Current Genetics (1984) 8 : 333-340
~
~
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© Springer-Verlag 1984
Plasmid cloning and expression of the E. colipolA + gene in S. cerevisiae A. Spanos and S. G. Sedgwick Genetics Division, National Institute for Medical Research, Mill Hill, London NW7 1AA, Great Britain
Summary. The E. coli polA + gene has been subcloned from a specialised k transducing phage onto a low copy number plasmid. Plasmid-encoded DNA polymerase I was synthesised at 2 to 3 times the wild-type E. coli level, and was biochemically indistinguishable from chromosomallyencoded protein. It was able to counteract the radiosensitivity of polAI, polAexl, polAex2 and polA12 mutants, but no complementation of polAl07 mutants occurred, even though the plasmid polA + gene was expressed. S. cerevisiae ars-1 or 2 g replicative sequences were introduced into the polA + plasmid. Transformation of yeast with these constructs increased total DNA polymerase levels 2 - 2 0 times, depending upon assay conditions. The additional activity was discriminated from yeast DNA polymerases by its ability to use low concentrations of substrate, by its resistance to chemical inhibition, and by co-electrophoresis with pure DNA polymerase I and its proteolytic fragments. The polA + gene was expressed in yeast without the aid of yeast promotor sequences. However, deletion of cloned DNA more than 99 base pairs in front of the structural gene prevented expression in yeast but not in E. coli, indicating that the two organisms use different sequences for expression of the plasmid polA ÷ gene. Key words: polA + - DNA polymerase I - Cloning Yeast
Introduction Both E. coli and S. cerevisiae have extensive ranges of genetically well-defined mutations affecting DNA repair and metabolism. In E. coli a number of these genes ~' Offprint requests to: A. Spanos
products are also further extensively characterised at the biochemical and physiological levels. Amongst such well understood proteins is DNA polymerase I, the product of the polA ÷ gene. DNA polymerase is capable of 5 ' - 3 ' DNA polymerisation, and 3 ' - 5 ' and 5 ' - 3 ' exonuclease degradation, and has roles in both DNA replication and excision repair of damaged nucleotides (see Komberg (1980) for a detailed review of these properties). The nucleotide sequence of the polA + gene has also been established (Joyce et al. 1982) and the base changes of several alleles determined (Joyce and Grindley 1983). In contrast to E. coli, most yeast mutations are poorly understood at the molecular level. One way of probing molecular deficiencies in yeast might to be perform complementation analyses after transformation with foreign genes whose activities are established. As a first step in this approach we describe how theE. colipolA ÷ has been transferred to a plasmid and expressed in S. cerevisiae. An initial problem encountered by us and others (Kelley et al. 1977) was the inability to construct high copy number plasmids encoding a wild-type polA ÷ gene, presumably indicating that high levels of DNA polymerase are lethal. It appeared that only inactive (Joyce et al. 1982) or incomplete (Joyce and Grindley 1982) polA gene products could be encoded by plasmids. Extrachromosomal transfer of a functional polA + gene had been achieved, but with bacteriophage k (Kelly et al. 1977; Murray and Kelley 1979), an unsuitable vector for the type of interspecies complementation envisaged here. This communication describes how these problems were circumvented by cloning the polA + gene on plasmids with a low bacterial copy number. The ability of the plasmids to produce active DNA polymerase I and complement several E. coli polA mutations was first characterised. We then describe how such plasmids were converted into shuttle vectors capable of transforming S. cerevisiae and producing active DNA polymerase I therein.
A. Spanos and S. G. Sedgwick: E. coli DNA polymerase I in yeast
334 Table 1. E. coli strains
Strain
Relevant genotype
Ref/source
JGl13 JGl12 R $5052 RS5068 MM383 KMBL1789 MH2001
polA + thyA rpoL thiA polAI thyA rpoL thiA polAex 1 polAex2 poIA12 poIAl07 F + pMH101 deoC rpo +
Gross and Gross (1969) Gross and Gross (1969) Kourad and Lehman (1974) Lehman and Chien (1973) Monk and Kirtross (1972) Glickman et al. (1973) This work
Materials a n d m e t h o d s
Phages, plasmids, bacteria and yeast, hbio 11, which is red gam int (Manly et al. 1969), and hpapa orginated from Dr. R. Devoret's collection, h964 is c1857 nin5 Qam73 Sam7 polA + (Murray and Kelley 1979) and was very kindly provided by Dr. N. Murray. Plasmid pHSG415 (Timmis 1981) was from Dr. P. Strike and confers resistance to ampicillin, chloramphenicol and kanamycin. YEpl3 (Broach et al. 1979) and YRp7 (Struhl et al. 1979) were from Dr. G. Banks. E. coli strains are described in Table 1. Saccharomyces cerevisiae strain M1-2B originated from Dr. R. Davis and is ~ trp-289 ura3-52 gal2. A daughter strain C1-1 isura3-52 leu2 and was constructed by Charles White. Culture conditions. General bacterial and phage methodology were as described previously (Sedgwick and Yarranton 1982). Bacteria were cured of plasmids according to Miller (1972). Yeast was cultured in YPD (1% yeast extract, 2% Difco peptone and 2% glucose) or in YNB selective minimal medium (0.67% Difco yeast nitrogen base lacking amino acids and 2% glucose) supplemented appropriately with 20 #g/ml uracil, 20 #g/ml tryptophan or 30 #g/ml leucine. Tests for plasmid-encoded fl-lactamase on starch plates were done according to ChevaUier and Aigle (1978). TnlO00 mutagenesis. Mutagenesis of pMH10t with Tnl000 (formely known as h6) (Guyer 1978) was performed by mating MH2001 with J G l l 2 . Exconjugants which received pMH101 transposed with Tn!000 were selected on L agar containing 15 #g/ml chloramphenicol and 100 #g/mi streptomycin. DNA methodology. Plasmid DNA was prepared from cultures of E. coli grown overnight without amplification using the acetate precipitation method (Ish-Horowicz and Burke 1981). To deal with the larger ratio of cellular material to plasmid DNA, the volumes of reagents 1, 2 and 3 used per litre of culture was 100, 200 and 150 mls. ?~polA+ was produced by thermal induction of ?~polA+ lysogenic bacteria and DNA was prepared as described by Maniatis et al. (1982). Restriction endonuclease digestion of DNA, ligation, electrophoretic analysis and bacterial transformation were as previously described (Sedgwick and Yarranton 1982). Fragments of restricted DNA were purified by their affinity with glass (Vogelstein and Gillespie 1979). Yeast was transformed using the method of Hinnen et ai. (1978). Colony polymerase assays. Plate-grown bacteria were replicated onto discs of Whatman DE-81 paper. The discs were then gently moistened with 1 mg/ml lysozyme, 20 mM Tris-HC1 pH 7.5, and 1 mM EDTA and incubated 1 h at 37 ° to lyse the cells. The discs
were immersed a further hour at 37 ° in a DNA polymerase assay mixture consisting of 0.05 M Tris-HC1 pH 8.0, 7 mM MgC12, 10 mM N-ethylmaleamide (NEM), 50 uM of each dCTP, dGTP, dATP and 0.1 #M [c~-32p] dTTP (10 #Ci/nmol). After extensive washing in 5% TCA-I% Na4P207; and rinsing in 96% ethanol, the discs were dried and incorporation of [32p] dTMP into DNA by the polymerase present in the lysed colonies visualised by autoradiography. Pre-soaking the filter with activated calf thymus DNA or including the DNA template in the assay mixture was found unnecessary because the endogenous DNA present in the lysed cells was retained on the paper and served as a sufficient template.
Polymerase assays o f cell extracts. Crude extracts of E. coli were prepared by passage of cells through a French pressure cell in 0.05 M Tris-HC1, pH 7.5, 10% glycerol, 1 mM EDTA (Spanos et al. 1981). Yeast post-mitochondrial extracts were similarly prepared, except that 5 mM /~-mercaptoethanol was included. For the investigation of PCMB or NEM inhibition, fl-mercaptoethanol was omitted from the breakage buffer or, alternatively, dialysed out from the extract. The effect of p-chloromercurobenzoate (PCMB 500 #g/ml, NEM 1 mg/ml) and aphidicolin (100 gg/ml) was investigated by including the inhibitor in the assay mixture. Enzyme assays for DNA polymerase I activity (Uyemura and Lehman 1976) contained 10 mM NEM with activated calf thymus DNA as template. Incubation was for 20 min at 37 °C unless indicated otherwise. DNA polymerase activity in yeast extracts was also measured using the same template-primer (Wintersberger and Wintersberger 1970). Aphidicolin was a kind gift of Dr. A. H. Todd of I.C.I. Pharmaceuticals Division. Estimation of protein in extracts was determined as described by Bradford (1976). Sephadex G-200 chromatography Yeast extracts were fractionated on a Sephadex G-200 column (Chang 1977). A sample of each fraction was assayed for activity using the yeast polymerase assay (Wintersberger and Wintersberger 1970) in the absence or presence of aphidicolin. Half of each active fraction (3 ml) was then dialysed to remove ~-mercaptoethanol and then assayed in the presence or absence of PCMB (500/~g/ml) in the polymerase assay mixture. SDS-polyacrylamide gel electrophoresis. Investigation of [35S] methionine labelled E. eoli cell extracts and in situ polymerase assays of both E. coli and yeast extracts, in 7.5% polyacrylamide gels, was performed as described previously (Spanos et al. 1981 ; Spanos and Hiibscher 1983), except that fl-mercaptoethanol was omitted and replaced by 500/am p-chloromercurobenzoate.
A. Spanos and S. G. Sedgwick: E. coli DNA polymerase I in yeast
335 UV DOSE Jim 2
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Fig. 2a, b. Radio-protection of polAI E. coli by pMH101. The UV-survivalof polAI E. coli JG 112, (a), and polA +E. coli JG 113, b, was determined before, - e - , and after transformation with pMH101, - e - ; PHSG415, -_A_; and pMH191, - x - . b also shows polAI E. coli containing pMH101 polA::TnlO00, D; or pMH101 b/a::Tnl000, •
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Fig. 1. A map of polA + plasmid pMH101 and its derivatives. Vector pHSG415 DNA, - - ; 5.1 kbp HindllI fragment encoding the polA+ gene, V---l,and flanking sequences, l . Sites of transposition by Tnl000 are shown by, Tn -~. Broken lines indicate sites of in vitro deletion, or insertion of either yeast 2 # or ars-1 segments (see text). Abbreviations: B, BamH1, Bg, BgllI, P, Pstl ; R, EcoR1
Results Cloning and expression o f the polA ÷ gene in E. coil
A 5.1 kbp HindlII fragment of XpolA + has been previously identified as a sequence encoding DNA polymerase I (Kelley et al. 1977). This fragment was purified and several unsuccessful attempts were made to clone it into the high copy number vector, pBR322. However, it was possible to introduce it into the unique HindlII site in the kanamycin-resistance gene of a low copy number plasmid, pHSG415. Initial screening in polAI E. coli JG112 was for kanamycin-sensitive derivatives of pHSG415, which upon purification and digestion with HindlII, were seen to contain a 5.1 kbp insert. In all isolates the insertion was orientated in the transcription direction shown in Fig. 1, which, from the work of Joyce et al. (1982), is in the opposite direction to that in the interrupted kanamycin-resistance gene. A representative isolate was designated pMH101 and was tested for corn-
plementation of its poIAI host. Preliminary half-plate UV-irradiation tests showed that pMH101 restored radiation-resistance to JG 112. Quantitation of this effect shows that resistance is at wild-type levels (Fig. 2). MMSresistance was similarly restored to polA[ hosts by pMH101 (data not shown). Radio-resistance was not found after transformation of J G I I 2 with pHSG415 (Fig. 2a) and was lost when J G l l 2 pMH101 was cured of plasmid with acridine orange (data not shown). Furthermore, insertion of Tnl000 into the centre of the cloned insert ofpMH101 (Fig. 1) eliminated radioprotection (Fig. 2b), whereas transpositional mutagenesis of the b/a gene (Fig. 1) did not (Fig. 2b). These results show that it was the 5.1 kbp fragment cloned into pHSG415 which conferred radio-resistance to polAI bacteria. Preliminary assays for DNA polymerase activity were performed with a replica plating technique where [32p] dTMP incorporation in colonies, replicated and lysed on DEAE paper discs, was visualised by autoradiography. All pMH101 transformants of JG112 gave a positive result in this assay (Fig. 3). Subsequent quantitative assays confirmed that introducing pMH101 into polAI or poIA + bacteria caused a 2- to 3-fold increase in DNA polymerase activity (Table 2). No increases were found after transformation with vector pHSG415 (Table 2), or after transformation with pMH101 polA::TnlO00 (Table 3). The reduced levels ofpolymerase activity directed by pMH101 bla::Tn 1000 (Table 3) might be caused by the plasmid's
A. Spanos and S. G. Sedgwick: E. coli DNA polymerase I in yeast
336
Table 3. Relative DNA polymerase I activity in extracts ofpolAI E. coli JG112 containing the plasmid indicated
Fig. 3. Replica test for pMH101-encoded DNA polymerase I. The left panel shows an agar plate with streaks of polAI E. coli JGl12 before and after transformation with pMH101. The right panel is an autoradiogram of a DE81 paper disc replicated from this plate and assayed for DNA polymerase activity, as described in Materials and methods
Table 2. Relative DNA polymerase I activity in pMH101 and pHSG415 transformants of wild-type and polA-defective E. coli Strain
Percentage of wild-type activity Plasmid
polA+ polAI polA12a polAexl polAex2 polA107
pHSG415
pMH101
98
~
~
~
© Springer-Verlag 1984
Plasmid cloning and expression of the E. colipolA + gene in S. cerevisiae A. Spanos and S. G. Sedgwick Genetics Division, National Institute for Medical Research, Mill Hill, London NW7 1AA, Great Britain
Summary. The E. coli polA + gene has been subcloned from a specialised k transducing phage onto a low copy number plasmid. Plasmid-encoded DNA polymerase I was synthesised at 2 to 3 times the wild-type E. coli level, and was biochemically indistinguishable from chromosomallyencoded protein. It was able to counteract the radiosensitivity of polAI, polAexl, polAex2 and polA12 mutants, but no complementation of polAl07 mutants occurred, even though the plasmid polA + gene was expressed. S. cerevisiae ars-1 or 2 g replicative sequences were introduced into the polA + plasmid. Transformation of yeast with these constructs increased total DNA polymerase levels 2 - 2 0 times, depending upon assay conditions. The additional activity was discriminated from yeast DNA polymerases by its ability to use low concentrations of substrate, by its resistance to chemical inhibition, and by co-electrophoresis with pure DNA polymerase I and its proteolytic fragments. The polA + gene was expressed in yeast without the aid of yeast promotor sequences. However, deletion of cloned DNA more than 99 base pairs in front of the structural gene prevented expression in yeast but not in E. coli, indicating that the two organisms use different sequences for expression of the plasmid polA ÷ gene. Key words: polA + - DNA polymerase I - Cloning Yeast
Introduction Both E. coli and S. cerevisiae have extensive ranges of genetically well-defined mutations affecting DNA repair and metabolism. In E. coli a number of these genes ~' Offprint requests to: A. Spanos
products are also further extensively characterised at the biochemical and physiological levels. Amongst such well understood proteins is DNA polymerase I, the product of the polA ÷ gene. DNA polymerase is capable of 5 ' - 3 ' DNA polymerisation, and 3 ' - 5 ' and 5 ' - 3 ' exonuclease degradation, and has roles in both DNA replication and excision repair of damaged nucleotides (see Komberg (1980) for a detailed review of these properties). The nucleotide sequence of the polA + gene has also been established (Joyce et al. 1982) and the base changes of several alleles determined (Joyce and Grindley 1983). In contrast to E. coli, most yeast mutations are poorly understood at the molecular level. One way of probing molecular deficiencies in yeast might to be perform complementation analyses after transformation with foreign genes whose activities are established. As a first step in this approach we describe how theE. colipolA ÷ has been transferred to a plasmid and expressed in S. cerevisiae. An initial problem encountered by us and others (Kelley et al. 1977) was the inability to construct high copy number plasmids encoding a wild-type polA ÷ gene, presumably indicating that high levels of DNA polymerase are lethal. It appeared that only inactive (Joyce et al. 1982) or incomplete (Joyce and Grindley 1982) polA gene products could be encoded by plasmids. Extrachromosomal transfer of a functional polA + gene had been achieved, but with bacteriophage k (Kelly et al. 1977; Murray and Kelley 1979), an unsuitable vector for the type of interspecies complementation envisaged here. This communication describes how these problems were circumvented by cloning the polA + gene on plasmids with a low bacterial copy number. The ability of the plasmids to produce active DNA polymerase I and complement several E. coli polA mutations was first characterised. We then describe how such plasmids were converted into shuttle vectors capable of transforming S. cerevisiae and producing active DNA polymerase I therein.
A. Spanos and S. G. Sedgwick: E. coli DNA polymerase I in yeast
334 Table 1. E. coli strains
Strain
Relevant genotype
Ref/source
JGl13 JGl12 R $5052 RS5068 MM383 KMBL1789 MH2001
polA + thyA rpoL thiA polAI thyA rpoL thiA polAex 1 polAex2 poIA12 poIAl07 F + pMH101 deoC rpo +
Gross and Gross (1969) Gross and Gross (1969) Kourad and Lehman (1974) Lehman and Chien (1973) Monk and Kirtross (1972) Glickman et al. (1973) This work
Materials a n d m e t h o d s
Phages, plasmids, bacteria and yeast, hbio 11, which is red gam int (Manly et al. 1969), and hpapa orginated from Dr. R. Devoret's collection, h964 is c1857 nin5 Qam73 Sam7 polA + (Murray and Kelley 1979) and was very kindly provided by Dr. N. Murray. Plasmid pHSG415 (Timmis 1981) was from Dr. P. Strike and confers resistance to ampicillin, chloramphenicol and kanamycin. YEpl3 (Broach et al. 1979) and YRp7 (Struhl et al. 1979) were from Dr. G. Banks. E. coli strains are described in Table 1. Saccharomyces cerevisiae strain M1-2B originated from Dr. R. Davis and is ~ trp-289 ura3-52 gal2. A daughter strain C1-1 isura3-52 leu2 and was constructed by Charles White. Culture conditions. General bacterial and phage methodology were as described previously (Sedgwick and Yarranton 1982). Bacteria were cured of plasmids according to Miller (1972). Yeast was cultured in YPD (1% yeast extract, 2% Difco peptone and 2% glucose) or in YNB selective minimal medium (0.67% Difco yeast nitrogen base lacking amino acids and 2% glucose) supplemented appropriately with 20 #g/ml uracil, 20 #g/ml tryptophan or 30 #g/ml leucine. Tests for plasmid-encoded fl-lactamase on starch plates were done according to ChevaUier and Aigle (1978). TnlO00 mutagenesis. Mutagenesis of pMH10t with Tnl000 (formely known as h6) (Guyer 1978) was performed by mating MH2001 with J G l l 2 . Exconjugants which received pMH101 transposed with Tn!000 were selected on L agar containing 15 #g/ml chloramphenicol and 100 #g/mi streptomycin. DNA methodology. Plasmid DNA was prepared from cultures of E. coli grown overnight without amplification using the acetate precipitation method (Ish-Horowicz and Burke 1981). To deal with the larger ratio of cellular material to plasmid DNA, the volumes of reagents 1, 2 and 3 used per litre of culture was 100, 200 and 150 mls. ?~polA+ was produced by thermal induction of ?~polA+ lysogenic bacteria and DNA was prepared as described by Maniatis et al. (1982). Restriction endonuclease digestion of DNA, ligation, electrophoretic analysis and bacterial transformation were as previously described (Sedgwick and Yarranton 1982). Fragments of restricted DNA were purified by their affinity with glass (Vogelstein and Gillespie 1979). Yeast was transformed using the method of Hinnen et ai. (1978). Colony polymerase assays. Plate-grown bacteria were replicated onto discs of Whatman DE-81 paper. The discs were then gently moistened with 1 mg/ml lysozyme, 20 mM Tris-HC1 pH 7.5, and 1 mM EDTA and incubated 1 h at 37 ° to lyse the cells. The discs
were immersed a further hour at 37 ° in a DNA polymerase assay mixture consisting of 0.05 M Tris-HC1 pH 8.0, 7 mM MgC12, 10 mM N-ethylmaleamide (NEM), 50 uM of each dCTP, dGTP, dATP and 0.1 #M [c~-32p] dTTP (10 #Ci/nmol). After extensive washing in 5% TCA-I% Na4P207; and rinsing in 96% ethanol, the discs were dried and incorporation of [32p] dTMP into DNA by the polymerase present in the lysed colonies visualised by autoradiography. Pre-soaking the filter with activated calf thymus DNA or including the DNA template in the assay mixture was found unnecessary because the endogenous DNA present in the lysed cells was retained on the paper and served as a sufficient template.
Polymerase assays o f cell extracts. Crude extracts of E. coli were prepared by passage of cells through a French pressure cell in 0.05 M Tris-HC1, pH 7.5, 10% glycerol, 1 mM EDTA (Spanos et al. 1981). Yeast post-mitochondrial extracts were similarly prepared, except that 5 mM /~-mercaptoethanol was included. For the investigation of PCMB or NEM inhibition, fl-mercaptoethanol was omitted from the breakage buffer or, alternatively, dialysed out from the extract. The effect of p-chloromercurobenzoate (PCMB 500 #g/ml, NEM 1 mg/ml) and aphidicolin (100 gg/ml) was investigated by including the inhibitor in the assay mixture. Enzyme assays for DNA polymerase I activity (Uyemura and Lehman 1976) contained 10 mM NEM with activated calf thymus DNA as template. Incubation was for 20 min at 37 °C unless indicated otherwise. DNA polymerase activity in yeast extracts was also measured using the same template-primer (Wintersberger and Wintersberger 1970). Aphidicolin was a kind gift of Dr. A. H. Todd of I.C.I. Pharmaceuticals Division. Estimation of protein in extracts was determined as described by Bradford (1976). Sephadex G-200 chromatography Yeast extracts were fractionated on a Sephadex G-200 column (Chang 1977). A sample of each fraction was assayed for activity using the yeast polymerase assay (Wintersberger and Wintersberger 1970) in the absence or presence of aphidicolin. Half of each active fraction (3 ml) was then dialysed to remove ~-mercaptoethanol and then assayed in the presence or absence of PCMB (500/~g/ml) in the polymerase assay mixture. SDS-polyacrylamide gel electrophoresis. Investigation of [35S] methionine labelled E. eoli cell extracts and in situ polymerase assays of both E. coli and yeast extracts, in 7.5% polyacrylamide gels, was performed as described previously (Spanos et al. 1981 ; Spanos and Hiibscher 1983), except that fl-mercaptoethanol was omitted and replaced by 500/am p-chloromercurobenzoate.
A. Spanos and S. G. Sedgwick: E. coli DNA polymerase I in yeast
335 UV DOSE Jim 2
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20
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10
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10 C - t
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H
z 101
.x2
163
Tn
1~4
H
IB J
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I 1
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Fig. 2a, b. Radio-protection of polAI E. coli by pMH101. The UV-survivalof polAI E. coli JG 112, (a), and polA +E. coli JG 113, b, was determined before, - e - , and after transformation with pMH101, - e - ; PHSG415, -_A_; and pMH191, - x - . b also shows polAI E. coli containing pMH101 polA::TnlO00, D; or pMH101 b/a::Tnl000, •
t
8
2~-L~ ~ ~
Fig. 1. A map of polA + plasmid pMH101 and its derivatives. Vector pHSG415 DNA, - - ; 5.1 kbp HindllI fragment encoding the polA+ gene, V---l,and flanking sequences, l . Sites of transposition by Tnl000 are shown by, Tn -~. Broken lines indicate sites of in vitro deletion, or insertion of either yeast 2 # or ars-1 segments (see text). Abbreviations: B, BamH1, Bg, BgllI, P, Pstl ; R, EcoR1
Results Cloning and expression o f the polA ÷ gene in E. coil
A 5.1 kbp HindlII fragment of XpolA + has been previously identified as a sequence encoding DNA polymerase I (Kelley et al. 1977). This fragment was purified and several unsuccessful attempts were made to clone it into the high copy number vector, pBR322. However, it was possible to introduce it into the unique HindlII site in the kanamycin-resistance gene of a low copy number plasmid, pHSG415. Initial screening in polAI E. coli JG112 was for kanamycin-sensitive derivatives of pHSG415, which upon purification and digestion with HindlII, were seen to contain a 5.1 kbp insert. In all isolates the insertion was orientated in the transcription direction shown in Fig. 1, which, from the work of Joyce et al. (1982), is in the opposite direction to that in the interrupted kanamycin-resistance gene. A representative isolate was designated pMH101 and was tested for corn-
plementation of its poIAI host. Preliminary half-plate UV-irradiation tests showed that pMH101 restored radiation-resistance to JG 112. Quantitation of this effect shows that resistance is at wild-type levels (Fig. 2). MMSresistance was similarly restored to polA[ hosts by pMH101 (data not shown). Radio-resistance was not found after transformation of J G I I 2 with pHSG415 (Fig. 2a) and was lost when J G l l 2 pMH101 was cured of plasmid with acridine orange (data not shown). Furthermore, insertion of Tnl000 into the centre of the cloned insert ofpMH101 (Fig. 1) eliminated radioprotection (Fig. 2b), whereas transpositional mutagenesis of the b/a gene (Fig. 1) did not (Fig. 2b). These results show that it was the 5.1 kbp fragment cloned into pHSG415 which conferred radio-resistance to polAI bacteria. Preliminary assays for DNA polymerase activity were performed with a replica plating technique where [32p] dTMP incorporation in colonies, replicated and lysed on DEAE paper discs, was visualised by autoradiography. All pMH101 transformants of JG112 gave a positive result in this assay (Fig. 3). Subsequent quantitative assays confirmed that introducing pMH101 into polAI or poIA + bacteria caused a 2- to 3-fold increase in DNA polymerase activity (Table 2). No increases were found after transformation with vector pHSG415 (Table 2), or after transformation with pMH101 polA::TnlO00 (Table 3). The reduced levels ofpolymerase activity directed by pMH101 bla::Tn 1000 (Table 3) might be caused by the plasmid's
A. Spanos and S. G. Sedgwick: E. coli DNA polymerase I in yeast
336
Table 3. Relative DNA polymerase I activity in extracts ofpolAI E. coli JG112 containing the plasmid indicated
Fig. 3. Replica test for pMH101-encoded DNA polymerase I. The left panel shows an agar plate with streaks of polAI E. coli JGl12 before and after transformation with pMH101. The right panel is an autoradiogram of a DE81 paper disc replicated from this plate and assayed for DNA polymerase activity, as described in Materials and methods
Table 2. Relative DNA polymerase I activity in pMH101 and pHSG415 transformants of wild-type and polA-defective E. coli Strain
Percentage of wild-type activity Plasmid
polA+ polAI polA12a polAexl polAex2 polA107
pHSG415
pMH101
98
