Description:
RecognitionSite:

McrBCisanendonucleasewhichcleavesDNAcontainingmethylcytosine*ononeorbothstrands.McrBCwillnotactuponunmethylatedDNA(1).SitesontheDNArecognizedbyMcrBCconsistoftwohalf-sitesoftheform(G/A)mC.Thesehalf-sitescanbeseparatedbyupto3kb,buttheoptimalseparationis55-103basepairs(2,3).McrBCrequiresGTPforcleavage,butinthepresenceofanon-hydrolyzableanalogofGTP,theenzymewillbindtomethylatedDNAspecifically,withoutcleavage(4).
*5-methylcytosineor5-hydroxymethylcytosineorN4-methylcytosine(5).
Linearizedplasmid(methylatedorunmethylated),containingoneMcrBCsite,incubatedwithMcrBC.Lane1,unmethylatedDNA;Lane2,methylatedDNA;Lane3,Marker:LamBDaDNABstEIIdigest.
HumanandDrosophilagenomicDNAincubatedwithMcrBC.60-90%ofCpGsinvertebrateDNAareestimatedtobemethylated(6)whilemethylatedCpGsareextremelyrareinDrosophilaDNA(7).Lane1,HumanDNA;Lane2,HumanDNAincubatedwithMcrBC;Lane3,DrosophilaDNA;Lane4,DrosophilaDNAincubatedwithMcrBC;Lane4,Marker:LambdaDNABstEIIdigest.ProductSource
ThetwocomponentproteinsarepurifiedseparatelyfromE.coliK-12strainscontainingplasmidsencodingMcrBandMcrC(1).ReagentsSupplied
Thefollowingreagentsaresuppliedwiththisproduct:
| Storeat(°C) | Concentration |
NEBuffer™2 | -20 | 10X |
PlasmidDNAforMcrBCLinearizedmethylated(20µl) | | 100μg/ml |
GTP | -20 | 100X |
PurifiedBSA | -20 | 10X |
Notes:
McrBCmakesonecutbetweeneachpairofhalf-sites,cuttingclosetoonehalf-siteortheother,butcleavagepositionsaredistributedoverseveralbasepairsapproximately30basepairsfromthemethylatedbase(2).Therefore,theenzymedoesnotproducedefinedDNAendsuponcleavage.Also,whenmultipleMcrBChalf-sitesarepresentinDNA(asisthecasewithcytosine-methylatedgenomicDNA)theflexIBLenatureoftherecognitionsequenceresultsinanoverlapofsites,andsoasmearedratherthanasharpbandingpatternisproduced.McrBCcleavageofthesupplied4.3kblinear,methylatedcontrolplasmidDNAproducesseveralfragmentsbetweenapproximately700bpand2.3kbinsize.GTPismorelABIlethanothernucleotides.Werecommendaliquotingthe100mMsolutionsuppliedandthawinganddilutingasnecessary.