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DNA REPAIR
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DNA repair
DNA repair is a cellular mechanism to correctdamage to DNA before it becomes fixed as amutation or chromosomal aberration, which
may lead to deleterious results such as celldeath or tumorigenesis. Mechanism of DNArepair is important for reducing the risk ofcancer as well as developing more effective
cancer therapies.
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MUTASI
Mutasi terbagi atas:
Mutasi spontan, fotoliase DNA
dan sinar tampak Mutasi induksi
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Types of Damage Repair
Photolyase
De-alkylation proteins (not catalytic)
(menghilangkan gugus alkil)
Base Excision Repair
Nucleotide ExcisionRepair (GG and TC)
MismatchRepair(dipotong bila psgnselingkuh)
Error-prone Repair or SOS (thp lanjut,lbhparah)
Double Strand Break Repair (keduanya
putus)
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Repairing Damaged asesDamaged bases can be repaired byseveral mechanisms:
Direct chemical reversal (langsung)
Excision Repair. There are three modes
of excision repair, each of which employs
specializedsets of enzymes.
BaseExcision Repair(BER)
NucleotideExcision Repair(NER)
MismatchRepair(MMR)
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Direct repair
Observation:
UV-
Photoreactivation
requires DNAphotolyases
requires visible light at 300500 nm
lightrepairperbaikan terang
Contrast: dark repair (BER, NER, mismatchrepair)
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DNA photolyases
Structure Generally contain 2 chromophores
Chromophore No. 1: always FADH -
Chromophore No. 2: folate(in E. coliand yeast) N5,N10-methenyltetrahydrofolylpolyglutamate
(mengikat molekul dimer, diuraikan, dikembalikan ke
mol asal yg monomer)
Function
bind to pyrimidine dimers
resolve pyrimidine dimers into original bases
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UV responsive photolyases
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Direct reversal (de-alkylating proteins)
B E i i R i (
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Base Excision Repair (secara
tdk langsung)
not restricted to a short time post replicationsimilar in most organisms (bacteria
mammals)
recognizes abnormal bases in the DNA,memotong, membuang basa)
requires four enzymes :
1.DNA glycosylases
2.AP-endonucleases
3.DNA polymerase I
4.DNA ligase
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DNA glycosylases Relatively small
enzymes (2030 KDa)
Recognize abnormal bases deaminatedbases
alkylatedbases Remove base via cleavage
at the glycosidicbondbetween the deoxyribose
and the base Cleavage creates apurinic
and apyrimidinic (AP sites)
UOP
G
OP
AOP
O
GOP
AOP
P
Before After
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APirimidine-endonucleases
recognize AP-sites
cleave
phosphodiester(ikatan
antara gula pentosa)
bonds near the AP siteand generate a 5
phosphate and 3-
hydroxyl
In E. colithis enzymealso has 3-5
exonuclease activity
The 3-OH functions asa rimer
P
A
P
G
P
G
P
C
P
T
P P
C
P
P
A
P
G
P
C
P
T
P
C
P
GC
P
A
P
G
P
G
P
C
P
T
P
P
A
P
G
P
C
P
C
P
GC
AP endonuclease
5
3
5
3
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The steps of BER:
1. removal of the damaged base (estimated to occur some 20,000times a day in each cell in our body!) by a DNA glycosylase. Wehave at least 8 genes encoding different DNA glycosylaseseach enzyme responsible for identifying and removing a specifickind of base damage.
2. removal of its deoxyribose phosphate in the backbone,producing a gap. We have two genes encoding enzymes withthis function.
3. replacement with the correct nucleotide. This relies on DNApolymerase, one of at least 11 DNA polymerases encoded byour genes.
4. ligation of the break in the strand. Two enzymes are known thatcan do this; both require ATP to provide the needed energy.
Base Excision Repair
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Base Excision Repair
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Nucleotide Excision Repair
Recognizes large distortions in the DNAstructure
Repairs UV-damaged DNA
Cleaves twophosphodiester
Generally generates fragments of 12 to 13nucleotides
Requires four different enzymes
1.Exonuclease
2.DNA helicase3.DNA polymerase4.DNA ligase
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Nucleotide Excision Repair (E.coli)
Enzyme Protein Function
UvrA (MW= 104,000) scans DNA, binds to UvrB
UvrB (MW = 78,000)
scanner; binds DNA cleaves
phosphate bond at 3' end, 5positions downstream of lesion
UvrC (MW = 68,000)
binds UvrB & DNA cleaves
phosphate bond at 5' end, 8
positions upstream of lesion
DNA helicase UvrD removes DNA fragmentDNA
polymerase
DNA polymerase I
(= PolA )fills emerging gap
DNA ligase Lig seal nick
Exinuclease
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Nucleotide Excision Repair (E.coli)
N l tid E i i R i
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exinuclease
UvrD DNA
helicase
DNA pol. I DNA ligase
ATP
PP
POH
PP
UvrAUvrB
UvrAMechanism The (UvrA)2:UvrBcomplex
scans DNA
UvrAdimer dissociates from
pryimidine dimer. UvrBbindsDNA and cuts at 3 end.
UvrCassociates with UvrB and
cuts DNA at 5 end of the
pyrimidine dimer
UvrDDNA helicase removes
the DNA fragment
DNA polymerase I fills the gap
DNA ligase seals the remaining
nick.
Nucleotide Excision Repair
in E. coli
N l tid E i i R i
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Nucleotide Excision Repair
(Global Genome Repair -Humans)
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Nucleotide Excision Repair
(Transcription Coupled -Humans)
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Genes Encoding Enzymes of Mismatch Repair
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MISMATCH
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Mismatch repair
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Mismatch repair in E. coliScenario 1
Mismatch is at the 5 endofcleavage site
Unmethylated DNA is
unwound via DNA helicase II
The 3-5 exonucleaseactivityof exonuclease I or exo X
degrades DNA through the
mismatch
DNA polymerase III
synthesizes the new DNA
strand
DNA ligase closes the
remaining nick.
CH CH
ATP
ADP+Pi
MutS-MutL
DNA helicase II
exonuclease I
or
exonuclease X
CH CH
CH CH
5
3
5
3
5
3
3
5
3
5
3
5
DNA polymerase III
SSBs
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Mismatch repair in E. coli
CH CH
ATP
ADP+Pi
MutS-MutL
DNA helicase II
exonuclease VII
orRecJ nuclease
CH CH
CH CH
Scenario 2
Mismatch is at the 3 endofcleavage site
Unmethylated DNA is
unwound via DNA helicase II
The 5-3 exonucleaseactivityof exonuclease VII or RecJ
nuclease degrades DNA
through the mismatch
DNA polymerase IIIsynthesizes the new DNA
strand.
DNA ligase closes the
remaining nick.
5
3
5
3
5
3
3
5
3
5
3
5
DNA polymerase III
SSBs
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E i (SOS)
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Error-prone repair (SOS) Activated upon:
severeDNA damage disruption of DNA replication
SOS-response
Inaccurate repair mechanism Requires at least 14 proteins in E. coli
Din proteins (damage induced)
Rec poteins (recombination)
Umu proteins (UV-mutagenesis)
Uvr proteins (UV-resistance)
Others: SulA, HimA, Ssb, and PolB
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Error Prone Bypass (E. co li)
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Double Breaks Strand
DSB Repair Double-strand breaks (DSBs)
are perhaps the most serious form of
DNA damage because they pose
problems for transcription, replication,and chromosome segregation. Damage
of this type is caused by a variety of
sources including exogenous agents
such as ionizing radiation, genotoxicchemicals, and mechanical stress on the
chromosomes.
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THANKS.