The EMBO Journal vol.10 no.9 pp.2689-2694, 1991
Endonuclease activity of Escherichia coli DNA helicase I directed against the transfer origin of the F factor
Ursula Reygers, Rainer Wessel1, Holger Muller and Hartmut Hoffmann-Berling Max-Planck-Institut fur Medizinische Forschung, D-6900 Heidelberg, FRG
'Present address: Fakultiit fuir Biologie, Universitat Konstanz, D-7750 Konstanz, FRG Communicated by H.Hoffmann-Berling
DNA helicase I, the traI gene product of the Escherichia coli F factor, was shown to be associated with endonuclease activity specific for the transfer origin of the F plasmid, onT. In the presence of Mg2+, the purified enzyme forms a complex, stable in the presence of sodium dodecylsulfate (SDS) with a negatively superhelical chineric plasmid containing onT. The enzyme nicks and, after this, apparently binds to the 5' nick terminus when this complex is heated in the presence of SDS and/or EDTA or treated with proteinase K. Dideoxy sequencing locates the nick site in the F DNA strand transferred during bacterial conjugation after nucleotide 138 clockwise of the mid-point of the Bgl site at 66.7 kb of the F genetic map. A sequencing stop after nucleotide 137 of this strand (where oriT-nicking seems to occur in vivo) is possibly an artefact caused by helicase I protein attached to the 5' terminal nucleotide. Deletion in the amino-terminal part of the tral polypeptide abolishes the onT-nicking activity while leaving the strand-separating activity intact. These results confirm the prediction from genetic studies that helicase I is bifunctional with site-specific endonuclease and strandseparating activities. Key words: DNA helicase I/endonuclease/oriT
mutations affecting oriT-nicking can be located in traI (Traxler and Minkley, 1988). The authors thus propose that the function ascribed to TraZ is one of TraI and that helicase I is bifunctional with oriT-nicking and strand-separating activities. The following results confirm this. Previous investigators have mapped oriT by propagating the cloned oriT sequence in cells expressing F Tra functions (Everett and Willets, 1980). With DNA sequencing techniques a single nick site was found in the DNA of a chimeric plasmid (at the position indicated below; Thompson et al., 1989) and a cluster of nick sites in the DNA of a XoriT phage. The nicked X DNA was possibly processed before packaging (Thompson et al., 1984). Besides helicase I, we have studied helicase I del29 (Benz and Muller, 1990), an MS2 replicase fusion protein comprising the carboxy-terminal 86% of the 192 kDa Tral polypeptide (Bradshaw et al., 1990). Despite Tralphenotype, the truncated gene product retains strand
separating activity.
Results Helicase I nicks F DNA in the oriT region The transfer origin of F is located in a 1.08 kb Bgll fragment near the BglII site at 66.7 kb of the F genetic map (Thompson and Achtman, 1978). Plasmid pEMBL8-onT carries this fragment inserted in the polylinker region of
pEMBL8 (Figure 1). 1
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Introduction The conjugal transfer of F DNA between Escherichia coli cells is initiated by strand-specific cleavage of the F plasmid at oriT, the transfer origin. The interrupted strand is subsequently exported to the recipient cell and, after recircularization, converted by DNA synthesis to a double strand (for reviews, see Willets and Wilkins, 1984; IppenIhler and Minkley, 1986). The endonuclease responsible for nicking at onT was until recently thought to be the product of the F transfer genes traY and traZ (Everett and Willets, 1980). Gene traZ and the promoter-proximal tral (which encodes DNA helicase I; Abdel-Monem et al., 1983) form the most distal part of the F transfer operon (Manning et al., 1982). Traxler and Minkley (1987) have recently found that tral is located 1000 bp nearer to the distal end of the tra operon compared to the position proposed previously and that a 94 kDa protein ascribed to traZ is actually an in-frame translational restart product of traL. They also showed that -
© Oxford University Press
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Fig. 1. Physical map of pEMBL8-oriT. (a) Restriction map (A, HaeII; E, EcoRI; H, HindIll; P, PvulI; S, SalI). The thick line indicates the inserted BglII fragment, the arrowhead oriT (the DNA sequence of the F oriT region was determined by Thompson et al., 1984). (b) Enlarged map of the oriT region. The duplex insert (thick lines) is shown with a nicked lower strand (the strand transferred during conjugation; Thompson et al., 1984). Oligonucleotide primers (open arrows pointing in the direction of primer extension) are drawn besides the sequences to which they are complementary. Distances are in basepairs from the mid-point of the BglII site at 66.7 kb of F. H, HindIIl; S, Safl.
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U.Reygers et al.
Plasmid pEMBL8-onT was incubated with helicase I, the mixture was treated with SDS, EDTA and proteinase K and the DNA was studied by agarose gel electrophoresis. Most of the superhelical plasmid was converted to the relaxed form. Relaxation was not observed with pEMBL8 and not when helicase I was replaced by helicase I de129, the genetically modified enzyme described in the Introduction (Figure 2). Relaxation was further achieved with pBE274 and not with pKTO43, the onT-free vector plasmid. Helicase I is thus associated with onT-specific endonuclease activity, a property lacking in helicase I de129.
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Fig. 2. Treatment with helicase I converts superhelical pEMBL8-ornT to the relaxed form. The reaction mixtures contained pEMBL8-onT or pEMBL8 and helicase I or helicase I de129. After incubation, the DNA was electrophoresed in a 0.6% agarose gel. Lanes 1-6, pEMBL8-onT. Larte 1, reaction without helicase; lanes 2-4, reactions with 350, 700 ng and 1.4 ytg helicase I, respectively; lane 5, with 1.4 jig helicase I de129; lane 6, EcoRI-linearized pEMBL8-ornT. Lanes 7,8, pEMBL8. Lane 7, reaction without helicase; lane 8, with 1.4 ltg helicase I.
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Fig. 3. Relaxed pEMBL8-ornT is nicked in the ornT region. The superhelical plasmid was sequentially treated with the enzymes indicated below and then electrophoresed in a 1 % agarose gel. Lane 1, pEMBL8-onT (enzymes omitted); lane 2, plasmid treated with helicase I; lane 3, with helicase I and topoisomerase I; lane 4, with helicase I, topoisomerase I and phosphodiesterase; lane 5, with helicase I, topoisomerase I, phosphodiesterase and EcoRI; lane 6, EcoRIlinearized pEMBL8-onT. In lane 5 the full-length linear DNA derives in part from incompletely relaxed covalently closed circles which, as a smear, are difficult to see in lanes 3 and 4. The linearized DNA migrates differently from the control in lane 6, a consequence probably of the high concentrations of EDTA (10 mM) and Mg2+ (20 mM) added to irreversibly inactivate the phophodiesterase and to prepare for EcoRI cleavage. The 1 kb band in lane 5 is smeared because of the presence of attached protein.
2690
The optimum ionic conditions for cleavage were 7 mM
Mg2+ and pH 7.2-8.8. Ca2+ or Mn2+ did not replace Mg2+, and excess EDTA was an inhibitor. Neither was ATP required nor did 1 mM adenosine 5'-[Lythio]triphosphate (which interferes with the strand separating activity) inhibit. A maximum of relaxed molecules was obtained by treatment with 55-110 helicase I/DNA (Figure 2). The relaxed plasmid was subsequently characterized by secondary cleavage with venom phophodiesterase and cleavage at the single EcoRl site. Phosphodiesterase degrades preferentially single-stranded DNA including the DNA opposite a nick. To avoid cleavage of the remaining superhelical plasmid molecules (Kowalski and Sanford, 1982), the helicase I product was first treated with topoisomerase I to produce relaxed covalently closed rings. After the subsequent phosphodiesterase treatment unit-length linear DNA molecules were the main product and after the treatment also with EcoRI fragments of approximately 4 kb and 1 kb (Figure 3). Attempts to linearize the relaxed plasmid with nuclease SI (which has difficulties in cleaving opposite a nick) were unsuccessful. The results suggest that the relaxed plasmid is nicked and that the interruption is located in the oriT region (cf. Figure 1). Helicase I failed to cleave when the superhelical plasmid was first treated with topoisomerase I. Linearized DNA was not detectable after treatment of the helicase I product with phosphodiesterase. Upon treatment with EcoRI also, linears, and not fragments, appeared (Figure 4). Thus, helicase I does not accept relaxed DNA as the nicking substrate. Studies using electron microscopy showed that helicase I remains attached to the DNA after nicking. The DNA was freed from SDS and EDTA and then linearized with restriction enzyme. The dense structures visible in the centers of the micrographs in Figure 5 are the denatured helicase protein which, after the removal of SDS, has formed aggregates. Attached to these structures are linear DNA duplexes of 82 + 3 % and 18 :4 3 % unit length, respectively, when the DNA was cut with EcoRl and of approximately unit length when the DNA was cut with HindIII. The protein is thus bound to the DNA in the oriT region.
Fig. 4. Relaxed pEMBL8-oriT is not cleaved. Lane 1, pEMBL8-oriT (enzymes omitted); lane 2, plasmid treated with topoisomerase I; lane 3, with topoisomerase I and helicase I; lane 4, with topoisomerase I, helicase I and phosphodiesterase; lane 5, with topoisomerase I, helicase I, phosphodiesterase and EcoRI.
Endonuclease activity of helicase I
Sequence specificity of the nick and character of the nick termini To map the location of the nick precisely the relaxed plasmid was cut with HindlIl (outside the BglII insert 20 bp away from the BglII site at 66.7 kb). The 3' ends of the linearized DNA were labeled with 32p, and the DNA was electrophoresed in a denaturing gel. The autoradiographic pattern in Figure 6a-a fragment of - 160 nucleotides besides very long fragments-is that expected if the F DNA strand transferred during conjugation was cleaved near the position known to represent oriT. The autoradiographic band near the 160mer marker is not sharp. The results were therefore checked by subjecting the nicked plasmid to dideoxy sequencing analysis using the primers described under Materials and methods. Interruption at a defined position of the template strand should lead to the appearance of an intense band in all four lanes (G, A, T and C). When the strand transferred during conjugation was sequenced, an intense band in all four lanes appeared at the position of nucleotide 138 (counted clockwise, in the 3' to 5' direction of the transferred strand, from the middle of the BglII site at 66.7 kb of F) and a less intense band in all four lanes at the position 137. No intense bands in all four lanes were visible at other positions nor were they seen when the opposite strand of the nicked plasmid or the strands of the unnicked plasmid were sequenced (Figure 6b). Using dideoxy sequencing techniques, Thompson et al. (1989) have located the in iivo nick site after nucleotide 137 of the transferred F DNA strand. The 5' strand terminus flanking the nick was characterized by secondary cleavage with SalI followed by alkali denaturation of the DNA and transfer of 32p label to its 5' ends. Plasmid nicked after nucleotide 138 of the transferred strand should yield radioactive fragments of 146, 389 and 535 nucleotides length, respectively, as well as much longer radioactive fragments. The autoradiogram in Figure 6c
A
confirms this expectation except for the shortest fragment which is missing. Since this fragment is that expected to carry the 5' nick terminus it appears that the latter is masked. Most likely the denatured helicase protein is bound at this site. Further studies showed that the nicked plasmid is accessible to degradation with exonuclease Ill suggesting that its 3' strand terminus is a free hydroxyl or free phosphate. After the treatment with exonuclease, the DNA was saturated with E. coli single strand binding protein (SSB) and vizualized using electron microscopy. Of 118 plasmid molecules counted, 83 were relaxed rings with a single stretch of SSBcovered DNA (not shown). Nicking is triggered by rigorous protein-denaturing treatment To characterize the influence of the nicking assay procedure, the treatment with proteinase K was omitted and the helicase I/pEMBL8-oriT mixture was either directly applied to an electrophoretic gel or first heated at 56°C for 10 min (Figure 7a). In a series of similar experiments the DNA was, in addition, digested with PvuII (Figure 7b). PvuII cuts pEMBL8-oriT into a 1.37 kb fragment containing the oriT region and a 3.7 kb fragment. Before electrophoresis, all mixtures were brought to the same concentration of SDS/EDTA. When the helicase/plasmid mixture was not heated, the DNA migrated as a diffuse band to positions intermediate between that of the superhelical plasmid and that of the relaxed plasmid. Band shift of the short PvuII fragment showed that the DNA is associated with protein in the oriT region. When the mixture was heated before the addition of SDS/EDTA the plasmid remained superhelical and the DNA was free of protein as unaltered mobility of the short PvuII fragment showed. The results were the same when the enzyme/DNA mixture was incubated in the presence of 1 mM ATP and then heated in the absence of SDS/EDTA (the PvuII product is not shown). On the other hand, when
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Fig. 5. Relaxed pEMBL8-oriT as seen in the electron microscope. (A) The relaxed plasmid (proteinase K was omitted) was linearized with EcoRI or (B) HindIll. The bar represents 0.5 Am. 2691
U.Reygers et al.
the heat treatment was carried out in the presence of SDS/EDTA, relaxed plasmid was the main product as in the experiment of Figure 2. The mobility of the short PvuII fragment was lowered except when the DNA was treated with proteinase K. The complex between helicase I and its superhelical substrate thus resists treatment with SDS/EDTA at room temperature. Nicking occurs when this complex is heated in the presence of SDS/EDTA and not when it is heated in the absence of SDS/EDTA. The enzyme then dissociates from the DNA leaving the plasmid superhelical. Since partially relaxed molecules are not detectable it appears that helicase I does not nick and seal under our conditions, i.e. does not act as a DNA topoisomerase. Heating in the presence of either SDS or EDTA led to essentially the same results as heating in the presence of SDS plus EDTA. Relaxation of approximately half of the plasmid molecules was also achieved by exposing the helicase I/pEMBL8oriT mixture to proteinase K (in the absence of SDS/EDTA).
Discussion Helicase I is associated with oriT-nicking activity as these experiments show. Using dideoxy sequencing techniques we find a major nick site after nucleotide 138 of the F DNA strand transferred during conjugation (in the notation specified) and a minor nick site after nucleotide 137 where, according to the sequencing studies of Thompson et al. (1989), ornT-nicking occurs in vivo (Thompson et al., 1989). Since the in vivo product was treated with SDS, a possible explanation of the discrepancy in the results is that protein attached to the 5' nick terminus prevented the sequencing enzyme from copying the 5' terminal nucleotide, an effect
less pronounced under our conditions. Evidence for a 5' terminal protein complex comes from the negative result of the end-labeling experiment in Figure 6c and the results in Figure 6a. The HindlIl fragment carrying the 5' nick terminus migrates as a diffuse band in a denaturing gel suggesting that the material in this band is not homogeneous (Figure 6a). The authentic nick site is thus probably after
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Fig. 6. Mapping of onT and characterization of the 5' nick terminus. (a) Restriction-mapping. The nicked plasmid linearized with HindlIl, the 3' ends were labeled with 32p, and the DNA was electrophoresed in a denaturing 6% polyacrylamide gel (lane 1). pBR322-HpaII fragments used as markers (lane 2). (b) Dideoxy sequencing reactions. The (+) strand primer was used (lanes marked +) the (-) strand primer (lanes marked -) together with nicked or unnicked pEMBL8-oriT. Filled triangles refer to lanes 1 -4 and indicate the positions of intense bands in all four lanes. Open triangles (lanes 9- 16) indicate the corresponding positions in the opposite strand (the sequence of the interrupted strand is 5'-GGGTGTGGTG-3' between the positions 143 and 134). (c) The 5' nick terminus is inaccessible to labeling with 32p. The nicked plasmid cleaved with Sall, alkali-denatured, and after transfer of 32p label to the 5' ends, electrophoresed in a DNA sequencing gel (lane 1). Lane 2 shows X DNA HindlIl fragments. was
were
or
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2692
Endonuclease activity of helicase I
nucleotide 138 of the transferred F DNA strand. Helicase I requires superhelical DNA as the endonuclease substrate as do other site-specific DNA-nicking enzymes [for instance the gene A protein of phage OX 174 (van der Ende et al. 1981) and the gene 2 protein of phage fd (Meyer and Geider, 1979)]. Helicase I does not even bind to an oriTcontaining DNA fragment as previous studies showed (Lahue and Matson, 1990) suggesting that local unwinding of the DNA, supported by negative superhelicity of the duplex, is required for the formation of a stable complex with oriT. Nicking occurs when this complex is subjected to rigorous protein-denaturing treatment, a reaction also observed with a type I DNA topoisomerase (Liu and Wang, 1979). An auxiliary function, probably that of TraY, is therefore needed to incise F plasmid for the conjugal transfer of DNA. TraY, a 17 kDa polypeptide, binds to DNA in the oriT region (Lahue and Matson, 1990) and, together with Tral (TraZ), is needed for oriT-nicking in vivo (Everett and Willets, 1980). In addition to its effect on the nicking activity TraY might enable the endonuclease to switch to the DNA unwinding function. Since helicase I unwinds processively in the 5' to 3' direction of the bound DNA strand, and since the interrupted F DNA strand is exported with its 5' end first, the enzyme should move along the strand to be transferred leaving its displaced 5' end free. Alternatively, the nicking enzyme might bind to the 5' strand terminus and, with the latter, enter the recipient cell where the complex between enzyme and 5' strand terminus might later serve to recircularize the transferred F DNA strand. The DNA unwinding function would then be left to other helicase I molecules, possibly such assembled prior to nicking near oriT. Helicase I binds cooperatively to single-stranded DNA
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(Wessel et al., 1990). It might also do so in a reaction involving local DNA denaturation. Functions as discussed here have been observed with other site specific DNA-binding enzymes. The OX174 gene A protein (van der Ende et al., 1981) and thefd gene 2 protein (Meyer and Geider, 1979) are both capable of ligating the specifically nicked DNA strand. The large tumour antigen of SV40 virus, a replicative helicase, initiates unwinding by binding to a continuous DNA double strand (Dodson et al., 1987; Scheffner et al., 1989). Finally, the primase of an I-like plasmid is an example of a protein that is quite likely to be exchanged during conjugation between the bacteria (Lanka et al., 1979). As the results obtained with helicase I de129 show, a functional amino-terminal part of the TraI polypeptide is needed for the oriT-nicking function although not for the strand-separating function. The bifunctional character of helicase I further explains why the tral products of the related plasmids F and R100 are not interchangeable (Willets and Maule, 1979). These plasmids differ at two positions of their oriT sequences (Finlay et al., 1986).
Materials and methods Enzymes and plasmids Helicase I and helicase I de129 were the preparations electrophoretically >95% homogeneous described previously (Benz and Muller, 1990). Calf thymus DNA topoisomerase I and SSB were kind gifts from H.P.Vosberg and K.Geider, respectively (this institute). Other enzymes were from Boehringer-Mannheim (FRG) except for Sequenase Version 2.0 (United States Biochemical Corporation. OH, USA). The plasmid pBE274 (kindly provided by M.Achtman, Max-Planck-Institut fur Genetik, Berlin) is pKTO43, a pBR322 derivative, with a 1.08 kb F DNA BglII fragment, containing orMT, cloned into the unique BglII site. pBE274 is efficiently mobilized by F Tra functions (Thompson and Achtman, 1978). pEMBL8-oriT was constructed in this laboratory by subcloning the 1.08 kb Bgll fragment from pBE274 into the unique BamHI site of pEMBL8 (Dente et al., 1983) thereby modifying the BglI and BamHI sites. Plasmids grown in F- recAl cells were purified by banding twice in CsCI -ethidium bromide. To prepare relaxed covalently closed pEMBL8-oriT the plasmid was treated with 1 jg DNA topoisomerase I/3.5 Ag DNA in nicking assay buffer at 35°C for 10 min. The DNA was then extracted with phenol.
DNA nicking assay i
-
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The standard reaction mixture (20 jlI; scaled up if necessary) contained in buffer (50 mM Tris-HCI, 20 mM KCI, 7 mM MgCI2, 1 mM dithiothreitol, 80 gg/ml bovine serum albumin; pH 7.6) 1.4 tg helicase I and 225 ng pEMBL8-oriT. After incubation at 35°C for 12 min, 2 /1l SDS/EDTA (5% SDS, 0.125 M Na3-EDTA, 50 % (v/v) glycerol) and 1 proteinase K were added. The mixture was heated at 45°C for 30 min and, after this, either applied to an agarose gel [electrophoresed in TAE buffer (Maniatis et al., 1982) at I V/cm or, in the experiment of Figure 7b, 5 V/cm] or, for further analysis of the DNA, extracted with phenol. Gels were stained with I tg/ml ethidium bromide.
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Characterization of the nicked plasmid
Fig. 7. Nicking is triggered by heat treatment of the helicase I/pEMBL8-oriT complex in the presence of SDS/EDTA. (a) The nicking assay mixtures were either heated or not heated as indicated below. SDS/EDTA was added before or after the heat treatment and, in addition, to the non-heated mixture. Electrophoresis was in a 1 % agarose gel. Lane 1, reaction without helicase I; lane 2, non-heated mixture; lane 3, mixture heated in the absence of SDS/EDTA; lane 4, mixture heated in the presence of SDS/EDTA; lane 5, 1 mM ATP was present and the mixture was heated in the absence of SDS/EDTA. (b) DNA gel mobility shift assay. The experiments were carried out as in (a). The helicase I-treated DNA was spin-filtered to remove SDS and EDTA and then cleaved with PvuII. Lane 1, reaction without helicase I; lane 2, non-heated mixture; lane 3, mixture heated in the absence of SDS/EDTA; lane 4, mixture heated in the presence of SDS/EDTA; lane 5, the product shown in lane 4 was treated with proteinase K.
Venom phosphodiesterase (from Crotalus durissimus) and restriction endonuclease were sequentially applied as described by Kowalski and Sanford (1982). HindU-linearized pEMBL8-onT was labeled at the recessed 3' ends by a fill-in reaction using DNA polymerase I-large fragment together with [a-32P]dATP and three non-labeled dNTPs (Maxam and Gilbert, 1980). The 5' ends of alkali-denatured DNA were labeled by treatment with calf intestine alkaline phosphatase and T4 polynucleotide kinase in the presence of [_y-32P]ATP (Maxam and Gilbert, 1980). Dideoxy sequencing (Sanger et al., 1977) was performed using oligonucleotide primers synthesized with the phosphoramidite method in a DNA synthesizer (Applied Biosystems, model 380B). The primers were then eluted from Sep-Pak Cartridges (Waters, Milford, MA). The (+) strand primer is a 20mer complementary to the positions 55-74 of the transferred F DNA strand, and the (-) strand primer is a 20mer complementary to the positions 216-197 of the opposite strand (Figure 1). Sequenase Version 2.0 was used together with [a-35SIdATP (Amersham Buchler,
2693
U.Reygers et al.
Braunschweig, FRG) as the labeled dNTP. The protocol was that of the manufacturer of Sequenase. Electron microscopy The nicking assay mixture was supplemented with 0.33 vol dimethylsulfoxide to completely dissolve SDS. The DNA was spin-filtered through a 300 ,ul Biogel A-0.5m column (Silhavy et al. 1984) and then treated with restriction enzyme or exonuclease III. Exonuclease IlI-treated DNA was saturated with SSB. The fixed samples were spread with the alkyl-benzyl-dimethylammonium chloride method, rotary-shadowed on the grids with tungsten and then viewed in a Philips EM 400T electron microscope. The techniques have been described previously (Wessel et al., 1990).
Acknowledgements We thank Steffi Haupenthal for the synthesis of oligonucleotide primers and Elke Stein for help with the sequencing gels.
References Abdel-Monem,M., Taucher-Scholz,G. and Klinkert,M.-Q. (1983) Proc. Naitl. Acad. Sci. USA, 80, 4659-4663. Benz,I. and Muller,H. (1990) Eur. J. Biochemn., 189, 267-276. Bradshaw,H.D., Traxler,B.A., Minkley,E.G.Jr., Nester.E.W. and Gordon,M.P. (1990) J. Bactetriol., 172, 4127-4131. Dente,L., Cesarini.G. and Cortese,R. (1983) Nuc-leic Acids Res., 11, 1645- 1655. Dodson,M., Dean.F.B., Bullock,P.. Echols,H. and Hurwitz,J. (1987) Science, 238, 964-967. Everett,R. and Willets,N. (1980) J. Mol. Biol., 136, 129-150. Finlay,B.B., Frost,L.S. and Paranchych,W. (1986) J. Bacteriol., 168, 132- 139. Ippen-lhler,K.A. and Minkley,E.G.Jr. (1986) Anonii. Rev. Geniet., 20, 593 -624. Kowalski,D. and Sanford,J.P. (1982) J. Biol. Clhemi., 257, 7820-7825. Lahue,E.E. and Matson,S.E. (1990) J. Bacteriol., 172, 1385 - 1391. Lanka,E., Scherzinger,E., Gunther,E. and Schuster.H. (1979) Proc. Natl. Acad. Sci. USA, 76, 3632-3636. Liu,L. F. and Wang,J.C. (1979) J. Biol. Chem., 254, 11082 - 11088. Maniatis,T., Fritsch,E. and Sambrook,J. (1982) Molecular Cloning, A Laiboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 150-157. Manning,P.A., Kusecek,B., Morelli,G., Fisseau,C. and Achtman,M. (1982) J. Bacteriol., 150, 76-88. Maxam,A. and Gilbert,W. (1980) Methods E:zovinol, 65, 500-560. Meyer,T. F. and Geider,K. (1 979) J. Biol. Clhem., 254, 12642-1 2646. Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Nail. Acad. Sci. USA, 74, 5463-5467. Scheffner,M., Wessel,R. and Stahl,H. (1989) Nucleic Acids Res., 17, 93-106 Silhavy,T., Berman,M. and Enquist,L. (1984) In Evperimeitts in Genie Fusion. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 203-204. Thompson,R. and Achtman,M. (1978) Mol. Geni. Genet., 165, 295-304. Thompson,R., Taylor,L., Kelly.K., Everett,R. and Willets,N. (1984) EMBO J., 3, 1175-1180. Thompson,T.L.. Centola.M.B. and Deonier,R.C. (1989) J. Mol. Biol., 207, 505 -512. Traxler,B.A. and Minkley,E.G.Jr. (1987) J. Bacteriol., 169, 3251 -3259. Traxler,B.A. and Minkley,E.G.Jr. (1988) J. Mol. Biol., 204, 205-209. van der Ende,A., Langeveld,S.A., Teerstra,R., van Arkel,G.A. and Weisbeck,P.J. (1981) Nucleic Acids Res., 9, 2037-2053. Wessel,R., Muller,H. and Hoffmann-Berling,H. (1990) Eur. J. Biochein.. 189, 277-285. Willets,N. and MauleJ. (1979) Mol. Gen. Geniet, 169, 325-336. Willets,N. and Wilkins,B. (1984) Microbiol. Rev., 48, 24-41.
Received on1 March 18, 1991; revised on Max' 15, 1991
2694
Endonuclease activity of Escherichia coli DNA helicase I directed against the transfer origin of the F factor
Ursula Reygers, Rainer Wessel1, Holger Muller and Hartmut Hoffmann-Berling Max-Planck-Institut fur Medizinische Forschung, D-6900 Heidelberg, FRG
'Present address: Fakultiit fuir Biologie, Universitat Konstanz, D-7750 Konstanz, FRG Communicated by H.Hoffmann-Berling
DNA helicase I, the traI gene product of the Escherichia coli F factor, was shown to be associated with endonuclease activity specific for the transfer origin of the F plasmid, onT. In the presence of Mg2+, the purified enzyme forms a complex, stable in the presence of sodium dodecylsulfate (SDS) with a negatively superhelical chineric plasmid containing onT. The enzyme nicks and, after this, apparently binds to the 5' nick terminus when this complex is heated in the presence of SDS and/or EDTA or treated with proteinase K. Dideoxy sequencing locates the nick site in the F DNA strand transferred during bacterial conjugation after nucleotide 138 clockwise of the mid-point of the Bgl site at 66.7 kb of the F genetic map. A sequencing stop after nucleotide 137 of this strand (where oriT-nicking seems to occur in vivo) is possibly an artefact caused by helicase I protein attached to the 5' terminal nucleotide. Deletion in the amino-terminal part of the tral polypeptide abolishes the onT-nicking activity while leaving the strand-separating activity intact. These results confirm the prediction from genetic studies that helicase I is bifunctional with site-specific endonuclease and strandseparating activities. Key words: DNA helicase I/endonuclease/oriT
mutations affecting oriT-nicking can be located in traI (Traxler and Minkley, 1988). The authors thus propose that the function ascribed to TraZ is one of TraI and that helicase I is bifunctional with oriT-nicking and strand-separating activities. The following results confirm this. Previous investigators have mapped oriT by propagating the cloned oriT sequence in cells expressing F Tra functions (Everett and Willets, 1980). With DNA sequencing techniques a single nick site was found in the DNA of a chimeric plasmid (at the position indicated below; Thompson et al., 1989) and a cluster of nick sites in the DNA of a XoriT phage. The nicked X DNA was possibly processed before packaging (Thompson et al., 1984). Besides helicase I, we have studied helicase I del29 (Benz and Muller, 1990), an MS2 replicase fusion protein comprising the carboxy-terminal 86% of the 192 kDa Tral polypeptide (Bradshaw et al., 1990). Despite Tralphenotype, the truncated gene product retains strand
separating activity.
Results Helicase I nicks F DNA in the oriT region The transfer origin of F is located in a 1.08 kb Bgll fragment near the BglII site at 66.7 kb of the F genetic map (Thompson and Achtman, 1978). Plasmid pEMBL8-onT carries this fragment inserted in the polylinker region of
pEMBL8 (Figure 1). 1
0
2
3
4
5
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Introduction The conjugal transfer of F DNA between Escherichia coli cells is initiated by strand-specific cleavage of the F plasmid at oriT, the transfer origin. The interrupted strand is subsequently exported to the recipient cell and, after recircularization, converted by DNA synthesis to a double strand (for reviews, see Willets and Wilkins, 1984; IppenIhler and Minkley, 1986). The endonuclease responsible for nicking at onT was until recently thought to be the product of the F transfer genes traY and traZ (Everett and Willets, 1980). Gene traZ and the promoter-proximal tral (which encodes DNA helicase I; Abdel-Monem et al., 1983) form the most distal part of the F transfer operon (Manning et al., 1982). Traxler and Minkley (1987) have recently found that tral is located 1000 bp nearer to the distal end of the tra operon compared to the position proposed previously and that a 94 kDa protein ascribed to traZ is actually an in-frame translational restart product of traL. They also showed that -
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Fig. 1. Physical map of pEMBL8-oriT. (a) Restriction map (A, HaeII; E, EcoRI; H, HindIll; P, PvulI; S, SalI). The thick line indicates the inserted BglII fragment, the arrowhead oriT (the DNA sequence of the F oriT region was determined by Thompson et al., 1984). (b) Enlarged map of the oriT region. The duplex insert (thick lines) is shown with a nicked lower strand (the strand transferred during conjugation; Thompson et al., 1984). Oligonucleotide primers (open arrows pointing in the direction of primer extension) are drawn besides the sequences to which they are complementary. Distances are in basepairs from the mid-point of the BglII site at 66.7 kb of F. H, HindIIl; S, Safl.
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U.Reygers et al.
Plasmid pEMBL8-onT was incubated with helicase I, the mixture was treated with SDS, EDTA and proteinase K and the DNA was studied by agarose gel electrophoresis. Most of the superhelical plasmid was converted to the relaxed form. Relaxation was not observed with pEMBL8 and not when helicase I was replaced by helicase I de129, the genetically modified enzyme described in the Introduction (Figure 2). Relaxation was further achieved with pBE274 and not with pKTO43, the onT-free vector plasmid. Helicase I is thus associated with onT-specific endonuclease activity, a property lacking in helicase I de129.
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Fig. 2. Treatment with helicase I converts superhelical pEMBL8-ornT to the relaxed form. The reaction mixtures contained pEMBL8-onT or pEMBL8 and helicase I or helicase I de129. After incubation, the DNA was electrophoresed in a 0.6% agarose gel. Lanes 1-6, pEMBL8-onT. Larte 1, reaction without helicase; lanes 2-4, reactions with 350, 700 ng and 1.4 ytg helicase I, respectively; lane 5, with 1.4 jig helicase I de129; lane 6, EcoRI-linearized pEMBL8-ornT. Lanes 7,8, pEMBL8. Lane 7, reaction without helicase; lane 8, with 1.4 ltg helicase I.
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Fig. 3. Relaxed pEMBL8-ornT is nicked in the ornT region. The superhelical plasmid was sequentially treated with the enzymes indicated below and then electrophoresed in a 1 % agarose gel. Lane 1, pEMBL8-onT (enzymes omitted); lane 2, plasmid treated with helicase I; lane 3, with helicase I and topoisomerase I; lane 4, with helicase I, topoisomerase I and phosphodiesterase; lane 5, with helicase I, topoisomerase I, phosphodiesterase and EcoRI; lane 6, EcoRIlinearized pEMBL8-onT. In lane 5 the full-length linear DNA derives in part from incompletely relaxed covalently closed circles which, as a smear, are difficult to see in lanes 3 and 4. The linearized DNA migrates differently from the control in lane 6, a consequence probably of the high concentrations of EDTA (10 mM) and Mg2+ (20 mM) added to irreversibly inactivate the phophodiesterase and to prepare for EcoRI cleavage. The 1 kb band in lane 5 is smeared because of the presence of attached protein.
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The optimum ionic conditions for cleavage were 7 mM
Mg2+ and pH 7.2-8.8. Ca2+ or Mn2+ did not replace Mg2+, and excess EDTA was an inhibitor. Neither was ATP required nor did 1 mM adenosine 5'-[Lythio]triphosphate (which interferes with the strand separating activity) inhibit. A maximum of relaxed molecules was obtained by treatment with 55-110 helicase I/DNA (Figure 2). The relaxed plasmid was subsequently characterized by secondary cleavage with venom phophodiesterase and cleavage at the single EcoRl site. Phosphodiesterase degrades preferentially single-stranded DNA including the DNA opposite a nick. To avoid cleavage of the remaining superhelical plasmid molecules (Kowalski and Sanford, 1982), the helicase I product was first treated with topoisomerase I to produce relaxed covalently closed rings. After the subsequent phosphodiesterase treatment unit-length linear DNA molecules were the main product and after the treatment also with EcoRI fragments of approximately 4 kb and 1 kb (Figure 3). Attempts to linearize the relaxed plasmid with nuclease SI (which has difficulties in cleaving opposite a nick) were unsuccessful. The results suggest that the relaxed plasmid is nicked and that the interruption is located in the oriT region (cf. Figure 1). Helicase I failed to cleave when the superhelical plasmid was first treated with topoisomerase I. Linearized DNA was not detectable after treatment of the helicase I product with phosphodiesterase. Upon treatment with EcoRI also, linears, and not fragments, appeared (Figure 4). Thus, helicase I does not accept relaxed DNA as the nicking substrate. Studies using electron microscopy showed that helicase I remains attached to the DNA after nicking. The DNA was freed from SDS and EDTA and then linearized with restriction enzyme. The dense structures visible in the centers of the micrographs in Figure 5 are the denatured helicase protein which, after the removal of SDS, has formed aggregates. Attached to these structures are linear DNA duplexes of 82 + 3 % and 18 :4 3 % unit length, respectively, when the DNA was cut with EcoRl and of approximately unit length when the DNA was cut with HindIII. The protein is thus bound to the DNA in the oriT region.
Fig. 4. Relaxed pEMBL8-oriT is not cleaved. Lane 1, pEMBL8-oriT (enzymes omitted); lane 2, plasmid treated with topoisomerase I; lane 3, with topoisomerase I and helicase I; lane 4, with topoisomerase I, helicase I and phosphodiesterase; lane 5, with topoisomerase I, helicase I, phosphodiesterase and EcoRI.
Endonuclease activity of helicase I
Sequence specificity of the nick and character of the nick termini To map the location of the nick precisely the relaxed plasmid was cut with HindlIl (outside the BglII insert 20 bp away from the BglII site at 66.7 kb). The 3' ends of the linearized DNA were labeled with 32p, and the DNA was electrophoresed in a denaturing gel. The autoradiographic pattern in Figure 6a-a fragment of - 160 nucleotides besides very long fragments-is that expected if the F DNA strand transferred during conjugation was cleaved near the position known to represent oriT. The autoradiographic band near the 160mer marker is not sharp. The results were therefore checked by subjecting the nicked plasmid to dideoxy sequencing analysis using the primers described under Materials and methods. Interruption at a defined position of the template strand should lead to the appearance of an intense band in all four lanes (G, A, T and C). When the strand transferred during conjugation was sequenced, an intense band in all four lanes appeared at the position of nucleotide 138 (counted clockwise, in the 3' to 5' direction of the transferred strand, from the middle of the BglII site at 66.7 kb of F) and a less intense band in all four lanes at the position 137. No intense bands in all four lanes were visible at other positions nor were they seen when the opposite strand of the nicked plasmid or the strands of the unnicked plasmid were sequenced (Figure 6b). Using dideoxy sequencing techniques, Thompson et al. (1989) have located the in iivo nick site after nucleotide 137 of the transferred F DNA strand. The 5' strand terminus flanking the nick was characterized by secondary cleavage with SalI followed by alkali denaturation of the DNA and transfer of 32p label to its 5' ends. Plasmid nicked after nucleotide 138 of the transferred strand should yield radioactive fragments of 146, 389 and 535 nucleotides length, respectively, as well as much longer radioactive fragments. The autoradiogram in Figure 6c
A
confirms this expectation except for the shortest fragment which is missing. Since this fragment is that expected to carry the 5' nick terminus it appears that the latter is masked. Most likely the denatured helicase protein is bound at this site. Further studies showed that the nicked plasmid is accessible to degradation with exonuclease Ill suggesting that its 3' strand terminus is a free hydroxyl or free phosphate. After the treatment with exonuclease, the DNA was saturated with E. coli single strand binding protein (SSB) and vizualized using electron microscopy. Of 118 plasmid molecules counted, 83 were relaxed rings with a single stretch of SSBcovered DNA (not shown). Nicking is triggered by rigorous protein-denaturing treatment To characterize the influence of the nicking assay procedure, the treatment with proteinase K was omitted and the helicase I/pEMBL8-oriT mixture was either directly applied to an electrophoretic gel or first heated at 56°C for 10 min (Figure 7a). In a series of similar experiments the DNA was, in addition, digested with PvuII (Figure 7b). PvuII cuts pEMBL8-oriT into a 1.37 kb fragment containing the oriT region and a 3.7 kb fragment. Before electrophoresis, all mixtures were brought to the same concentration of SDS/EDTA. When the helicase/plasmid mixture was not heated, the DNA migrated as a diffuse band to positions intermediate between that of the superhelical plasmid and that of the relaxed plasmid. Band shift of the short PvuII fragment showed that the DNA is associated with protein in the oriT region. When the mixture was heated before the addition of SDS/EDTA the plasmid remained superhelical and the DNA was free of protein as unaltered mobility of the short PvuII fragment showed. The results were the same when the enzyme/DNA mixture was incubated in the presence of 1 mM ATP and then heated in the absence of SDS/EDTA (the PvuII product is not shown). On the other hand, when
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Fig. 5. Relaxed pEMBL8-oriT as seen in the electron microscope. (A) The relaxed plasmid (proteinase K was omitted) was linearized with EcoRI or (B) HindIll. The bar represents 0.5 Am. 2691
U.Reygers et al.
the heat treatment was carried out in the presence of SDS/EDTA, relaxed plasmid was the main product as in the experiment of Figure 2. The mobility of the short PvuII fragment was lowered except when the DNA was treated with proteinase K. The complex between helicase I and its superhelical substrate thus resists treatment with SDS/EDTA at room temperature. Nicking occurs when this complex is heated in the presence of SDS/EDTA and not when it is heated in the absence of SDS/EDTA. The enzyme then dissociates from the DNA leaving the plasmid superhelical. Since partially relaxed molecules are not detectable it appears that helicase I does not nick and seal under our conditions, i.e. does not act as a DNA topoisomerase. Heating in the presence of either SDS or EDTA led to essentially the same results as heating in the presence of SDS plus EDTA. Relaxation of approximately half of the plasmid molecules was also achieved by exposing the helicase I/pEMBL8oriT mixture to proteinase K (in the absence of SDS/EDTA).
Discussion Helicase I is associated with oriT-nicking activity as these experiments show. Using dideoxy sequencing techniques we find a major nick site after nucleotide 138 of the F DNA strand transferred during conjugation (in the notation specified) and a minor nick site after nucleotide 137 where, according to the sequencing studies of Thompson et al. (1989), ornT-nicking occurs in vivo (Thompson et al., 1989). Since the in vivo product was treated with SDS, a possible explanation of the discrepancy in the results is that protein attached to the 5' nick terminus prevented the sequencing enzyme from copying the 5' terminal nucleotide, an effect
less pronounced under our conditions. Evidence for a 5' terminal protein complex comes from the negative result of the end-labeling experiment in Figure 6c and the results in Figure 6a. The HindlIl fragment carrying the 5' nick terminus migrates as a diffuse band in a denaturing gel suggesting that the material in this band is not homogeneous (Figure 6a). The authentic nick site is thus probably after
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Fig. 6. Mapping of onT and characterization of the 5' nick terminus. (a) Restriction-mapping. The nicked plasmid linearized with HindlIl, the 3' ends were labeled with 32p, and the DNA was electrophoresed in a denaturing 6% polyacrylamide gel (lane 1). pBR322-HpaII fragments used as markers (lane 2). (b) Dideoxy sequencing reactions. The (+) strand primer was used (lanes marked +) the (-) strand primer (lanes marked -) together with nicked or unnicked pEMBL8-oriT. Filled triangles refer to lanes 1 -4 and indicate the positions of intense bands in all four lanes. Open triangles (lanes 9- 16) indicate the corresponding positions in the opposite strand (the sequence of the interrupted strand is 5'-GGGTGTGGTG-3' between the positions 143 and 134). (c) The 5' nick terminus is inaccessible to labeling with 32p. The nicked plasmid cleaved with Sall, alkali-denatured, and after transfer of 32p label to the 5' ends, electrophoresed in a DNA sequencing gel (lane 1). Lane 2 shows X DNA HindlIl fragments. was
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Endonuclease activity of helicase I
nucleotide 138 of the transferred F DNA strand. Helicase I requires superhelical DNA as the endonuclease substrate as do other site-specific DNA-nicking enzymes [for instance the gene A protein of phage OX 174 (van der Ende et al. 1981) and the gene 2 protein of phage fd (Meyer and Geider, 1979)]. Helicase I does not even bind to an oriTcontaining DNA fragment as previous studies showed (Lahue and Matson, 1990) suggesting that local unwinding of the DNA, supported by negative superhelicity of the duplex, is required for the formation of a stable complex with oriT. Nicking occurs when this complex is subjected to rigorous protein-denaturing treatment, a reaction also observed with a type I DNA topoisomerase (Liu and Wang, 1979). An auxiliary function, probably that of TraY, is therefore needed to incise F plasmid for the conjugal transfer of DNA. TraY, a 17 kDa polypeptide, binds to DNA in the oriT region (Lahue and Matson, 1990) and, together with Tral (TraZ), is needed for oriT-nicking in vivo (Everett and Willets, 1980). In addition to its effect on the nicking activity TraY might enable the endonuclease to switch to the DNA unwinding function. Since helicase I unwinds processively in the 5' to 3' direction of the bound DNA strand, and since the interrupted F DNA strand is exported with its 5' end first, the enzyme should move along the strand to be transferred leaving its displaced 5' end free. Alternatively, the nicking enzyme might bind to the 5' strand terminus and, with the latter, enter the recipient cell where the complex between enzyme and 5' strand terminus might later serve to recircularize the transferred F DNA strand. The DNA unwinding function would then be left to other helicase I molecules, possibly such assembled prior to nicking near oriT. Helicase I binds cooperatively to single-stranded DNA
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(Wessel et al., 1990). It might also do so in a reaction involving local DNA denaturation. Functions as discussed here have been observed with other site specific DNA-binding enzymes. The OX174 gene A protein (van der Ende et al., 1981) and thefd gene 2 protein (Meyer and Geider, 1979) are both capable of ligating the specifically nicked DNA strand. The large tumour antigen of SV40 virus, a replicative helicase, initiates unwinding by binding to a continuous DNA double strand (Dodson et al., 1987; Scheffner et al., 1989). Finally, the primase of an I-like plasmid is an example of a protein that is quite likely to be exchanged during conjugation between the bacteria (Lanka et al., 1979). As the results obtained with helicase I de129 show, a functional amino-terminal part of the TraI polypeptide is needed for the oriT-nicking function although not for the strand-separating function. The bifunctional character of helicase I further explains why the tral products of the related plasmids F and R100 are not interchangeable (Willets and Maule, 1979). These plasmids differ at two positions of their oriT sequences (Finlay et al., 1986).
Materials and methods Enzymes and plasmids Helicase I and helicase I de129 were the preparations electrophoretically >95% homogeneous described previously (Benz and Muller, 1990). Calf thymus DNA topoisomerase I and SSB were kind gifts from H.P.Vosberg and K.Geider, respectively (this institute). Other enzymes were from Boehringer-Mannheim (FRG) except for Sequenase Version 2.0 (United States Biochemical Corporation. OH, USA). The plasmid pBE274 (kindly provided by M.Achtman, Max-Planck-Institut fur Genetik, Berlin) is pKTO43, a pBR322 derivative, with a 1.08 kb F DNA BglII fragment, containing orMT, cloned into the unique BglII site. pBE274 is efficiently mobilized by F Tra functions (Thompson and Achtman, 1978). pEMBL8-oriT was constructed in this laboratory by subcloning the 1.08 kb Bgll fragment from pBE274 into the unique BamHI site of pEMBL8 (Dente et al., 1983) thereby modifying the BglI and BamHI sites. Plasmids grown in F- recAl cells were purified by banding twice in CsCI -ethidium bromide. To prepare relaxed covalently closed pEMBL8-oriT the plasmid was treated with 1 jg DNA topoisomerase I/3.5 Ag DNA in nicking assay buffer at 35°C for 10 min. The DNA was then extracted with phenol.
DNA nicking assay i
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The standard reaction mixture (20 jlI; scaled up if necessary) contained in buffer (50 mM Tris-HCI, 20 mM KCI, 7 mM MgCI2, 1 mM dithiothreitol, 80 gg/ml bovine serum albumin; pH 7.6) 1.4 tg helicase I and 225 ng pEMBL8-oriT. After incubation at 35°C for 12 min, 2 /1l SDS/EDTA (5% SDS, 0.125 M Na3-EDTA, 50 % (v/v) glycerol) and 1 proteinase K were added. The mixture was heated at 45°C for 30 min and, after this, either applied to an agarose gel [electrophoresed in TAE buffer (Maniatis et al., 1982) at I V/cm or, in the experiment of Figure 7b, 5 V/cm] or, for further analysis of the DNA, extracted with phenol. Gels were stained with I tg/ml ethidium bromide.
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(a)
Characterization of the nicked plasmid
Fig. 7. Nicking is triggered by heat treatment of the helicase I/pEMBL8-oriT complex in the presence of SDS/EDTA. (a) The nicking assay mixtures were either heated or not heated as indicated below. SDS/EDTA was added before or after the heat treatment and, in addition, to the non-heated mixture. Electrophoresis was in a 1 % agarose gel. Lane 1, reaction without helicase I; lane 2, non-heated mixture; lane 3, mixture heated in the absence of SDS/EDTA; lane 4, mixture heated in the presence of SDS/EDTA; lane 5, 1 mM ATP was present and the mixture was heated in the absence of SDS/EDTA. (b) DNA gel mobility shift assay. The experiments were carried out as in (a). The helicase I-treated DNA was spin-filtered to remove SDS and EDTA and then cleaved with PvuII. Lane 1, reaction without helicase I; lane 2, non-heated mixture; lane 3, mixture heated in the absence of SDS/EDTA; lane 4, mixture heated in the presence of SDS/EDTA; lane 5, the product shown in lane 4 was treated with proteinase K.
Venom phosphodiesterase (from Crotalus durissimus) and restriction endonuclease were sequentially applied as described by Kowalski and Sanford (1982). HindU-linearized pEMBL8-onT was labeled at the recessed 3' ends by a fill-in reaction using DNA polymerase I-large fragment together with [a-32P]dATP and three non-labeled dNTPs (Maxam and Gilbert, 1980). The 5' ends of alkali-denatured DNA were labeled by treatment with calf intestine alkaline phosphatase and T4 polynucleotide kinase in the presence of [_y-32P]ATP (Maxam and Gilbert, 1980). Dideoxy sequencing (Sanger et al., 1977) was performed using oligonucleotide primers synthesized with the phosphoramidite method in a DNA synthesizer (Applied Biosystems, model 380B). The primers were then eluted from Sep-Pak Cartridges (Waters, Milford, MA). The (+) strand primer is a 20mer complementary to the positions 55-74 of the transferred F DNA strand, and the (-) strand primer is a 20mer complementary to the positions 216-197 of the opposite strand (Figure 1). Sequenase Version 2.0 was used together with [a-35SIdATP (Amersham Buchler,
2693
U.Reygers et al.
Braunschweig, FRG) as the labeled dNTP. The protocol was that of the manufacturer of Sequenase. Electron microscopy The nicking assay mixture was supplemented with 0.33 vol dimethylsulfoxide to completely dissolve SDS. The DNA was spin-filtered through a 300 ,ul Biogel A-0.5m column (Silhavy et al. 1984) and then treated with restriction enzyme or exonuclease III. Exonuclease IlI-treated DNA was saturated with SSB. The fixed samples were spread with the alkyl-benzyl-dimethylammonium chloride method, rotary-shadowed on the grids with tungsten and then viewed in a Philips EM 400T electron microscope. The techniques have been described previously (Wessel et al., 1990).
Acknowledgements We thank Steffi Haupenthal for the synthesis of oligonucleotide primers and Elke Stein for help with the sequencing gels.
References Abdel-Monem,M., Taucher-Scholz,G. and Klinkert,M.-Q. (1983) Proc. Naitl. Acad. Sci. USA, 80, 4659-4663. Benz,I. and Muller,H. (1990) Eur. J. Biochemn., 189, 267-276. Bradshaw,H.D., Traxler,B.A., Minkley,E.G.Jr., Nester.E.W. and Gordon,M.P. (1990) J. Bactetriol., 172, 4127-4131. Dente,L., Cesarini.G. and Cortese,R. (1983) Nuc-leic Acids Res., 11, 1645- 1655. Dodson,M., Dean.F.B., Bullock,P.. Echols,H. and Hurwitz,J. (1987) Science, 238, 964-967. Everett,R. and Willets,N. (1980) J. Mol. Biol., 136, 129-150. Finlay,B.B., Frost,L.S. and Paranchych,W. (1986) J. Bacteriol., 168, 132- 139. Ippen-lhler,K.A. and Minkley,E.G.Jr. (1986) Anonii. Rev. Geniet., 20, 593 -624. Kowalski,D. and Sanford,J.P. (1982) J. Biol. Clhemi., 257, 7820-7825. Lahue,E.E. and Matson,S.E. (1990) J. Bacteriol., 172, 1385 - 1391. Lanka,E., Scherzinger,E., Gunther,E. and Schuster.H. (1979) Proc. Natl. Acad. Sci. USA, 76, 3632-3636. Liu,L. F. and Wang,J.C. (1979) J. Biol. Chem., 254, 11082 - 11088. Maniatis,T., Fritsch,E. and Sambrook,J. (1982) Molecular Cloning, A Laiboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 150-157. Manning,P.A., Kusecek,B., Morelli,G., Fisseau,C. and Achtman,M. (1982) J. Bacteriol., 150, 76-88. Maxam,A. and Gilbert,W. (1980) Methods E:zovinol, 65, 500-560. Meyer,T. F. and Geider,K. (1 979) J. Biol. Clhem., 254, 12642-1 2646. Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Nail. Acad. Sci. USA, 74, 5463-5467. Scheffner,M., Wessel,R. and Stahl,H. (1989) Nucleic Acids Res., 17, 93-106 Silhavy,T., Berman,M. and Enquist,L. (1984) In Evperimeitts in Genie Fusion. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 203-204. Thompson,R. and Achtman,M. (1978) Mol. Geni. Genet., 165, 295-304. Thompson,R., Taylor,L., Kelly.K., Everett,R. and Willets,N. (1984) EMBO J., 3, 1175-1180. Thompson,T.L.. Centola.M.B. and Deonier,R.C. (1989) J. Mol. Biol., 207, 505 -512. Traxler,B.A. and Minkley,E.G.Jr. (1987) J. Bacteriol., 169, 3251 -3259. Traxler,B.A. and Minkley,E.G.Jr. (1988) J. Mol. Biol., 204, 205-209. van der Ende,A., Langeveld,S.A., Teerstra,R., van Arkel,G.A. and Weisbeck,P.J. (1981) Nucleic Acids Res., 9, 2037-2053. Wessel,R., Muller,H. and Hoffmann-Berling,H. (1990) Eur. J. Biochein.. 189, 277-285. Willets,N. and MauleJ. (1979) Mol. Gen. Geniet, 169, 325-336. Willets,N. and Wilkins,B. (1984) Microbiol. Rev., 48, 24-41.
Received on1 March 18, 1991; revised on Max' 15, 1991
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