Cleavage of DNA.RNA hybrids by Type II restriction enzymes

Cleavage of DNA.RNA hybrids by Type II restriction enzymes
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  Volume 8 Number 13 198 Nucleic cids  ese rch Cleavage of DNA RNA hybrids byType II restriction enzymes Peter L.Molloyand Robert H.Symons Department of Biochemistry, University of Adelaide, Adelaide, South Australia, 5001, Australia Received 7 May 1980 ABSTRACT The action of a number of restriction enzymes on DNA.RNA hybrids hasbeen examined using hybrids synthesised with RNAsof cucumbermosaic virus as templates.The enzymes EcoRI, HindII,SalI, MspI, HhaI, AluI, TaqI and HaeIII cleaved the DNA strandof the hybrids (andpossibly alsotheRNA strand)into specific fragments. For four of theseenzymes,HhaI, AluI, TaqI and HaeIII, comparison of therestriction fragments produced with the knownsequences of the viralRNAs confirmed that they were recognising and cleaving theDNA strandof the hybrids at theircorrect recognition sequences. It is likely that the ability to utiliseDNA.RNA hybrids as substrates is   general propertyof Type II restriction enzymes.INTRODUCTION The specific cleavageofduplex DNA by Type II restrictionenzymesisolated from a wide range of bacterialsourcesandthe propertiesof theseenzymes have been extensivelystudied (1,2). The action of these enzymesinvolves an initial recognitionof a specificsequence in a double-stranded DNA substrate, followed by cleavage of a phosphodiester bond in eachstrand, with this cleavageoccurringwithin the recognition siteformostenzymes. Single-stranded DNA has also beenfound to act as a substrate for a limited number of restriction enzymes, e.g., HaeIII (and its isoschitzomer BspI), HhaI,MboI,HinfIand HpaII (3,4,5). Of these, theenzymeHaeIII has beenmost widely usedfor the cleavage of single-stranded DNA. Analysisof the temperature dependence ofthe reaction led Blakesley et al. (6) to con-cludethat cleavagerequired the formation of duplexregions within single- stranded molecules and that the reaction wasnot, therefore, essentially dif- ferent to that on a double-stranded substrate. The cleavage of one strand only of a duplex DNA at a modified EcoRI site has also been reported (7) indicating that neither the recognition nor cleavage step requires the full double-stranded recognition site. In this © IRL Press Limited, 1 Falconberg Court, London W1V 5FG, U.K. Nucleic Acids Research olume 8 Number 13 1980 2939  Nucleic Acids Research casethe EcoRIsite contained a single base substitutionadjacent to the cleavage site in one strand; only thestrandcontaining the unmodified sequenceatthe cleavage sitewas cut. In this paper we report on the abilityof a numberofrestriction enzymes to utilise DNA.RNA hybrid duplexes as substrates.Theenzymesretain their sequence specificity in cutting theDNA strandof the duplex and possibly also specifically cleave theRNAstrand. MATERIALSAND METHODS RNA. Cucumbermosaic virus (CMV) RNA   andits satellite RNA (SAT RNA) wereprepared by a two-step polyacrylamide slab gel procedure (8) follow- ed bysucrose gradient centrifugation to remove contaminating acrylamide. Polyadenylation ofthe RNAs was as described by Gouldet al. (9). Enzymes. Restriction enzymeswere obtained fromNewEnglandBiolabs, with theexceptionof EcoRI,SalI and HindIIwhichwereprepared in the Department and kindlyprovided by S.J. Clark,Prof. G.E. Rogersand R.P. Har- vey, respectively. Digestions withrestrictionenzymeswere done in thebuf-fers recommended byN.E. Biolabs. Nuclease SI was prepared by the methodof Vogt (10) up to and including the DEAE-cellulose step. Reversetranscriptase from avian myeloblastosis virus was generouslyprovided by the Office ofPro-gram Resources andLogistics,National Cancer Institute, Bethesda, Maryland. Synthesisofcomplementary DNA. cDNA synthesis wasdone in a buffer containing 20mM Tris.HCl, pH 8.3, 70 mM KC1, 10 mM MgCl2, 4 mMNa4P207, 20 mM dithiothreitol, 500 pM each of dATP,dTTP,dGTPanddCTP in the presenceof 2 to 3 pCi/pl of a-32P-dCTP (11). Reactionmixturesof 50 pl also contained 2 to 5 pg of polyadenylated RNA, a tenfoldmolar excess of d(pT8G)(P/L Biochem- icals) and 10 units ofreverse transcriptase.After 45 min at 370Creactions were terminated by the addition ofEDTA to 15 mMand SDS to0.5 , extracted with phenolandthe cDNA.RNA hybrid recovered by gel filtrationoverSephadex G50followed by ethanol precipitation. When added to reactionmixes,Actino- myci n D was presentat 100 ig/ml   Gel electrophoresis and autoradiography.Polyacrylamide gels (4 or 5 ) were essentially as described by Sanger andCoulson (12); urea waspresent at 7 M in denaturing gels andabsent in non-denaturing gels. Autoradiography wasdoneat 40C or -700C using pre-flashedFuji-RX medicalX-ray film and Ilford fast tungstateintensifying screens (13). Nuclease SI digestions. The determination ofthe proportion of single- strandednucleicacid by nuclease SI digestion was as described by Gouldand 2940  NucleicAcidsResearch Symons (14). RESULTS Synthesis of cDNA.RNAHybridsSynthesis of cDNA using reverse transcriptase has been observed to give varying amountsofsingle-or double-stranded DNA aswell as cDNA still bound to its RNA template undersome syntheticconditions (15,16). This has been ascribed to the action ofRNAse H which degradesthe RNA strandof a DNA.RNAhybrid, thus producingregionsofsingle-stranded DNA andreleasing small RNA fragments which can act as primers forsynthesis of anticomplementary DNA onthe cDNAstrand (16). Theadditionofsodium pyrophosphate (4 mM)orhigh levels of deoxynucleotides wasfound to totallyinhibit the actionof RNAse H andunder theseconditions theRNAand product cDNA remained stably complexed (16). Synthesis ofcDNA was, therefore, done in the presenceof 4 mMsodiumpyrophosphateand 500 pM of each dNTP. TheRNAsused forthe synthesis of DNA.RNAhybridswereSATRNA and RNA 4 ofCMV which are, respectively, 336 and approximately 1000 nucleotides in length.Most of SAT RNA has been sequenced (unpublished results) and ithas beenfound tobe very similar to anothersatellite RNA of CMV, CARNA 5, sequenced by Richards et al. (17); the sequenceof the first 270 residuesfromthe 3'-end of CMV RNA 4 has been determined (18). As SATRNAandCMVRNA 4 terminate at the 3'-end in thebases -CC and -CCA, respectively (18,19 and unpublished results), it is possible following polyadenylation to specifically prime cDNA synthesis using d(pT8G), generatingcDNA.RNA hybridshaving speci- fic5'-termini ofthecDNA. A number of restrictionenzyme sitescan be pre- dicted from the known sequencesand the hybrids are therefore well suited for use in the analysis of the actionof restriction enzymes on DNA.RNA hybrids. DNA.RNA hybrids of both CMV RNA 4 andSATRNA were synthesised in the presence of c-32P-dCTP andtheir integrity examined by digestion withnuclease SI. The cDNA synthesised was 100 and 92 resistant to nuclease SI digestion,respectively, for the two RNAs; followingremoval of theRNAstrandsby treat- ment with 0.3 M NaOH for 3 h at 370C the residual cDNA resistant to digestion with nuclease SI was 19 and 11 , respectively. Survey of Action of a Number of RestrictionEnyzmes on cDNA.RNA Hybrids An initial survey of restriction enzymes was done by digesting aliquots of a cDNA.RNA 4 hybrid preparation in the appropriate buffers.Each20 pl reactioncontained 12 ngof hybrid and 0.1 to 0.2unitsof enzyme and was incubated for 2 h; this 20 to 50 fold excess of enzyme over that used with 2941  Nucleic AcidsResearch double-strand DNA is similar to thatusedfor digestionof single-stranded DNA with HaeIII (4) and has beenfound necessary withmostenzymes to completelydigest the DNA.RNA hybrids. The digested hybridswere denatured by boiling in 70 formamide for 5 min andthe radiolabelled cDNA-strandproductsanalysed by electrophoresis on a denaturing 4 polyacrylamide-urea gel (Fig. 1). Com- plete or near complete digestion of the cDNAstrand to discrete fragments was observed for the enzymes EcoRI,SalI, HindII, AluI, HhaI, TaqI,MspIandHae- BASES a a 800 650 570 350 p t 267 185 162 eq 128 ; bt:v   45 A B C D EF GH I FIGURE 1. RestrictionenzymedigestsofcDNA.CMV RNA 4 hybridsanalysed by electrophoresis on a denaturing 4 acrylamide, 7 M urea gel whichwas runat 20 mAfor 90 min (12). End-labelled fragmentsderivedfrom anMspI digest ofthe replicative form ofbacteriophageM13were used as sizemarkers (24). Track A, EcoRI digest; B, SalI; C, HindII; D, AluI; E, HhaI; F, TaqI; G, MspI; H,no enzyme; I, HaeIII.Bands markedwith an asterisk arethose predicted fromtheknown 3'-terminalsequenceof CMV RNA 4 (18). 2942  Nucleic AcidsResearch III. A numberof enzymes which wereused in thesurvey did not digest the hybridat all (MboI, BamHI,PstI, HindIII,SacI, XbaI and HpaI). From theknown 3'-terminal sequenceof270residuesof CMV RNA 4 (about 25 oftotal), no sites for theseenzymeswould be predicted so it is not known whether they are capable of cleaving a hybrid molecule if the correctsequenceswere pre- sent. The productionofspecific fragments by a numberof enzymessuggests that the cDNAwascutatsequence specific sites, presumably the recognition sites ofthe different enzymes.The appearance ofthesame sizefragment on digestionwithHindII (recognition sequence GTPyPuAC) and SalI (GTCGAC) sup-ports this.From the known sequenceof the 3'-end ofCMV RNA   (18) sites for three restriction enzymes werepredicted in the first 270residues.Fragments predicted from the sequence are thosemarked with an asterisk in Fig. 1; TaqI (142 bases), HhaI (221 bases) andAluI (89 and108 bases). This specificity of cleavage was further examined using cDNA.SAT RNAhybrids. Digestion of cDNA.SATRNA Hybrids Mostof thesequenceof SATRNA has been determined (Symons, unpublish- ed data) andfrom this sequence the restrictionsites for HaeIIIandTaqI, which are depicted in Fig. 2A,were expected. Hybrid cDNA.SAT RNA was digested with bothenzymes,the productsboiled in 70 formamide and subjected to electrophoresis on a 4 polyacrylamide-urea gel (Fig.2B). Digestion with TaqI yielded the expected cDNA fragments of 119and 225bases (including the d(pT8G)primer); digestion with HaeIII also gavethe expectedproducts and in addition the possible partial digestion productsof 197 and 221 bases could normally beseen. It is clear that bothenzymes specificallyrecognised the restrictionsites in the cDNA.RNAhybrid and specifically cleaved the cDNA strandofthe hybrid in the normal positions. To examine whether theRNAstrand ofthe hybrid was also cleaved by the restriction enzymes the digestion products were also analysed by electrophore- sis on a non-denaturing 5 polyacrylamide gel (tracks D and E, Fig. 2B). Discrete bands were again observed forthe digestion products ofbothenzymes, with thesizesof the double-stranded fragments being as expected from the known sequence. Fromthis result it is possible that theRNA strand of the hybridwasalso being specifically cleaved.However, it is also possible thatthenick- ing of the cDNAstrandpermits cleavageof theRNAat this site by non- specific RNases or thatthe RNA strand was already extensivelynicked leading to double-strand separation on nickingof the cDNA strand. Furtherexperi- 2943
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