dna repair-r.ppt

8/14/2019 DNA repair-r.ppt

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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.



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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.


in E. coli

N l tid E i i R i


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(Global Genome Repair -Humans)


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(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.

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