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
 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
UV-responsive photolyases
Direct reversal (de-alkylating
proteins)
 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
G P G
Relatively small 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
Before After
Cleavage creates apurinic
and apyrimidinic (AP sites)
 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
T C C G
a primer 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 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)
 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.
THANKS.