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CBG PANT Replication, transcription and translation > CBG Lecture 3: DNA Replication > Flashcards

CBG Lecture 3: DNA Replication Flashcards

1
Q

what must be overcome wrt DNA structure in order for DNA replication to proceed

A

high fidelity copy: as digital info
double helix needds to be melted
need to synthesis 5’-3’ and 3’-5’ simultaneously
very long molecule=avoid tangling

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2
Q

what does ease of melting DNA depend on

A

AT:CG ratio as C:G base pair 3 H bonds harder to melt

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3
Q

where is DNA replication initiated

A

at origins or replication

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4
Q

how is DNA replication initiated

A

by proteins melting AT rich origins of replication

at around 90degrees using helicase

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5
Q

where does replication occur

A

at repication forks, which contain 2 molecules of DNAP and a large number of other essenial proteins

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6
Q

what direction is DNA synthesized in

A

5’ to 3’ so the leading strand is synthesized continuously, the lagging strand is synthesized in Okazaki fragments

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7
Q

what are the main things replication should achieve

A 
digital info (high fidelity copy)
long molecule (avoid tangling)
doouble helix (must be melted, must synthesize 5'-3' and 3'-5' simultaneously
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8
Q

how many origins of replication are there on a prokaryotic chrom

A

one

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9
Q

how many origins of replication are there on eukaryotic chrom - why

A

have more than one ori per chromosome, as they have a larger genome

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10
Q

how to tell from microscope where DNA replication is happening

A

you can see a replication bubble, between two replication forks

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11
Q

what do initiator proteins allow

A

DNA to melt a

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12
Q

how is DNA replicated

A

semiconservatively by DNAP

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13
Q

what experiment showed DNA semiconservative replication

A

Meselson-Stahl
used density gradient centrifugation
15N grown E.coli transferred to 14N medium.
after 1 gen in 14N, single band of DNA between 15N and 14N density
after 2 gens, 2 bands, one at intermediate density,, and one at 14N band

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14
Q

what is the Klenow Fragment

give features of it

A

in prokaryotes
part of the bacterial DNAP1 repair polymerase (in E.coli)
it is the large fragment of DNAP1
present at high copy number (easy to extract as lots of it)
has a primer removing exonuclease domain removed by hydrolysis
shaped like a right hand
has 5’ to 3’ polymerase activity
has 3’ to 5’ exonuclease proofreading activity

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15
Q

outline structure of Klenow fragment

A

shaped like a right hand
has fingers, thumb and palm
has primer-removing exonuclease domain which can be removed by hydrolysis
palm region has 2 AS

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16
Q

what active sites are present in the Klenow fragment

A

has 2 active sites
polymerase site 5’ - 3’ which synthesizes DNA then has 3’ - 5’ exonuclease which breaks down DNA in opposite direction
secondary AS used for proofreading and removing incorrectly added nucleotides

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17
Q

why does DNAP1 have high fidelity rate

A

because Klenow fragment has a secondary active site used for proofreading and removing incorrectly added nuceotides

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18
Q

define lesion

A

A potential mutation

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19
Q

what mutations can be introduced in DNA synthesis

A

transient base tautomery

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20
Q

what occurs during transient base tautomery? How does this happen?

A

if a base is in the wrong tautomeric form during DNA synthesis, it will stick to the wrong base pair and become a lesion

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21
Q

how is transient base tautomery prevented

A

the Klenow fragment (part of the bacterial DNAP-1 repair polymerase has a proofreading 3’-5’ exonuclease site

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22
Q

which bases are prone to transient base tautomery

A

all 4 bases

as all are capable of either amino or imino or keto-enol tautomery

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23
Q

define tautomerization

A

The interconversion of two isomers that differ only in the position of protons (and, often, double bonds).

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24
Q

what is aminocytosine

A

guanine base pair

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25
Q

what is iminocytosine

A

adenine base pair

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26
Q

what form are the N- atoms attached to C G and A in

A

amino NH2 form

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27
Q

what form is the O atom in G and T in

A

keto form =O

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28
Q

what is imino form of N?

A

=NH

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29
Q

what types of rare base pairing are there?

A

A(amino)=C(imino)
A(imino)=C(amino)
G(keto)=(3dbl bond)T(enol)
G(enol)=(3dblbond)T(keto)

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30
Q

what does deamination of cytoisine make? what can this pair with?

A

Uracil

pairs with A

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31
Q

what does deamination of 5-Methylcytosine make

A

thymine

32
Q

how does exonuclease of DNAP work

A

it removes a mismatched base and exposes a ‘clean’ OH group, to which another dNTP can be attached, so polymerisation can immediately recommence after proofreading activity

33
Q

why does DNAP have to be unidirectional

A

because during 5’-3’ synthesis, it is the dNTPs that bring in the energy via the P-P-P group,, needed to make the new phosphodiester bond in the backbone of DNA
if the strand was synthesised 3’-5’, it would be the P-P-P already on the daughter strand that provides the energy for polymerisation.
removal of a mismatched base would result in a ‘clean’ 5’ OH group which would terminate polymerisation, so youd need some complex and inefficient system to readd the P-P-P and restart polymeisation

34
Q

give elongation eqtn for DNA syn

A

DNAn + dNTP -> DNAn+1 +PPi

35
Q

what direction do DNAPs synthesise in?

A

5’ -> 3’

36
Q

what error rate does binding of nucleotides by DNAP have

A

1x10-4 bp-1

37
Q

what is the mutation rate in inherited DNA

A

10-9 per base pair

38
Q

how does the proofreading site of DNAP work

A

Iminocytosine base pairs with A, but because its a tautomer IC quickly goes back to C, which is wrong pairing
this causes C to flip into the proofreading site, so gets removed

39
Q

what are the 2 strands of DNA termed in DNA synthesis

A

lagging and leading strand

40
Q

what direction is leading strand

A

5’ to 3’

41
Q

what direction is lagging strand

A

3’ to 5’

42
Q

how is the lagging strand synthesis different to leading strand synthesis

A

lagging strand is synthesised discontinuously in discrete Okozaki fragments (5’ to 3’ chuncks)
leading strand is synthesised continuously in 5’ to 3’

43
Q

what is the direction of Daughter DNA strand

A

5’ to 3’

44
Q

which DNA will DNAP bind

A

ssDNA

45
Q

how does DNAP bind ssDNA

A

through addition of erasable RNA primers

46
Q

why wont DNAP bind ssDNA

A

due to a conflict between being able to spot a mismatched base at 3’ end DNA and being able to initiate a new strand de novo

47
Q

on the 5’3’ strand, what way does DNAP bind and synthesise

A

it creates a 5’3’ daughter strand, therefore binds at end/bottom 3’ end working continuously upwards towards the 5’ end

48
Q

compare addition of RNA primers on leading/lagging strand

A

DNAP wont bind ssDNA so 1 erasable primer added on leading strand and MANY on lagging strand

49
Q

how long roughly is RNA primer

A

100bp

50
Q

what part of DNAP-1 is removed to make the Klenow fragment?

how is this done

A

the primer removing nuclease activity of the DNAP-1 is removed during hydrolysis to make the Klenow fragment

51
Q

what is primase

A

adds RNA primer to ssDNA so DNAP can extend the ssDNA

52
Q

what deletes the added RNA primer to ssDNA

A

RNAse, then DNA ligase stickes the two bits together

53
Q

define Okazaki fragment

A

short, newly synthesized DNA fragment formed on the lagging template strand during DNA replication

54
Q

what are the steps for the termination of DNA synthesis

A

remove primer through use of RNAse
DNA ligase
if molecule linear: TELOMERES

55
Q

define telomeres

A

piece of DNA at end of eukaryotic chromosomes that cant be replicated using DNAP
only occurs in eukaryotes with linear chromosomes, doesnt happen in prokaryotes with circular DNA

56
Q

what is a replisome

A

replicating machine to transcribe DNA, composed of numerous proteins

57
Q

what is helicase

what difficulties does helicase create?how is this overcome?

A

enzyme that melts DNA strands, breaks H bonds, creates replication bubble.

makes negative supercoiling behind it and positive supercoiling up stream, therefore have proteins upstream to combat this

58
Q

what is DNA topoisomerase

A

runs ahead of helicase and nicks one of the strands of DNA to allow strain release of DNA

59
Q

give some examples of proteins in the replisome

A 
sliding clamp:increased processivity
topoisomerase:strain relief
helicase: melt DNA
primase: add RNA primer
single stranded binding proteins: stabilise ssDNA of parent and prevent self annealing of ssDNA as it is sticky and can make hairpin loops on itself
60
Q

what are single strand binding proteins?

A

stabilise ssDNA of parent and prevent self annealing of ssDNA as it is sticky and can make hairpin loops on itself

61
Q

what does a sliding clamp do? where is it found?

A

found on leading clamp
hexomeric donut shope
increases processivity to stop DNAP falling off DNA
always behind DNA

62
Q

what is telomerase

A

a reverse transcriptase with an internal RNA template which extends telomeres in germline cells

63
Q

how are different DNAPs specialised in E.coli

A

DNApol 1 - repair and Okazaki clean up
DNApol 2 - repair
DNApol 3 - replicative polymerase

64
Q

how are different DNAPs specialised in humans

A

DNApol alpha: priming

beta: repair
gamma: mitochondrial
delta: lagging strand
epsilon: leading strand

65
Q

what base tautomery exists for T+G

A

keto-enol

66
Q

what base tautomery exists for A+C

A

amino-imino

67
Q

which bases have amino-imino base tautomery

A

A+C

68
Q

which bases have keto enol tautomery

A

T+G

69
Q

what is a primosome

A

primase and helicase, found in bacteria

70
Q

what four sections make up DNA replication, transcription and translation

A
  1. MACHINE
  2. INITIATION
  3. ELONGATION
  4. TERMINATION
71
Q

what effect does germ line cells having telomerase have

A

no hayflick limit therfore can replicate indefinitely

72
Q

name some cells that have their own telomerase

A

stem cells and cancer cells - can replicate indefinitely

73
Q

what is the Hayflick limit/constant

A

max number of divisions a cell can undergo before death, due to the absence of telomerase which extends the telomeres

74
Q

which nucleotides are telomeres rich in

A

GC

75
Q

what happens to length of telomeres over generations

A

constantly get cut off end of chromosomes and eventually runs into genes/coding DNA

76
Q

what does telomerase do

A

extends the telomeres -its a reverse transcriptase

CBG PANT Replication, transcription and translation (10 decks)

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