DNA REPAIR

DNA repairDNA 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.

DNA REPAIRperbaikan DNA adalah mekanisme seluler untuk memperbaiki kerusakan pada DNA sebelum menjadi tetap sebagai mutasi atau kelainan kromosom, yang dapat mengakibatkan hasil yang berbahaya seperti kematian sel atau tumorigenesis.Mekanisme perbaikan DNA sangat penting untuk mengurangi risiko kanker serta mengembangkan terapi kanker yang lebih efektif.

MUTASI Mutasi terbagi atas: Mutasi spontan Mutasi induksi

Types of Damage Repair PhotolyaseDe-alkylation proteins (not catalytic)

Base Excision RepairNucleotide Excision Repair (GG and TC)Mismatch Repair

Error-prone Repair or SOSDouble Strand Break Repair

Types of Damage Repair Photolyase De-alkilasi protein (tidak katalitik) Base eksisi Perbaikan Nukleotida eksisi Perbaikan (GG dan TC) Ketidaksesuaian Perbaikan Rawan kesalahan Perbaikan atau SOS Double Strand Break Perbaikan

Repairing Damaged BasesDamaged bases can be repaired by several mechanisms: Direct chemical reversalExcision 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)

Repairing Damge BasesBasisRusakdapatdiperbaikimelalui beberapa mekanisme:LangsungkimiapembalikanPerbaikaneksisi.Adatigamodeperbaikaneksisi,masing-masing yang mempekerjakan setkhususenzim. BaseeksisiPerbaikan(BER) NukleotidaeksisiPerbaikan(APM) Perbaikanketidaksesuaian(MMR)

Direct repairObservation:UV-

PhotoreactivationRequires(membutuhkan) DNA photolyasesrequires visible light (memerlukan cahaya) at 300 500 nmlight repair Contrast: dark repair (BER, NER, mismatch repair) =Kontras:perbaikangelap(BER,APM,perbaikanmismatch)

DNA photolyasesStructureGenerally contain 2 chromophoresChromophore No. 1: always FADH -Chromophore No. 2: folate (in E. coli and yeast) N5,N10-methenyltetrahydrofolylpolyglutamateFunctionbind to pyrimidine dimersresolve pyrimidine dimers into original bases

DNA PHOTOLYASESStrukturUmumnya mengandung2chromophoresKromoforNo1:selaluFADHKromoforNo2:folat(dalamE.colidanragi) N5,N10-methenyltetrahydrofolylpolyglutamateFungsimengikatuntukpirimidindimermenyelesaikan dimer pirimidin ke basis asli

UV-responsive photolyases

Direct reversal (de-alkylating proteins)

Base Excision Repairnot restricted to a short time post replicationsimilar in most organisms (bacteria mammals)recognizes abnormal bases in the DNArequires four enzymesDNA glycosylases AP-endonucleasesDNA polymerase IDNA ligase

Base Excision(memotong) Repairtidakterbataspadareplikasipostingwaktu singkatserupadisebagian besar organisme(bakteri-mamalia)mengakuibasisabnormaldalamDNA memerlukanempatenzim - DNAglycosylases - AP-endonuklease - DNApolimeraseI - DNAligase

DNA glycosylasesRelatively small enzymes (20 30 KDa)Recognize abnormal bases deaminated basesalkylated basesRemove base via cleavage at the glycosidic bond between the deoxyribose and the baseCleavage creates apurinic and apyrimidinic (AP sites)

DNA glycosylasesRelatif kecil enzim(20 - 30KDa)Kenaliabnormalbasis - deaminatedbasis - dialkilasibasisHapusdasarmelaluipembelahandiikatanglikosidikantaradeoksiribosa&basaPembelahanmenciptakanapurinicdanapyrimidinic(APsitus)

AP-endonucleasesrecognize AP-sitescleave phosphodiester bonds near the AP site and generate a 5 phosphate and 3-hydroxylIn E. coli this enzyme also has 3-5 exonuclease activityThe 3-OH functions as a primerGTCTCGCGTCGCAP endonuclease5353

AP-endonucleasesmengakuiAP-situsbersatufosfodiesterobligasidekatlokasiAPdanmenghasilkan5'-P dan3'-hidroksilPadaE. colienziminijugamemilikiaktivitasexonuclease3'-5The3'-OHberfungsisebagaiprimerGTCTCGCGTCGCAP endonuclease5353

The steps of BER: 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.

removal of its deoxyribose phosphate in the backbone, producing a gap. We have two genes encoding enzymes with this function.

replacement with the correct nucleotide. This relies on DNA polymerase, one of at least 11 DNA polymerases encoded by our genes.

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

BASE EXCISION REPAIRLangkah-langkahBER:Penghapusan basisrusak (diperkirakanterjadi sekitar20.000 kali seharidisetiapsel dalam tubuhkita!) olehglycosylaseDNA. Kami memilikisetidaknya8genpengkodeanDNAyang berbedasetiap glycosylasesenzimyang bertanggung jawabuntuk mengidentifikasi danmenghapusjenistertentukerusakandasar.penghapusandeoksiribosafosfatditulang punggung, menghasilkan kesenjangan.Kami memilikiduagenenzim pengkodeandenganfungsiini.penggantiandengannukleotidayang benar.Hal inibergantung padaDNApolimerase,salah satu darisedikitnya 11polimerase DNAdikodeolehgenkita.ligasiistirahatdalamrantai tersebut.Duaenzim yangdikenalyangdapatmelakukanhal ini,keduanya membutuhkan ATPuntukmenyediakanenergi yang dibutuhkan

Base Excision Repair

Nucleotide Excision RepairRecognizes large distortions in the DNA structureRepairs UV-damaged DNACleaves two phosphodiester Generally generates fragments of 12 to 13 nucleotidesRequires four different enzymes ExonucleaseDNA helicase DNA polymerase DNA ligase

MengenalibesardistorsidalamstrukturDNAUV-perbaikankerusakan DNAMemotongduafosfodiesterUmumnyamenghasilkanfragmendari12 sampai 13nukleotidaMemerlukan empatberbedaenzim - Exonuclease - DNAhelikase - DNApolimerase - DNAligase

Nucleotide Excision Repair (E.coli)

Sheet1

EnzymeProteinFunction

ExinucleaseUvrA (MW= 104,000)scans DNA, binds to UvrB

UvrB (MW = 78,000)scanner; binds DNA cleaves phosphate bond at 3' end, 5 positions downstream of lesion

UvrC (MW = 68,000)binds UvrB & DNA cleaves phosphate bond at 5' end, 8 positions upstream of lesion

DNA helicaseUvrDremoves DNA fragment

DNA polymeraseDNA polymerase I (= PolA )fills emerging gap

DNA ligaseLigseal nick

Sheet2

Sheet3

Nucleotide Excision Repair (E.coli)

MechanismThe (UvrA)2:UvrB complex scans DNAUvrA dimer dissociates from pryimidine dimer. UvrB binds DNA and cuts at 3 end.UvrC associates with UvrB and cuts DNA at 5 end of the pyrimidine dimerUvrD DNA helicase removes the DNA fragmentDNA polymerase I fills the gapDNA ligase seals the remaining nick.Nucleotide Excision Repair in E. coli

MekanismeThe(UvrA)2:kompleksUvrBDNAscanDimerUvrAmemisahkandaridimerpryimidine.UvrBmengikatDNAdanlukadiakhir3'.UvrCasosiasidenganUvrB&DNAlukadiujung5'daridimerpirimidinUvrDhelikaseDNAmenghilangkanfragmenDNADNA polimerase Sayamengisi kesenjanganDNAligasesegelnicktersisa.Nucleotide Excision Repair in E. coli

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. coliScenario 1Mismatch is at the 5 end of cleavage siteUnmethylated DNA is unwound via DNA helicase II

The 3-5 exonuclease 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.

CH3CH3ATPADP+PiMutS-MutLDNA helicase IIexonuclease Iorexonuclease XCH3CH3CH3CH3535353353535DNA polymerase IIISSBs

Mismatch repair in E. coliScenario 1Ketidaksesuaianadalahpadaujung 5 'situspembelahanUnmethylatedDNAdibatalkanmelaluiDNAhelikaseII The3'-5'exonucleasekegiatanexonucleaseIatauexoXDNAmenurunkanmelalui ketidakcocokan DNApolimeraseIIIsintesisuntaiDNAbaru DNAligasemenutupnicktersisa.

CH3CH3ATPADP+PiMutS-MutLDNA helicase IIexonuclease Iorexonuclease XCH3CH3CH3CH3535353353535DNA polymerase IIISSBs

Mismatch repair in E. coliCH3CH3ATPADP+PiMutS-MutLDNA helicase IIexonuclease VIIorRecJ nucleaseCH3CH3CH3CH3Scenario 2Mismatch is at the 3 end of cleavage siteUnmethylated DNA is unwound via DNA helicase II

The 5-3 exonuclease activity of exonuclease VII or RecJ nuclease degrades DNA through the mismatch

DNA polymerase III synthesizes the new DNA strand.

DNA ligase closes the remaining nick.

535353353535DNA polymerase IIISSBs

Mismatch repair in E. coliCH3CH3ATPADP+PiMutS-MutLDNA helicase IIexonuclease VIIorRecJ nucleaseCH3CH3CH3CH3Scenario 2Ketidaksesuaianadalahpadaujung 3'situspembelahanUnmethylatedDNAdibatalkanmelaluiDNAhelikaseII The5'-3'exonucleasekegiatanexonucleaseVIIatauRecJnukleaseDNAmenurunkanmelaluiketidakcocokan DNApolimeraseIIIsintesisuntaiDNAbaru. DNAligasemenutupnicktersisa.535353353535DNA polymerase IIISSBs

SOS ResponseSOS 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.

SOS RESPONSESOSperbaikanterjadiketika sel-selkewalahanolehkerusakanUV-inimemungkinkanseluntukbertahan hiduptapi padabiayamutagenesis.SOSresponhanyadipicuketikasistemperbaikanlainnyakewalahan olehjumlahkerusakanDNAdiperbaikisehinggaterakumulasidalam sel.

Error-prone repair (SOS)Activated upon: severe DNA damagedisruption of DNA replicationSOS-responseInaccurate repair mechanismRequires at least 14 proteins in E. coliDin proteins (damage induced)Rec poteins (recombination)Umu proteins (UV-mutagenesis)Uvr proteins (UV-resistance)Others: SulA, HimA, Ssb, and PolB

Error-prone repair (SOS)Diaktifkanpada:parahkerusakanDNAgangguanreplikasiDNA"SOS-responTidak akuratperbaikanmekanismeMemerlukansetidaknya14proteindalamE. coliDinprotein(kerusakaninduksi)Recpoteins(rekombinasi)Umuprotein(UV-mutagenesis)UVRprotein(UV-hambatan)Lainnya:Sula,Himawan,SSB,danPolB

Error Prone Bypass (E. coli)

Double Breaks StrandDSB 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.

Double Breaks StrandDSBPerbaikanistirahatDouble-untai (DSBs) mungkin adalahbentukpaling serius kerusakan DNAkarenamereka menimbulkanmasalahbagitranskripsi,replikasi,dansegregasikromosom.Kerusakanjenisinidisebabkanolehberbagaisumbertermasukageneksogensepertiradiasipengion,bahan kimiagenotoksik,dan stres mekanispadakromosom.

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