| Description | ExonucleaseIcleavessingle-strandedDNAinthe3'to5'direction,releasing5'-mono/di-nucleotidesandleavingdouble-strandedDNAmoleculesandthe5'-terminusintact.Theenzymeisprocessivethoughdigestionisinhibitedbythepresenceofa3'-terminalphosphate.ThisenzymedoesnotcleaveDNAstrandswithterminal3'-OHgroupsblockedbyphosphoryloracetylgroups |
| Applications | - PrimerremovalfromPCRmixtures:
- priortoPCRproductsequencing(2)
- forone-tube"megaprimer"PCRmutagenesis(3)
- Removalofsingle-strandedDNAcontaininga3'-hydroxylterminusfromnucleicacidmixtures.
- Assayforthepresenceofsingle-strandedDNAwitha3'-hydroxylterminus(4)
- IntheprescenceoflamBDaexonuclease,ExoIiseffectiveinremovinglinearDNAfromplasmidpreparations.
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| Source | AnE.colistrainthatcarriestheclonedEcolisbcBgene |
| UnitDefinition | Oneunitoftheenzymecatalyzesthereleaseof10nmolofacidsolublenucleotidesin30minat37°C. |
| MolecularWeight | 54.5kDamonomer. |
| Components | - EndonucleaseI:20,000U/mlin10mMTris-HCl,pH7.5@25°C,100mMNaCl,1mMDTT,0.5mMEDTA,50%glycerol
- 10XReactionBuffer:670mMGlycine-KOH,pH9.5@25°C,67mMMgCl2,10mMDTT.
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| QualityControl | Theabsenceofotherendo-,exodeoxyribonucleases,andribonucleasesconfirmedbyappropriatequalitytests. |
| StorageCondition | -20°C |
| References | I.R.Lehman,A.L.Nussbaum,ThedeoxyribonucleasesofEscherichiacoli.V.OnthespecificityofexonucleaseI(phosphodiesterase).J.Biol.Chem.239,2628-2636(1964). E.Werleetal.,Convenientsingle-step,onetubepurificationofPCRproductsfordirectsequencing.NucleicAcidsRes.22,4354-4355(1994). S.Nabavi,R.N.Nazar,Simplifiedone-tube“megaprimer”polymerasechainreactionmutagenesis.Anal.Biochem.2,346-348(2005). J.Rosamondetal.,ModulationoftheactionoftherecBCenzymeofEscherichiacoliK-12byCa2+.J.Biol.Chem.254,8646-8652(1979). Y.Sasaki,D.Miyoshi,N.Sugimoto,RegulationofDNAnucleasesbymolecularcrowding.NucleicAcidsRes.35,4086-4093(2007).
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