79
Q

what cleaves a catanene

A

topoisomerase IV

80
Q

what is the end result of semi-discontinuous replication

A

two non-identical dsDNA molecules

81
Q

how did okazaki investigate the synthesis of DNA using radioactivity

A

E.coli culture infected with
T4 bacteriophage
add 3H-TTP (tritiated thymidine)
new DNA strands will be radioactive
map out sizes of radioactive ssDNA over time

82
Q

what happens to the 3H-TTP

A

after 2s its removed
‘chased’ by normal TTP
the radioactivity moves down the tube by 60s

83
Q

how do we know that okazaki fragments also contain a RNA primer

A

because tiny fragments are left over after using DNase on okazaki fragments

84
Q

what do eukaryotic DNA polymerase not contain

A

5’-3’ exonuclease

85
Q

where do origins of replication arise from

A

autonomously replicating sequence

86
Q

name sequence repair mechanisms

A

direct repair
base excision repair (BER)
nucleotide excision repair (NER)
mismatch repair (MMR)

87
Q

name molecular repair mechanisms

A

homologous recombination
non-homologous end repair

88
Q

function of O6-methylguanine-DNA methyltransferase

A

transfers methyl/ethyl group from G to a Cys residue on itself
G is restored

89
Q

function of DNA photolyase

A

absorbs blue light and breaks T-T internucleotide bonds using FADH
2 T nucleotides restored

90
Q

function of DNA glycosylases

A

recognise abnormal bases and cleave them from the deoxyribose
creates an abasic site

91
Q

what remains attached to DNA after DNA glycosylase cleaves the sugar-base bond

A

UDGase

92
Q

what is UDGase responsible for

A

responsible for leading strand fragments

93
Q

what happens to a baseless nt

A

recognised and phosphodiester backbone is cleaved by AP endonuclease
leads to nicked DNA

94
Q

what happens to nicked DNA

A

Pol I nick translation restores T nt and DNA ligase seals nick

95
Q

what is nucleotide excision repair (NER)

A

removal of oligonucleotide fragments from one strand

96
Q

what is NER triggered by

A

physical changes in the duplex as a result of damage

97
Q

what enzyme drives NER

A

UvrABC exinuclease (in E.coli)

98
Q

how does NER excise a thymine dimer

A

UvrA and UvrB bind to the dimer
UvrA dissociates, UvrC binds to dimer
UvrC and UvrB move ~5nts away and cleave DNA
UvrC and UvrB dissociate
UvrD helicase displaces damaged DNA ~12/13nts

99
Q

step 1-3 of methyl-directed mismatch repair (MMR)

A

. MutH binds to unmethylated GATC at OriC,
identifying the daughter strand.
. MutS binds to a distorted site on the duplex
MutL binds to MutS

100
Q

steps 4-6 of MMR

A

MutL/MutS complex travels back to the origin and activates MutH
MutH cleaves daughter strand (nicked)
Specialized helicase and exonucleases remove nt until past the distortion

101
Q

step 7 of MMR

A

Pol III fills in missing nt. DNA ligase seals nick

102
Q

what protein do eukaryotes not have a homologue for in MMR and why

A

No homologues of MutH
they don’t use hemimethylation replication tags either

103
Q

outline the sequence repair multi-pronged approach

A

MutT recognises 8-oxo-GTP and hydrolyses it
MutM recognises 8-oxo-G in DNA and removes it, via BER
MutY recognises 8-oxo-G opposite A in DNA and removes the A, via BER

104
Q

outline the homologous recombination method for molecular repair of two strand breaks

A

Following replication, while the sister chromatids are still joined one can be used as a template to repair the other

105
Q

outline non-homologous end-joining (NHEJ) to repair double strand breaks

A

A protein complex binds the naked ends of duplex fragments and recruits DNA ligase IV, which can ligate both strands – but it does it blindly, to any two pieces of DNA, with loss of some nt

106
Q

features of Xeroderma Pigmentosum

A

Individuals show dry, parchment-like skin (xeroderma) and many freckles (pigmentosum)
Increased sensitivity to UV light
1000-fold increased risk of skin cancer
Due to inherited defects in one of eight distinct genes responsible for components of the NER complex

107
Q

features of Hereditary non-polyposis colon cancer (HNPCC)

A

Individuals exhibit a predisposition to colon cancer (2-3% of all colon cancer cases)
Due to defects in the human equivalents of the MutS/L MMR system (MSH2 and MLH1)
Leads to the accumulation of mutations throughout the genome

108
Q

what is a down mutation

A

to decrease promoter efficiency usually decrease conformance to the consensus sequence

109
Q

structure of holoenzyme

A

2 α subunits
β, β’ (prime)
σ sigma (70 kD)

110
Q

what do the 2 α and 2 β form and what is their function in a holoenzyme

A

core enzyme
The core enzyme has a general affinity for DNA- this is known as loose binding
+vely charged (Mg2+ and Zn2+ bound ions which has affinity for the –vely charged DNA

111
Q

function of σ subunit

A

unit ensures RNA polymerase only binds at promoter sequences
1000 X binding strength

Biomed 2 (18 decks)

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