EndonucleaseVIIIfromE.coliactsasbothanN-glycosylaseandanAP-lyase.TheN-glycosylaseactivityreleasesdamagedpyrimidinesfromdouble-strandedDNA,generatinganapurinic(APsite).TheAP-lyaseactivitycleaves3´and5´totheAPsiteleavinga5´phosphateanda3´phosphate.DamagedbasesrecognizedandremovedbyEndonucleaseVIIIincludeurea,5,6-dihydroxythymine,thymineglycol,5-hydroxy-5-methylhydanton,uracilglycol,6-hydroxy-5,6-dihydrothymineandmethyltartronylurea(1,2).WhileEndonucleaseVIIIissimilartoEndonucleaseIII,EndonucleaseVIIIhasβandδlyaseactivitywhileEndonucleaseIIIhasβlyaseactivity.

• Singlecellgelelectrophoresis(Cometassay)(3,4,5)
• Alkalineelution(6)
• Alkalineunwinding(7)

Oneunitisdefinedastheamountofenzymerequiredtocleave1pmolofa34meroligonucleotideduplexcontainingasingleAPsite*inatotalreactionvolumeof10µlin1hourat37°Cin1XEndonucleaseVIIIReactionBuffercontaining10pmoloffluorescentlylabeledoligonucleotideduplex.

*AnAPsiteiscreatedbytreating10pmolofa34meroligonucleotideduplexcontainingasingleuracilresiduewith1unitofUracil-DNAGlycosylase(UDG)for2minutesat37°C.

• EndonucleaseVIII:10,000U/mlin10mMTris-HCl,pH8.0@25°C,250mMNaCl,0.1mMEDTA,50%glycerol
• 10XReactionBuffer:100mMTris-HCl,pH8.0@25°C,750mMNaCl,10mMEDTA

-20°C

1. Dizdaroglu,M.,Laval,J.andBoiteux,S.(1993).Biochemistry.32,12105-12111.
2. Hatahet,Z.etal.(1994).J.Biol.Chem..269,18814-18820.
3. Singh,N.etal.(1988).Exp.Cell.Res..175,184-191.
4. Collins,A.etal.(1993).Carcinogenesis.14,1733-1735.
5. Collins,A.etal.(1996).Environ.HealthPerspect.104,465-469.
6. Pflaum,M.etal.(1998).FreeRad.Res..29,585-594.
7. Hartwig,A.etal.(1996).Toxicol.Lett..88,85-90.
8. Marks,K.unpublishedobservation.