實(shí)驗(yàn)十一DNA酶切

實(shí)驗(yàn)十一

ＤＮＡ的酶切

RestrictionEndonucleases:

AnOverviewRestrictionenzymeswerediscoveredabout30yearsagoduringinvestigationsintothephenomenonofhost-specificrestrictionandmodificationofbacterialviruses.Bacteriainitiallyresistinfectionsbynewviruses,andthis"restriction"ofviralgrowthstemmedfromendonucleaseswithinthecellsthatdestroyforeignDNAmolecules.Amongthefirstofthese"restrictionenzymes"tobepurifiedwereEcoR

IandEcoRIIfromEscherichiacoli,andHindIIandHindIIIfromHaemophilusinfluenzae.TheseenzymeswerefoundtocleaveDNAatspecificsites,generatingdiscrete,gene-sizefragmentsthatcouldbere-joinedinthelaboratory.Researcherswerequicktorecognizethatrestrictionenzymesprovidedthemwitharemarkablenewtoolforinvestigatinggeneorganization,functionandexpression.Astheuseofrestrictionenzymesspreadamongmolecularbiologistsinthelate1970’s,companiessuchasNewEnglandBiolabsbegantosearchformore.Exceptforcertainviruses,restrictionenzymeswerefoundonlywithinprokaryotes.Manythousandsofbacteriaandarchaehavenowbeenscreenedfortheirpresence.Analysisofsequencedprokaryoticgenomesindicatesthattheyarecommon--allfree-livingbacteriaandarchaeaappeartocodeforthem.Restrictionenzymesareexceedinglyvaried;theyrangeinsizefromthediminutivePvuII(157aminoacids)tothegiantCjeI(1250aminoacids)andbeyond.Amongover3,000activitiesthathavebeenpurifiedandcharacterized,morethan250differentsequence-specificitieshavebeendiscovered.Ofthese,over30%werediscoveredandcharacterizedatNewEnglandBiolabs.Thesearchfornewspecificitiescontinues,bothbiochemically,bytheanalysisofcell-extracts,andcomputationally,bytheanalysisofsequencedgenomes.Althoughmostactivitiesencounteredtodayturnouttobeduplicates--isoschizomers--ofexistingspecificities,restrictionenzymeswithnewspecificitiesarefoundwithregularity.Beginningintheearly1980’s,NewEnglandBiolabsembarkedonaprogramtocloneandoverexpressthegenesforrestrictionenzymes.Cloningimprovesenzymepuritybyseparatingenzymesfromcontaminatingactivitiespresentinthesamecells.Italsoimprovesenzymeyieldsandgreatlysimplifiespurification,anditprovidesthegenesforsequencingandanalysis,andtheproteinsforx-raycrystallography.Restrictionenzymesprotectbacteriafrominfectionsbyviruses,anditisgenerallyacceptedthatthisistheirroleinnature.Theyfunctionasmicrobialimmunesystems.WhenastrainofE.colilackingarestrictionenzymeisinfectedwithavirus,mostvirusparticlescaninitiateasuccessfulinfection.Whenthesamestraincontainsarestrictionenzyme,however,theprobabilityofsuccessfulinfectionplummets.Thepresenceofadditionalenzymeshasamultiplicativeeffect;acellwithfourorfiveindependentrestrictionenzymescouldbevirtuallyimpregnable.Restrictionenzymesaretraditionallyclassifiedintothreetypesonthebasisofsubunitcomposition,cleavageposition,sequence-specificityandcofactor-requirements.However,aminoacidsequencinghasuncoveredextraordinaryvarietyamongrestrictionenzymesandrevealedthatatthemolecularleveltherearemanymorethanthreedifferentkinds.TypeIenzymesarecomplex,multisubunit,combinationrestriction-and-modificationenzymesthatcutDNAatrandomfarfromtheirrecognitionsequences.Originallythoughttoberare,wenowknowfromtheanalysisofsequencedgenomesthattheyarecommon.TypeIenzymesareofconsiderablebiochemicalinterestbuttheyhavelittlepracticalvaluesincetheydonotproducediscreterestrictionfragmentsordistinctgel-bandingpatterns.TypeIIenzymescutDNAatdefinedpositionsclosetoorwithintheirrecognitionsequences.Theyproducediscreterestrictionfragmentsanddistinctgelbandingpatterns,andtheyaretheonlyclassusedinthelaboratoryforDNAanalysisandgenecloning.Ratherthenformingasinglefamilyofrelatedproteins,type

IIenzymesareacollectionofunrelatedproteinsofmanydifferentsorts.TypeIIenzymesfrequentlydiffersoutterlyinaminoacidsequencefromoneanother,andindeedfromeveryotherknownprotein,thattheylikelyaroseindependentlyinthecourseofevolutionratherthandivergingfromcommonancestors.ThemostcommontypeIIenzymesarethoselikeHha

I,HindIIIandNotIthatcleaveDNAwithintheirrecognitionsequences.Enzymesofthiskindaretheprincipleonesavailablecommercially.MostrecognizeDNAsequencesthataresymmetricbecausetheybindtoDNAashomodimers,butafew,(e.g.,BbvCI:CCTCAGC)recognizeasymmetricDNAsequencesbecausetheybindasheterodimers.Someenzymesrecognizecontinuoussequences(e.g.,

EcoR

I:GAATTC)inwhichthetwohalf-sitesoftherecognitionsequenceareadjacent,whileothersrecognizediscontinuoussequences(e.g.,BglI:GCCNNNNNGGC)inwhichthehalf-sitesareseparated.Cleavageleavesa3′-hydroxylononesideofeachcutanda5′-phosphateontheother.TheyrequireonlymagnesiumforactivityandthecorrespondingmodificationenzymesrequireonlyS-adenosylmethionine.Theytendtobesmall,withsubunitsinthe200–350aminoacidrange.ThenextmostcommontypeIIenzymes,usuallyreferredtoas‘typeIIs"arethoselikeFokIandAlwIthatcleaveoutsideoftheirrecognitionsequencetooneside.

Theseenzymesareintermediateinsize,400–650aminoacidsinlength,andtheyrecognizesequencesthatarecontinuousandasymmetric.Theycomprisetwodistinctdomains,oneforDNAbinding,theotherforDNAcleavage.TheyarethoughttobindtoDNAasmonomersforthemostpart,buttocleaveDNAcooperatively,throughdimerizationofthecleavagedomainsofadjacentenzymemolecules.Forthisreason,sometypeIIsenzymesaremuchmoreactiveonDNAmoleculesthatcontainmultiplerecognitionsites.ThethirdmajorkindoftypeIIenzyme,moreproperlyreferredtoas"typeIV"arelarge,combinationrestriction-and-modificationenzymes,850–1250aminoacidsinlength,inwhichthetwoenzymaticactivitiesresideinthesameproteinchain.Theseenzymescleaveoutsideoftheirrecognitionsequences;thosethatrecognizecontinuoussequences(e.g.,Eco57I:CTGAAG)cleaveonjustoneside;thosethatrecognizediscontinuoussequences(e.g.,BcgI:CGANNNNNNTGC)cleaveonbothsidesreleasingasmallfragmentcontainingtherecognitionsequence.Theaminoacidsequencesoftheseenzymesarevariedbuttheirorganizationareconsistent.TheycompriseanN-terminalDNA-cleavagedomainjoinedtoaDNA-modificationdomainandoneortwoDNAsequence-specificitydomainsformingtheC-terminus,orpresentasaseparatesubunit.Whentheseenzymesbindtotheirsubstrates,theyswitchintoeitherrestrictionmodetocleavetheDNA,ormodificationmodetomethylateit.一．實(shí)驗(yàn)?zāi)康募氨尘?/p>

核酸限制性內(nèi)切酶是一類能識別雙鏈ＤＮＡ中特定堿基順序的核酸水解酶，這些酶都是從原核生物中發(fā)現(xiàn)，它們的功能猶似高等功物的免疫系統(tǒng)，用于抗擊外來ＤＮＡ的侵襲。限制性內(nèi)切酶以內(nèi)切方式水解核酸鏈中的磷酸二酯鍵，產(chǎn)生的ＤＮＡ片段５’端為Ｐ，３’端為ＯＨ。限制酶的類型根據(jù)限制酶的識別切割特性，催化條件及是否具有修飾酶活性可分為Ⅰ、Ⅱ、Ⅲ型三大類。Ⅰ類和Ⅲ類限制性內(nèi)切酶，在同一蛋白分子中兼有甲基化作用及依賴ATP的限制性內(nèi)切酶活性。Ⅰ類限制性內(nèi)切酶結(jié)合于特定識別位點(diǎn)，且沒有特定的切割位點(diǎn)，酶對其識別位點(diǎn)進(jìn)行隨機(jī)切割，很難形成穩(wěn)定的特異性切割末端。Ⅲ類限制性內(nèi)切酶在識別位點(diǎn)上切割，然后從底物上解離下來。故Ⅰ類和Ⅲ類酶在基因工程中基本不用。Ⅱ型酶Ⅱ型酶就是通常指的ＤＮＡ限制性內(nèi)切酶.它們能識別雙鏈ＤＮＡ的特異順序，并在這個順序內(nèi)進(jìn)行切割，產(chǎn)生特異的ＤＮＡ片段；Ⅱ型酶分子量較小，僅需Mg2+作為催化反應(yīng)的輔助因子，識別順序一般為４～６個堿基對的反轉(zhuǎn)重復(fù)順序；Ⅱ型內(nèi)切酶切割雙鏈ＤＮＡ產(chǎn)生３種不同的切口－－５’端突出；３’端突出和平末端。正是得益于限制性的內(nèi)切酶的發(fā)現(xiàn)和應(yīng)用，才使得人們能在體外有目的地對遺傳物質(zhì)ＤＮＡ進(jìn)行改造，從而極大地推動了分子生物學(xué)的興旺和發(fā)展。２．ＤＮＡ：作為內(nèi)切酶底物，ＤＮＡ應(yīng)該具備一定的純度，其溶