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DNA Replication
• DNA Replication
– Polymerases
• Activities
• Structure
• Mechanism
• Replication/polymerase fidelity
– Exonuclease Activities
– Mismatch repair
– Post-replication repair
• Reversal
• Base Excision repair
• Nucleotide Excision repair
• DNA Sequencing
• PCR
DNA Replication
Nucleicacidfunction:CentralDogma
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DNA Replication
Addition at 3’ end
DNA Polymerases Require 3 things:
Pyrophosphatase
dNTP
2Pi
Comparison of Polymerases
*
(a4)
?à
?à
Processivity
adNTP
polymerizedmin-1 at37°C.
10010,000
500,000
b
b
not includingOkasaki fragments
*Inacomplexwith10proteins(26subunits)of>900kD.Coreistrimer ofa, q, e
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DNAPolymeraseStructure
DNA/RNA polymerase mechanism
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DNA/RNA polymerase mechanism
Metal Ion Role
Proximity Role
a
b
g
Asp
=
O
C
Asp
DNA Replication
• DNA Replication
– Polymerases
• Activities
• Structure
• Mechanism
• Replication/polymerase fidelity
– Exonuclease Activities
– Mismatch repair
– Post-replication repair
• Reversal
• Base Excision repair
• Nucleotide Excision repair
• DNA Sequencing
• PCR
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Replication/polymerase Fidelity
open
closed
2
3
m inorgroove
2
3
m inorgroove
E+dNTP à E*(open)•dNTP à E**(closed)•dNTP(correct) à E***(translocation)•PPi à E+PPi
slow
Replication/polymerase Fidelity
DNA Polymerases have 3’ à 5’
Exonuclease Activity
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Replication/polymerase Fidelity
DNA Polymerases have 3’ à 5’ Exonuclease Activity
The 3’ à 5’ exonuclease
domain
correct base
wrong base
Phosphodiester bond hydrolyzed
Phosphodiester bond made
Phosphodiester bond made
Detected by 3’ à 5’
exonuclease activity (part
of DNA polymerase)
ReplicationFidelity:Proofreading
Replication/polymerase Fidelity
Comparison of Polymerases
*
(a4)
?à
Processivity
adNTP polymerizedmin-1 at37°C.
10010,0000
500,000b
b
not includingOkasaki fragments
*Inacomplexwith10proteins(26subunits)of>900kD.Coreistrimer ofa, q, e
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DNA Replication Process
Pol I Has 5’ à 3’ Exonuclease Activity
Nick Translation
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Replication/polymerase Fidelity
Polymerases make mistakes in synthesis (polymerase
5’à3’ activity) = 1/10,000
DNA polymerases have proofreading ability (3’à5’
exonuclease activity) = 1/10,000
OverallforReplication =1/1010
After replication fork, any mistakes are corrected by
mismatch repair mechanism. This misses = 1/100
3x109 bp/haploidgenomex1/1010 mistakes/bp =0.3mistakes/haploidgenome
Plus DNA can be damaged in living cells. These damages are
repaired by other mechanisms called Post-replication DNA
Repair:
•
•
•
Directreversalofmodification
Base-excisionrepair
Nucleotideexcisionrepair
Replication/polymerase Fidelity
Mechanism of Mismatch Repair
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Replication/polymerase Fidelity
Mechanism of Mismatch Repair
InE.coli amismatchisrecognizedbyMutS,which
thenrecruitsMutL.
InanATPdependentmanner,thecomplex
translocates alongthedoublestrandedDNAto
generatealoop.
Onencounteringahemi-methylatedGATCsequence,
itrecruitsMutH (anendonuclease)andMutH cuts
theunmethylated DNA.
Ahelicase(UvrD)thenseparatethetwostrandsand
theexonucleasecompletelyremovesthedefected
strand.
ThegapisthenfilledbyDNApolymeraseandDNA
ligase.
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Replication/polymerase Fidelity
Mechanism of Mismatch Repair
InE.coli amismatchisrecognizedbyMutS,which
thenrecruitsMutL.
InanATPdependentmanner,thecomplex
translocates alongthedoublestrandedDNAto
generatealoop.
Onencounteringahemi-methylatedGATCsequence,
itrecruitsMutH (anendonuclease)andMutH cuts
theunmethylated DNA.
Ahelicase(UvrD)thenseparatethetwostrandsand
theexonucleasecompletelyremovesthedefected
strand.
ThegapisthenfilledbyDNApolymeraseandDNA
ligase.
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DNA Repair
Polymerases make mistakes in synthesis (polymerase
5’à3’ activity) = 1/10,000
DNA polymerases have proofreading ability (3’à5’
exonuclease activity) = 1/10,000
OverallforReplication =1/1010
After replication fork, any mistakes are corrected by
mismatch repair mechanism. This misses = 1/100
3x109 bp/haploidgenomex1/1010 mistakes/bp =0.3mistakes/haploidgenome
Plus DNA can be damaged in living cells. These damages are
repaired by other mechanisms called Post-replication DNA
Repair:
•
•
•
Directreversalofmodification(I)
Base-excisionrepair(II)
Nucleotideexcisionrepair(III)
DNARepair(I):DirectReversalofModification
UV-crosslinking:pyrimidinedimers
DNAphotolyase
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Anotherexampleofdirectreversal:
O6-alkylguaninemodificationcanbereversedby“alkyltransferase”(alkB)
A. nidulans DNA photolyase
PDBid 1TEZ
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RECALL:
and other bases
Oxidations/Alkylations:
•
Methylation of G at O6
can base-pair with C or T,
which causes G à A
mutations
DNARepair(II):BaseexcisionRepair
abasic endo-nuclease mechanism
1
Base is changed
in a small way
2
3
5’
3’
5’
3’
5’
4
5
• Hydrolysis of phosphodiester 5’-side of abasic site
• Asp acts as a general base, abstracts proton from
water to make it a better nucleophile.
• Pentavalent phosphate transition state formed from
attack of water
• Mg2+ stabilizes the charge that forms on the 3’
oxygen when the phosphate bond is cleaved.
1.
2.
3.
4.
5.
Glycosylase removesdamagedbasebycleavage
ofglycosidic bond
Abasic-siteendonucleasecleaves
phosphodiesterbond
Exonuclease removesabasic deoxyribose
Polymeraserequiredtoaddnewnucleotide
Ligasecondensesphosphodiester bond
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DNARepair(II):BaseexcisionRepair
Depends on there being a specific Glycosylase
STEP1
Oxo-guanineglycosylase
BendsDNAhelixandextrudes
damagedbaseintoenzymeactivesite
UracilDNAglycosylase
FlipsoutbaseandfillsholewithArg
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DNARepair(III):NucleotideExcisionRepair
Most common and versatile mechanism of repair
Bulky alkylation or
other distortion of the
double helix
UvrABC endonuclease
(ATP-driven)
(A2B2 tetramer bends
DNA at damage)
P
(helicase)
(UvrC is the endonuclease)
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Problem:
ExplainwhymethylationatO6 ofguanidineismutagenic
whereasC5methylationofcytosineisnot
O6-methylguanine- causesG•Cà A•Tmutations
Me
(m5)
5-Mecytosinewillnotinterferewithbasepairing– thisisavery
commonepigenetic modification– aheritablefactoraffecting
genetranscriptionthatisnot basedonthenucleotidesequence
(ATGC)
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