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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, fotoliase DNA dan sinar tampak Mutasi induksi

Types of Damage Repair


Photolyase De-alkylation proteins (not catalytic) (menghilangkan gugus alkil) Base Excision Repair Nucleotide Excision Repair (GG and TC) Mismatch Repair(dipotong bila psgn selingkuh) Error-prone Repair or SOS (thp lanjut,lbh parah) Double Strand Break Repair (keduanya putus)

Repairing Damaged Bases


Damaged bases can be repaired by several mechanisms: Direct chemical reversal (langsung) 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: UVPhotoreactivation

requires DNA photolyases requires visible light at 300 500 nm light repair perbaikan terang 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 (mengikat molekul dimer, diuraikan, dikembalikan ke mol asal yg monomer)

Function
bind to pyrimidine dimers resolve pyrimidine dimers into original bases

UV-responsive photolyases

Direct reversal (de-alkylating proteins)

Base Excision Repair (secara tdk langsung)


not restricted to a short time post replication similar 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

DNA glycosylases
Relatively small P enzymes (20 30 KDa) Recognize abnormal bases
deaminated bases alkylated bases
P

G P O U
O

G
O

Remove base via cleavage at the glycosidic bond between the deoxyribose and the base Cleavage creates apurinic and apyrimidinic (AP sites)

A
O

A
O

Before

After

APirimidine-endonucleases
5 P P P P P P P recognize AP-sites A G G C A G C cleave phosphodiester(ikatan T C C T C G antara gula pentosa) 3 P P P P P P P bonds near the AP site AP endonuclease andgeneratea5 phosphateand3hydroxyl P P P P P P P In E. coli this enzyme A G G C A G C alsohas3-5 T C C G exonuclease activity 5 P P The3-OH functions as 3 P P a primer

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
UvrA (MW= 104,000) UvrB (MW = 78,000) Exinuclease UvrC (MW = 68,000) DNA helicase DNA polymerase DNA ligase UvrD DNA polymerase I (= PolA ) Lig

Function
scans DNA, binds to UvrB scanner; binds DNA cleaves phosphate bond at 3' end, 5 positions downstream of lesion binds UvrB & DNA cleaves phosphate bond at 5' end, 8 positions upstream of lesion removes DNA fragment fills emerging gap seal nick

Nucleotide Excision Repair (E.coli)

Nucleotide Excision Repair in E. coli


Mechanism The (UvrA)2:UvrB complex scans DNA UvrA dimer dissociates from pryimidine dimer. UvrB binds DNAandcutsat3end. UvrC associates with UvrB and cutsDNAat5endofthe P pyrimidine dimer UvrD DNA helicase removes the DNA fragment DNA polymerase I fills the gap DNA ligase seals the remaining nick.
UvrA UvrA

UvrB

exinuclease P

ATP
P

P
OH DNA pol. I

UvrD DNA helicase P

DNA ligase

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 Mismatch is at the 5end of cleavage site Unmethylated DNA is unwound via DNA helicase II
The 3-5exonuclease activity of exonuclease I or exo X degrades DNA through the mismatch DNA polymerase III synthesizes the new DNA strand DNA ligase closes the remaining nick. 5 3 5 3
CH3 CH3

ATP ADP+Pi
5 3
CH3

MutS-MutL DNA helicase II exonuclease I or exonuclease X


CH3

3 5

3 5 DNA polymerase III SSBs


CH3

CH3

3 5

Mismatch repair in E. coli


Scenario 2 Mismatch is at the 3end of cleavage site Unmethylated DNA is unwound via DNA helicase II The 5-3exonuclease activity of exonuclease VII or RecJ nuclease degrades DNA through the mismatch DNA polymerase III synthesizes the new DNA strand. 5 3
CH3 CH3

ATP ADP+Pi
5 3
CH3

MutS-MutL DNA helicase II exonuclease VII or RecJ nuclease


CH3

3 5

3 5 DNA polymerase III SSBs


CH3

DNA ligase closes the remaining nick.

5 3

CH3

3 5

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.

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