实验十一酶切

实验十一酶切第1页，共19页，2023年，2月20日，星期六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.第2页，共19页，2023年，2月20日，星期六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.第3页，共19页，2023年，2月20日，星期六

Restrictionenzymesusuallyoccurincombinationwithoneortwomodificationenzymes(DNA-methyltransferases)thatprotectthecell’sownDNAfromcleavagebytherestrictionenzyme.ModificationenzymesrecognizethesameDNAsequenceastherestrictionenzymethattheyaccompany,butinsteadofcleavingthesequence,theymethylateoneofthebasesineachoftheDNAstrands.ThemethylgroupsprotrudeintothemajorgrooveofDNAatthebindingsiteandpreventtherestrictionenzymefromactinguponit.Together,arestrictionenzymeandits"cognate"modificationenzyme(s)formarestriction-modification(R-M)system.InsomeR-Msystemstherestrictionenzymeandthemodificationenzyme(s)areseparateproteinsthatactindependentlyofeachother.Inothersystems,thetwoactivitiesoccurasseparatesubunits,orasseparatedomains,ofalarger,combined,restriction-and-modificationenzyme.第4页，共19页，2023年，2月20日，星期六Restrictionenzymesaretraditionallyclassifiedintothreetypesonthebasisofsubunitcomposition,cleavageposition,sequence-specificityandcofactor-requirements.However,aminoacidsequencinghasuncoveredextraordinaryvarietyamongrestrictionenzymesandrevealedthatatthemolecularleveltherearemanymorethanthreedifferentkinds.TypeIenzymesarecomplex,multisubunit,combinationrestriction-and-modificationenzymesthatcutDNAatrandomfarfromtheirrecognitionsequences.Originallythoughttoberare,wenowknowfromtheanalysisofsequencedgenomesthattheyarecommon.TypeIenzymesareofconsiderablebiochemicalinterestbuttheyhavelittlepracticalvaluesincetheydonotproducediscreterestrictionfragmentsordistinctgel-bandingpatterns.第5页，共19页，2023年，2月20日，星期六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.第6页，共19页，2023年，2月20日，星期六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.第7页，共19页，2023年，2月20日，星期六TypeIIIenzymesTypeIIIenzymesarealsolargecombinationrestriction-and-modificationenzymes.TheycleaveoutsideoftheirrecognitionsequencesandrequiretwosuchsequencesinoppositeorientationswithinthesameDNAmoleculetoaccomplishcleavage;theyrarelygivecompletedigests.Nolaboratoryuseshavebeendevisedforthem,andnoneareavailablecommercially.

第8页，共19页，2023年，2月20日，星期六一．实验目的及背景

核酸限制性内切酶是一类能识别双链ＤＮＡ中特定碱基顺序的核酸水解酶，这些酶都是从原核生物中发现，它们的功能犹似高等功物的免疫系统，用于抗击外来ＤＮＡ的侵袭。限制性内切酶以内切方式水解核酸链中的磷酸二酯键，产生的ＤＮＡ片段５’端为Ｐ，３’端为ＯＨ。第9页，共19页，2023年，2月20日，星期六限制酶的类型根据限制酶的识别切割特性，催化条件及是否具有修饰酶活性可分为Ⅰ、Ⅱ、Ⅲ型三大类。Ⅰ类和Ⅲ类限制性内切酶，在同一蛋白分子中兼有甲基化作用及依赖ATP的限制性内切酶活性。Ⅰ类限制性内切酶结合于特定识别位点，且没有特定的切割位点，酶对其识别位点进行随机切割，很难形成稳定的特异性切割末端。Ⅲ类限制性内切酶在识别位点上切割，然后从底物上解离下来。故Ⅰ类和Ⅲ类酶在基因工程中基本不用。第10页，共19页，2023年，2月20日，星期六Ⅱ型酶Ⅱ型酶就是通常指的ＤＮＡ限制性内切酶.它们能识别双链ＤＮＡ的特异顺序，并在这个顺序内进行切割，产生特异的ＤＮＡ片段；Ⅱ型酶分子量较小，仅需Mg2+作为催化反应的辅助因子，识别顺序一般为４～６个碱基对的反转重复顺序；Ⅱ型内切酶切割双链ＤＮＡ产生３种不同的切口－－５’端突出；３’端突出和平末端。正是得益于限制性的内切酶的发现和应用，才使得人们能在体外有目的地对遗传物质ＤＮＡ进行改造，从而极大地推动了分子生物学的兴旺和发展。第11页，共19页，2023年，2月20日，星期六酶切反应中应注意以下几个问题：１．内切酶：不应混有其它杂蛋白特别是其它内切酶或外切酶的污染；注意内切酶的识别位点及形成的粘性末端；内切酶的用量根据内切酶单位和ＤＮＡ用量而定，通常1u指在适当条件下，1小时内完全酶解１ug特定ＤＮＡ底物所需要的限制性内切酶量，使用中一般以１ugDNA对２－３u酶短时间为宜。同时内切酶体积不能超过反应体系１０％，因内切酶中含５０％甘油，体系中甘油超过５％会抑制内切酶活力；内切酶操作应在低温下进行（冰上）；使用时防止操作中对内切酶的污染。第12页，共19页，2023年，2月20日，星期六２．ＤＮＡ：作为内切酶底物，ＤＮＡ应该具备一定的纯度，其溶液中不能含酚、氯仿、乙醚、SDS、EDTA、高盐浓度、酒精等，这些因素的存在均不同程度影响限制性内切酶的