DNA REPAIR

DNA repair
DNA repair is a cellular mechanism to correct
damage to DNA before it becomes fixed as a
mutation or chromosomal aberration, which
may lead to deleterious results such as cell
death or tumorigenesis. Mechanism of DNA
repair is important for reducing the risk of
cancer as well as developing more effective
cancer therapies.
 MUTASI
Mutasi terbagi atas:
• Mutasi spontan
• Mutasi induksi
Terbentuknya struktur Thymine dimer
Terbentuknya struktur Thymine dimer
 Types of Damage Repair
• Photolyase
• De-alkylation proteins (not catalytic)

• Base Excision Repair

• Nucleotide Excision Repair (GG and TC)
• Mismatch Repair

• Error-prone Repair or SOS

• Double Strand Break Repair
 Repairing Damaged Bases
Damaged bases can be repaired by
several mechanisms:
• Direct chemical reversal
• Excision Repair. There are three modes
of excision repair, each of which employs
specialized sets of enzymes.
Base Excision Repair (BER)
Nucleotide Excision Repair (NER)
Mismatch Repair (MMR)
 Direct repair
• Observation:
– UV-

Photoreactivation
– requires DNA photolyases
– requires visible light at 300 – 500 nm
– “light repair”
– Contrast: dark repair (BER, NER, mismatch
repair)
 DNA photolyases
• Structure
– Generally contain 2 chromophores
– Chromophore No. 1: always FADH -
– Chromophore No. 2: folate (in E. coli and yeast)
• N5,N10-methenyltetrahydrofolylpolyglutamate
• Function
– bind to pyrimidine dimers
– resolve pyrimidine dimers into original bases
DNA Photolyase

Memperbaiki

Thymine dimer
UV-responsive photolyases
Direct reversal (de-alkylating proteins)
Excision repair
 Base Excision Repair
not restricted to a short time post replication
similar in most organisms (bacteria –
mammals)
recognizes abnormal bases in the DNA
requires four enzymes
1.DNA glycosylases
2.AP-endonucleases
3.DNA polymerase I
4.DNA ligase
 DNA glycosylases
• Relatively small G P G
P O O
enzymes (20 – 30 KDa)
• Recognize abnormal bases U P
P O
– deaminated bases O
– alkylated bases
A
• Remove base via cleavage A P O
P O
at the glycosidic bond
between the deoxyribose
and the base
• Cleavage creates apurinic Before After
and apyrimidinic (AP sites)
 DNA repair:
DNA uracil glycosylase
 AP-endonucleases
• recognize AP-sites 5’ P P P P P P P
• cleave phosphodiester A G G C A G C
bonds near the AP site T C C T C G
and generate a 5’ –
3’ P P P P P P P
phosphate and 3’-
hydroxyl AP endonuclease

• In E. coli this enzyme

also has 3’-5’ P P P P P P P
exonuclease activity
A G G C A G C
• The 3’-OH functions as
a primer T C C G
5’
P P 3’ P P
 Base Excision Repair
The steps of BER:
1. removal of the damaged base (estimated to occur some 20,000
times a day in each cell in our body!) by a DNA glycosylase. We
have at least 8 genes encoding different DNA glycosylases
each enzyme responsible for identifying and removing a specific
kind of base damage.

2. removal of its deoxyribose phosphate in the backbone,

producing a gap. We have two genes encoding enzymes with
this function.

3. replacement with the correct nucleotide. This relies on DNA

polymerase, one of at least 11 DNA polymerases encoded by
our genes.

4. ligation of the break in the strand. Two enzymes are known that
can do this; both require ATP to provide the needed energy.
Base Excision Repair
 Nucleotide Excision Repair
Recognizes large distortions in the DNA
structure
Repairs UV-damaged DNA
Cleaves two phosphodiester
Generally generates fragments of 12 to 13
nucleotides
Requires four different enzymes
1.Exonuclease
2.DNA helicase
3.DNA polymerase
4.DNA ligase
Nucleotide Excision Repair (E.coli)

Enzyme Protein Function

UvrA (MW= 104,000) scans DNA, binds to UvrB
scanner; binds DNA cleaves
UvrB (MW = 78,000) phosphate bond at 3' end, 5
Exinuclease positions downstream of lesion
binds UvrB & DNA cleaves
UvrC (MW = 68,000) phosphate bond at 5' end, 8
positions upstream of lesion
DNA helicase UvrD removes DNA fragment
DNA DNA polymerase I
fills emerging gap
polymerase (= PolA )
DNA ligase Lig seal nick
Nucleotide Excision Repair (E.coli)
 Nucleotide Excision Repair
in E. coli
UvrA
UvrB
Mechanism UvrA
• The (UvrA)2:UvrB complex
scans DNA ATP
exinuclease
• UvrA dimer dissociates from
pryimidine dimer. UvrB binds P P
DNA and cuts at 3’ end.
• UvrC associates with UvrB and
cuts DNA at 5’ end of the P P UvrD DNA
helicase
pyrimidine dimer
OH P
• UvrD DNA helicase removes
the DNA fragment
• DNA polymerase I fills the gap DNA pol. I DNA ligase
• DNA ligase seals the remaining
nick.
 Nucleotide Excision Repair
(Global Genome Repair -Humans)
 Nucleotide Excision Repair
(Transcription Coupled -Humans)
Genes Encoding Enzymes of Mismatch Repair
MISMATCH
Mismatch repair
 Mismatch repair in E. coli
Scenario 1
CH3 CH3
Mismatch is at the 5’ end of 5’ 3’
cleavage site 3’ MutS-MutL 5’
• Unmethylated DNA is
unwound via DNA helicase II DNA helicase II
ATP exonuclease I
• The 3’-5’ exonuclease activity ADP+Pi or
of exonuclease I or exo X exonuclease X
CH3 CH3
degrades DNA through the
5’ 3’
mismatch
3’ 5’
• DNA polymerase III DNA polymerase III
synthesizes the new DNA SSBs
strand CH3 CH3
5’ 3’
• DNA ligase closes the 3’ 5’
remaining nick.
 Mismatch repair in E. coli
Scenario 2
CH3 CH3
Mismatch is at the 3’ end of
5’ 3’
cleavage site
3’ MutS-MutL 5’
• Unmethylated DNA is
unwound via DNA helicase II DNA helicase II
ATP exonuclease VII
• The 5’-3’ exonuclease activity ADP+Pi or
of exonuclease VII or RecJ RecJ nuclease
CH3 CH3
nuclease degrades DNA
5’ 3’
through the mismatch
3’ 5’
• DNA polymerase III DNA polymerase III
synthesizes the new DNA SSBs
strand. CH3 CH3
5’ 3’
• DNA ligase closes the 3’ 5’
remaining nick.
 SOS Response
SOS repair occurs when cells are
overwhelmed by UV damage - this allows
the cell to survive but at the cost of
mutagenesis.
SOS response only triggered when
other repair systems are overwhelmed
by amount of damage so that unrepaired
DNA accumulates in the cell.
 Error-prone repair (SOS)
• Activated upon:
– severe DNA 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
Error Prone Bypass (E. coli)
Postreplication repair
 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, genotoxic
chemicals, and mechanical stress on the
chromosomes.