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Plant Molecular and Cellular Biology
Lecture 2: Fidelity of DNA
Replication
Gary Peter
Learning Objectives
1. List and explain the
mechanisms that insure
high-fidelity DNA replication
2. Explain why the fidelity of
DNA replication is so
important for survival and
for recombinant DNA work
Importance of High-Fidelity
DNA Replication
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If 1 mutation occurs
per 109 bp
Then what's the
mutation rate of a
1000 bp gene in a
population of 106
bacteria?
What’s the frequency
of the mutant in the
population?
Generation 1
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106 copies of 1000 bp gene or 109 bp to
be copied
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Generation 2
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2x106 copies of 1000 bp gene or 2x109
bp to be copied
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4x106 copies of 1000 bp gene or 4x109
bp to be copied
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2 new mutants/ 4x106 cells= 5x10-7
Frequency in pop.=4/(4x106)=1x10-6
Generation 3
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1 new mutant/ 2 x106 cells= 5x10-7
Frequency in pop.=1/(2x106)=5x10-7
4 new mutants/ 8x106 cells= 5x10-7
Frequency in pop.=12/(8x106)=1.5x10-6
Mutation rate is constant at 5x10-7
Frequency of mutants in the population
increases
Consequences of High
Mutation Rates
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Mutator strain
propagated for
various cycles 40
cycles = 1000
generations
Funchain et al., 2000 Genetics 154: 959-970
What Processes and
Pathways are Important for
DNA Replication?
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Monomer biosynthesis
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De novo and salvage pathways for nucleotide
biosynthesis
Purine and pyrimidine biosynthesis
Nucleoside monophosphate conversion to triphosphate
Polymer biosynthesis
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Template
Primer
DNA polymerases
Other enzymes?????
Method for Measuring the in Vitro
Fidelity of DNA Polymerases
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Why use M13?
What does the host
need?
Can this assay be
used to measure the
fidelity of all
polymerases?
What do you expect
the results to be?
Kunkel, 1985 JBC 260 (9):5787-5796
Results of Assay
Kunkel, 1985 JBC 260 (9):5787-5796
Frame shifts and substitutions
are similar in frequency
Kunkel, 1985 JBC 260 (9):5787-5796
Why M13?
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Facilitates creation of gapped duplex
Non essential gene and α-complementation
already present
Large dsDNA region contains adenine
methylation which should help limit the mismatch
repair of the mutations arising during in vitro
synthesis
Easy to score large numbers of plaques
Single stranded phage are readily sequenced
What does the host strain
need?
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The rest of the lac Z coding sequence for the
intracistronic complementation to work
This is located on the F’ plasmid to insure that
susceptibility to infection by M13 phage
Can this assay be used to
measure the fidelity of all
polymerases?
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Only those
polymerases that
can use gapped
templates
Template
Primer
Fidelity of Various
Thermostable DNA Polymerases
DNA Polymerase
Error Rate x 10-6
(Mutation rate per bp)
Accuracy x 105
(Error rate-1)*
Pfu
1.3 (0.2)
7.7
Deep Vent
2.7 (0.2)
3.7
Tli (VentR)
2.8 (0.9)
3.6
Taq
8.0 (3.9)
1.3
UlTma
55.3 (2.0)
0.2
*Accuracy is the average number of bases duplicated before an error is made
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What does this mean for your recombinant
DNA work?
The Impact of Error Rate on %
of clones with sequence errors
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Error rate of PfuUltra is 4x10-6 and Phusion is
1.4x10-6,
The number of mutations per bp equals the (error
rate)*number of doublings
Mutation frequency is MF=ER*bp*doublings
Size
PfuUltra
Phusion
1 kb
0.7%
2.4%
5 kb
3.4%
11.9%
10 kb
6.8%
23.8%
Impact of Reverse Transcriptase
Errors on cDNA Sequence
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AccuScript RT
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ER = 1.6 x 10-5
So for a 1 kb cDNA expect 1.6% of the clones
to have an error
After 20 doublings (106 amplification) expect
Enzyme
Error Rate (10-6)
% Mutant Clones
PfuUltra
0.43
2.5%
PicoMaxx
4.0
9.6%
What’s the Normal Mutation
Rate
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The probability of a mutation occurring due to
random chemical decomposition of nucleotides is
estimated at 1/10,000 bp
The standard fidelity of E. coli DNA replication is 1
error in 1 billion base pairs
Four Mechanisms that Control
the Fidelity of DNA Replication
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Complementary base pairing
Conformational change of polymerase
which delays addition to a growing chain
allowing additional time for the incorrect
nucleotide to dissociate
Removal of incorrect nucleotides at the 3’
terminus of the growing chain by the 3’ to
5’ exonuclease function of the DNA
polymerase
Removal of mispaired nucleotides by the
mismatch repair system
Summary
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The fidelity of DNA replication is due to the
structure of DNA, the 3’-5’ proofreading
activity of DNA polymerases and DNA repair
enzymes
DNA replication requires the concerted
activity of multiple enzymes which bind
together at the replication fork into the
repliosome
Nucleotide Levels in Cells
DNA
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Amount (mM)
RNA
Amount (mM)
dNMP
43
rNMP
270
dATP
0.18 (0.013)
ATP
3.0 (2.8)
dGTP
0.12 (0.005)
GTP
0.92 (0.48)
dCTP
0.07 (0.022)
CTP
0.52 (0.21)
dTTP
0.08 (0.025)
UTP
0.89 (0.48)
The supply of deoxyribonucleotides in the E. coli is
~1% of that which is needed for replication
In mammalian cells the supply is significantly less
Why keep the levels so low?
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Inadequate supplies of dNTPs are lethal
Too high a concentration is mutagenic
Nucleotide Synthesis
SALVAGE
Deoxyribonucleotides
DE NOVO
Deoxyribonucleotides
Bases
Ribonucleotides
Ribonucleotides
Ribose, amino acids,
CO2, NH3
De Novo Purine Biosynthesis
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Pathway is highly
conserved from
bacteria to mammals
What about plants?
First Committed Step in Purine
Biosynthesis
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First step in de novo purine biosynthesis is
catalyzed by 5-phophoribosyl pyrophosphate
synthase
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PRPP synthase is feedback regulated – inhibitors
include IMP, GMP, AMP (end products)
Pyrimidine Biosynthesis
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In contrast to purine
biosynthesis,
pyrimidine
biosynthesis starts
with the formation of
the pyrimidine
skeleton and then
ribose is attached