| Description | ExonucleaseIII(ExoIII)exhibitsfourcatalyticactivities.The3'=>5'exodeoxyribonucleaseactivityofExoIIIisspecificfordouble-strandedDNA.ExoIIIdegradesdsDNAfrombluntends,5'-overhangsornicks,releases5'-mononucleotidesfromthe3'-endsofDNAstrandsandproducesstretchesofsingle-strandedDNA.Alimitednumberofnucleotidesareremovedduringeachbindingevent,resultingincoordinatedprogressivedeletionswithinthepopulationofDNAmolecules.Itisnotactiveon3'-overhangendsofDNAthatareatleastfour-baseslonganddonotcarrya3'-terminalC-residueonsingle-strandedDNA,oronphosphorothioate-linkednucleotides.Thispropertycanbeexploitedtoproduceunidirectionaldeletionsfromalinearmoleculewithoneresistant(3´-overhang)andonesusceptIBLe(bluntor5´-overhang)terminus. ExoIII3'-phosphataseactivityremovesthe3'-terminalphosphate,generatinga3'-OHgroup.ExoIIIRNaseHactivityexonucleolyticallydegradestheRNAstrandinRNA-DNAhybrids.ExoIIIapurinic/apyrimidinic(AP)endonucleaseactivitycleavesphosphodiesterbondsatapurinicorapyrimidinicsitestoproduce5'-terminithatarebase-freedeoxyribose5'-phosphateresidues. |
| Applications | - CreationofunidirectionaldeletionsinDNAfragmentsinconjunctionwithS1Nuclease.
- Generationofasingle-strandedtemplatefordideoxy-sequencingofDNA.
- Site-directedmutagenesis.
- CloningofPCRproducts.
- Preparationofstrand-specificprobesfordideoxysequencing
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| Source | AnE.colistrainthatcarriestheclonedEcolixthgene |
| UnitDefinition | Oneunitoftheenzymecatalyzesthereleaseof1nmolofacidsolublereactionproductsfromE.coli[3H]-DNAin30minat37°C. |
| MolecularWeight | 31kDamonomer. |
| Components | - EndonucleaseIII:100,000units/mlin25mMTris-HCl,pH8.0@25°C,50mMKCl,1.0mMDTT,0.1%mMEDTA,50%Glycerol
- 10XReactionBuffer:100mMTris-HCl,pH7.0@25°C,100mMMgCl2,10mMDTT.
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| QualityControl | - Theabsenceofendodeoxyribonucleasesconfirmedbyappropriatequalitytest.
- FunctionallytestedforcreationofunidirectionaldeletionsinDNAfragments.
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| StorageCondition | -20°C |
| References | - S.G.Rogers,B.Weiss,ExonucleaseIIIofEscherichiacoliK-12,anAPendonuclease.MethodsEnzymol.65,201-211(1980).
- J.D.Hoheisel,OntheActivitiesofEscherichiacoliExonucleaseIII.Anal.Biochem.209,238-246(1993).
- S.Henikoff,UnidirectionaldigestionwithexonucleaseIIIcreatestargetedbreakpointsforDNAsequencing.Gene.28,351-359(1984).
- L-H.Guo,R.Wu,NewrapidmethodsforDNAsequencingbasedonexonucleaseIIIdigestionfollowedbyrepairsynthesis.NucleicAcidsRes.10,2065-2084(1982).
- M.A.Vandeyaretal.,Asimpleandrapidmethodfortheselectionofoligodeoxynucleotide-directedmutants.Gene.65,129-133(1988).
- C.Li,R.M.Evans,Ligationindependentcloningirrespectiveofrestrictionsitecompatibility.NucleicAcidsRes.25,4165-4166(1997).
- H.M.Eun,EnzymologyPrimerforRecombinantDNATechnology(AcademicPressInc.,SanDiego,California,1996).
- J.F.Tomb,G.J.Barcak,Regulatingthe3’-5’activityofexonucleaseIIIbyvaryingthesodiumchlorideconcentration.BioTechniques.7,932-933(1989).
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