| Description | EndonucleaseIV(EndoIV)recognizesapurinic/apyrimidinic(AP)sitesofdsDNAandcleavesthephosphodiesterbond5'tothelesiongeneratingahydroxylgroupatthe3'-terminus.Theenzymecanalsoactasa3'-diesterasethatisabletorelease3'-phosphoglycolateor3'-phosphatefromthedamagedendsofdsDNA.EndoIVpossessesalsoa3'=>5'exonucleaseactivity.Itsprogressiononsubstratesissensitivetoionicstrength,metalions,EDTA,andreducingconditions.Substrateswith3'-recessedendsarepreferredsubstratesforthe3'=>5'exonucleaseactivity. |
| Applications | - StudiesofDNAdamageandrepair
- Singlecellelectrophoresis(cometassay)
- Antitumordrugresearch
- DNAstructureresearch
- SNPanalysis
- DNADetectionUsingRecombinasePolymeraseAmplification(RPA)
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| Source | AnE.colistrainwhichcarriestheclonedEndonucleaseIV(nfo)gene |
| UnitDefinition | Oneunitisdefinedastheamountofenzymerequiredtocleave1pmolofa34-meroligonucleotideduplexcontainingasingleAPsite*inatotalreactionvolumeof10µlin1hourat37°C. *AnAPsiteiscreatedbytreating10pmolofa34meroligonucleotideduplexcontainingasingleuracilresiduewith1unitofUracil-DNAGlycosylase(UDG)for2minutesat37°C. |
| Components | - EndonucleaseIV:10,000U/mlinstoragebuffercontaining50mMTris-HCl(pH7.5),0.1MNaCl,1mMDTT,0.05%(v/v)TritonX-100,and50%(v/v)glycerol.
- 10XReactionBuffer:1000mMNaCl,500mMTris-HCl,100mMMgCl2,10mMDTT,pH7.9@25°C
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| QualityControl | Theabsenceofotherendo-,exodeoxyribonucleases,andribonucleasesconfirmedbyappropriatequalitytests. |
| StorageCondition | -20°C |
| References | - J.D.Levin,HomogeneousEscherichiacoliendonucleaseIV.CharacterizationofanenzymethatrecognizesoxidativedamageinDNA.J.Biol.Chem.263,8066-8071(1988).
- S.M.Kerins,etal.,CharacterizationofanendonucleaseIV3’-5’exonucleaseactivity.J.BiolChem.278(5),3048-3054(31January2003).
- B.Demple,L.Harrison,RepairofoxidativedamagetoDNA:enzymologyandBIOLOGy.Annu.Rev.Biochem.63,915-948(1994).
- J.D.Levin,B.Demple,InvitrodetectionofendonucleaseIV–specificDNAdamageformedbybleomycininvivo.NucleicAcidsRes.24,885-889(1996)
- J.N.Patro,etal.,ProbingtheconfigurationsofformamidopyrimidinelesionsFapy.dAandFapy.dGinDNAusingendonucleaseIV.Biochemistry.43,13397-13403(2004).
- A.R.Collins,ThecometassayforDNAdamageandrepair:principles,applications,andlimitations.Molec.Biotechnol.26,249-261(2004).
- D.J.Hosfieldetal.,StructureoftheDNArepairenzymeendonucleaseIVanditsDNAcomplex:double-nucleotideflippingatabasicsitesandthree-metal-ioncatalysis.Cell.98,397-408(1999).
- I.V.Kutyavinetal.,AnovelendonucleaseIVpost-PCRgenotypingsystem.NucleicAcidsRes.29,1-9(2006).
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