A Carboxyl-terminal Cys2/His2-type Zinc-finger Motif in DNA Primase Influences DNA Content in Synechococcus PCC 7942*

Amanda J. BirdDagger §, Jennifer S. Turner-CavetDagger , Jeremy H. Lakey, and Nigel J. Robinson

From the Department of Biochemistry and Genetics, The Medical School, University of Newcastle, Newcastle NE2 4HH, United Kingdom

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The DNA primase gene, dnaG, has been isolated from the cyanobacterium Synechococcus PCC 7942. It is not part of a macromolecular synthesis operon but is co-transcribed with pheT and located adjacent to the metallothionein divergon, smt. At the carboxyl terminus of this DnaG is a Cys2/His2 zinc-finger motif. The carboxyl-terminal 91 residues bound 65Zn and 0.95 g atom of Zn2+ mol-1 were detected with 4-(2-pyridylazo)resorcinol. Following exposure to Cd2+, 0.95 g atom of Cd2+ was displaced by 2 equivalents of p-(hydroxymercuri) phenylsulfonate mol-1, while only 0.03 g atom of Cd2+ was displaced mol-1 polypeptide missing the carboxyl-terminal (residue 592 onward) zinc-finger motif. Zn2+ caused an increase in intensity, and a reduction in wavelength, of Trp fluorescence at the tip of the predicted zinc-finger, while EDTA caused the converse. Cells containing a single chromosomal codon substitution (C597S), altering the zinc-finger, were generated by exploiting Zn2+-sensitive smt mutants and the proximity of dnaG to smt. Cells in which smt and dnaG(C597S) had integrated into the chromosome were selected via restored Zn2+ tolerance. Synechococcus PCC 7942 and its dnaG(C597S) mutant grew at equivalent rates, but the latter had a reduced number of chromosomes.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Chromosome replication involves multiple, sequential, protein-protein and protein-DNA interactions. In Escherichia coli progress has been made in identifying what regulates the timing and duration of some of these interactions (1). In Synechococcus PCC 7942 there are differences in the mechanisms that regulate DNA synthesis compared with E. coli (2). A most obvious manifestation of modified regulation of DNA synthesis is a greater variability in DNA content with as many as 30 chromosomes in each cyanobacterial cell (3). It was hypothesized that in this organism a phenotype associated with modified rates of DNA replication would be modified DNA content.

DNA primase, encoded by dnaG, synthesizes primer RNA required to initiate leading strand (4) and to repeatedly reinitiate lagging strand (5) DNA synthesis. Each time primase is required it is recruited to the replication fork by protein-protein interaction with the DnaB helicase (6, 7). It is the strength of this interaction that "sets the replication fork clock," this being the principal regulator of the cycle of synthesis of Okazaki fragments (1). The carboxyl terminus of E. coli DNA primase is responsible for the interaction with DnaB helicase (6, 7). Deletions and alanine cluster mutagenesis have established that it is the extreme carboxyl-terminal 8 amino acids of E. coli DNA primase that interact with the DnaB helicase (8). In addition, mutations 12 and 15 residues away from the carboxyl terminus of DNA primase impair chromosome partitioning in E. coli (9), a phenotype which Tougu and Marians (8) have attributed to aberrant interaction with DnaB helicase.

Purified DNA primase from E. coli contains tightly bound Zn2+ with an estimated stoichiometry close to 1:1 (10). Zn2+ binds to a conserved motif which is near to the amino terminus of all known bacterial and bacteriophage DNA primase sequences (11-13). This amino-terminal Zn2+-binding motif is implicated in DNA recognition, although it is not the sole determinant of DNA sequence recognition (14).

Here we report the isolation of the dnaG gene from Synechococcus PCC 7942 which is not located within a normal bacterial MMS1 operon but is adjacent to the MT divergon, smt. The presence of a predicted zinc-finger at the extreme carboxyl terminus of the deduced DNA primase, in addition to the conserved amino-terminal Zn2+-binding motif found in other DNA primases, is of particular interest due to (i) the known roles of such structures in protein-protein (15) as well as protein-RNA and protein-DNA (16) interactions, (ii) the importance of the protein-protein interactions mediated by the equivalent region of E. coli DNA primase (which does not contain a carboxyl-terminal zinc-finger motif), (iii) the fact that such eukaryotic style zinc-fingers have not yet been structurally characterized in prokaryotes and furthermore it has been suggested that by avoiding such structures prokaryotes may have avoided the "hidden cost" of precise Zn2+ homeostasis (17), and (iv) the close proximity of the gene to the cyanobacterial MT gene, smtA, which is known to act as a regulator of the availability of endogenous Zn2+ in Synechococcus PCC 7942 (18). Experiments were therefore performed to test whether or not the carboxyl-terminal region of DNA primase from Synechococcus PCC 7942 does bind Zn2+ and if Zn2+ association induces the formation of the protein structure. These two predictions have been confirmed in vitro using recombinant polypeptides.

The functional significance of the zinc-finger motif was then tested in vivo. There are precedents for the analysis, both in vitro and in vivo, of proteins containing mutant zinc-fingers with impaired Zn2+ binding (19-21). Substitution of a Ser residue for either the second, or fourth, Cys in a Cys4-type zinc-finger within isoleucyl-tRNA synthetase, impaired (but did not abolish) Zn2+ binding in vitro and conferred Zn2+-dependent cell growth in E. coli (19). We have previously generated mutants deficient in smt via homologous recombination-mediated insertion of the chloramphenicol acetyltransferase gene, cat (18). These mutants are hypersensitive to Zn2+, and reintegration of smt into these cells has been used as a selectable marker since it restores normal Zn2+ tolerance (18, 22). It was hypothesized that a mutant 3' end of dnaG could be co-integrated with smt to create cells containing only a single codon substitution within the chromosomal region encoding the zinc-finger motif of DNA primase. It has indeed been possible to specifically, and exclusively, substitute a Ser codon for the second Cys codon within the part of dnaG encoding the zinc-finger motif within the chromosome of Synechococcus PCC 7942. These cells have a reduced DNA content showing that the thiol group on this residue, and hence the Cys2/His2-type zinc-finger motif, influences chromosome replication.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials and Bacterial Strains-- Cyanobacterial strains used were: R2-PIM8, a small plasmid-cured derivative of Synechococcus PCC 7942 (23), referred to herein as "wild-type"; and R2-PIM8(smt), an smt-deficient mutant (18). Strains were cultured as described previously (18) and supplemented with 5 µg of kanamycin ml-1 as appropriate. DNA restriction and modification enzymes were supplied by New England Biolabs, [alpha -32P]dCTP and nylon (Hybond N+) filters were obtained from Amersham International plc., and other reagents were purchased from Sigma.

Cloning and Sequencing of dnaG, Located Adjacent to the smt Divergon-- Plasmid pJSTNR4.1, containing smt- flanking sequences and pSU19 (24), was generated previously by plasmid recovery into E. coli, using DNA from R2-PIM8(smt) (18). Restriction fragments (~0.5 kb) of pJSTNR4.1 were recovered from agarose gels, subcloned into appropriate sites of pSU19 or pGEM3z (Promega), and sequenced as described previously (25) with reaction products analyzed using an Applied Biosystems 370A DNA sequence analyzer. For each insert, one strand was sequenced at least twice, and the complementary strand at least once.

Production and Purification of a Recombinant Carboxyl-terminal Region of DnaG, p10.5, and a Truncated Mutant, p7.5-- Plasmid pJSTNR4.1 was used as template DNA in PCR reactions with 5' primer I (5'-CTTGGATCCGGATGTATCAAGTCGAGCG-3'; Fig. 1) and an M13 forward primer. The PCR amplification product (0.71 kb), which contained the 3' end of dnaG, was ligated to pGEM-T (Promega) prior to subcloning into the BamHI/SmaI site of the glutathione S-transferase gene fusion vector pGEX-3X (Pharmacia Biotech Ltd) to create pGEXAB1.1, and checked by sequence analysis. Recombinant fusion protein was expressed and purified as described previously (26). The carboxyl-terminal 91 amino acids of DNA primase and 3 residues of glutathione S-transferase (p10.5) were released from glutathione-Sepharose-bound glutathione S-transferase by incubation overnight with factor Xa (Pharmacia). A truncated version of p10.5 (p7.5), lacking the carboxyl-terminal 24 amino acids of DNA primase, was generated by converting a CGC codon (Arg592) to a UGA stop codon. Primers 5'-GGTGGAACGACTGTGATGCGAAAAACGTTGC-3' and 5'-GCAACGTTTTTCGCATCACAGTCGTTCCACC-3' were used for site-directed mutagenesis via "Quik-change" (Stratagene) according to the manufacturer's protocols. The identity and purity of all recombinant proteins was confirmed by polyacrylamide gel electrophoresis.


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Fig. 1.   In vivo substitution of codon Cys597 to Ser in dnaG. A 1.8-kb HindIII/SalI fragment of DNA, containing the smt divergon, and a 3.7-kb BamHI (gained from the pGEM3z polylinker)/HindIII fragment, containing the 3' end of pheT and dnaG (see Fig. 2) were ligated into pSK+ to create pABNR2.1. The vector pABNR2.1 thus contains a 5.5-kb contiguous fragment of Synechococcus PCC 7942 DNA including the protein coding regions of smtA (diagonal shading), smtB (dark shading), and dnaG (light shading). This was used as template for site-directed mutagenic PCR to convert codon Cys597 to Ser in dnaG (panel A). The 5.5-kb contiguous genomic fragment was released with SalI and used to transform R2-PIM8(smt) to Zn2+ resistance. Transformation with the genomic fragment should result in homologous recombination, with cat being lost to restore the wild-type genotype, with the exception of the single nucleotide change converting Cys597 to Ser (panel B). The annealing positions of primers are indicated (short vertical lines, panel A).

Metal-binding Studies-- Recombinant p10.5, in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM CaCl2, was incubated at 4 °C for 2 h with ~17 kBq 65Zn before fractionation on Sephadex G-25. Fractions (0.5 ml) were collected, and aliquots (0.1 ml) were analyzed for protein content and radioactivity. An aliquot of the protein was hydrolyzed and analyzed for amino acid composition (Alta Bioscience, University of Birmingham) to allow calibration of colorimetric estimations of p10.5.

The amount of Zn2+ or Cd2+ bound to p10.5 or p7.5, purified from E. coli grown in the presence or absence of permissive concentrations of Zn2+ (200 µM) or Cd2+ (250 µM), was determined immediately upon elution from glutathione-Sepharose by addition of 0.1 mM PAR and incubation for 5 min with aliquots (5 µl) of 0.5 µM PMPS in the range of 0 to 6 equivalents. The metallochromic indicator PAR generates colored chelate compounds with Zn2+ (27) and Cd2+ (28). Metal ion release was monitored by the increase (subtracting values prior to the addition of PMPS) in absorbance at 500 nm (27) and values calibrated by reacting known amounts of Zn2+ or Cd2+ with PAR. In addition, an aliquot of p10.5, isolated from Cd2+ exposed cells, was incubated with a 2-fold molar excess of Cd2+ in vitro and unbound metal removed by gel filtration on Sephadex G-25 prior to determining metal content.

Fluorescence Measurements-- Fluorescence emission spectra were recorded at 20 °C using a SLM 8100 spectrofluorometer operating in the ratio mode with spectral bandwidths of 8 nm for both excitation and emission. Excitation wavelength was set to 280 nm. Intensities were measured between 296 and 430 nm before and after the addition of a 40-fold molar excess of Zn2+ or EDTA to ~50 µg ml-1 p10.5. The Raman scatter contribution was eliminated by subtraction of appropriate blanks.

Insertional Inactivation of dnaG on a Proportion of Chromosomes-- PCR was performed using pJSTNR4.1 as template with primers II (5'-ACTTGCATGCTTCTTGGCGGTGATC-3') and VIII (Fig. 1), and the amplification product (2.6 kb), containing dnaG and part of pheT, was ligated to pGEM-T. PCR was subsequently performed using primer III (5'-CTTGGATCCACGGTGACGACTTAG-3'; Fig. 1) with an M13 forward primer (which anneals within pSU19 sequences of pJSTNR4.1). This amplification product (0.7 kb), containing the 3' end of dnaG and downstream sequences, was ligated to pGEM-T prior to subcloning into the BamHI/SacI site of pSU19 containing neo (a kanamycin-resistance gene). A 2.2-kb SalI/SacI fragment, containing neo and the 3' end of dnaG, was subsequently released and subcloned into pGEM-T containing the initial 2.6-kb fragment. A 0.5-kb HincII fragment containing part of dnaG was then removed to generate pABNR1.1. A 4.3-kb ApaI/SacI fragment of pABNR1.1, containing the resulting 2.1- and 0.7-kb fragments of Synechococcus PCC 7942 genomic DNA separated by neo (1.5 kb), was used to transform Synechococcus PCC 7942 to kanamycin resistance.

DNA Isolation and Southern Analyses-- Genomic DNA was isolated as described previously (18), and Southern analyses were performed using standard protocols (29). Probes used to confirm inactivation of dnaG were: A, a 1.5-kb fragment of DNA containing neo; B, a 0.2-kb PCR product containing a region of dnaG deleted from pABNR1.1 generated using primers IV (5'-CTTCCATGGTCAGGCTAGC-3') and V (5'-GCTGCAAAGCCAAGGTGC-3') (Fig. 1); C, a 0.3-kb BamHI/HindIII fragment released from pABNR1.1 containing retained sequences to the 3' of dnaG; and D, a 0.7-kb PstI/HindIII fragment released from pABNR1.1 containing retained 5'-flanking sequences.

Determination of the DNA Content of Cyanobacterial Strains-- The DNA contents of logarithmically growing cultures were determined. Cells were subcultured at 48-h intervals, to an optical density at 595 nm of 0.03 (~1 × 106 cells ml-1), for a minimum of 2 weeks prior to analyses. DNA was extracted based on a method described by Mann and Carr (3) and quantified by the diphenylamine assay of Burton (30), calibrated with D-deoxyribose standards. Assays were carried out in triplicate, and the entire experiment was repeated on at least three separate occasions with independent cultures.

Scheme to Introduce a Single Nucleotide Change in dnaG within Synechococcus PCC 7942-- A 1.8-kb HindIII/SalI fragment of DNA was released from plasmid pJHNR49 (a product of a size fractionated Synechococcus PCC 7942 genomic library described in Huckle et al. (25)), and a 3.7-kb BamHI/HindIII fragment of DNA was released from plasmid pRCNR01 (a 3.7-kb SalI/HindIII genomic fragment in the vector pGEM3z). The fragments were ligated into the vector pSK+ (Stratagene) to create pABNR2.1 (Fig. 1). A single nucleotide change (C597S) was introduced using the primers 5'-GCAAATGGCGGGAACGTTTTTCGCAGC-3' and 5'-GCTGCGAAAAACGTTCCCGCCATTTGC-3' via Quik-change. However, due to the relatively large size of pABNR2.1 there is a high probability of single strand breaks and hence a low ratio of nascent:template DNA following DNA synthesis in the Quik-change protocol. This will increase the amount of hemimethylated plasmid DNA containing one strand of mutant, nascent DNA, and one strand of methylated template. Hemimethylated DNA is inefficiently digested with DpnI (17, 18), and such plasmids will be reverted to the original sequence by the mismatch repair system in E. coli. Hence products were introduced into a mismatch repair-deficient strain of E. coli, XL mutS (Stratagene), prior to introduction into Epicurian E. coli XL1-Blue (Stratagene). Plasmids containing the single nucleotide change were selected by sequence analysis and designated pABNR2.2. A 5.5-kb SalI fragment of Synechococcus PCC 7942 genomic DNA was released from pABNR2.2 (Fig. 1) and used to transform R2-PIM8(smt) to Zn2+ tolerance. Putative mutants were selected by growth on Allens plates supplemented with 15 µM Zn2+. DNA isolated from two Zn2+-resistant clones was digested with SalI, and integration of the 5.5-kb fragment was confirmed using Southern analysis. The probes used were a 0.8-kb SacII/NcoI fragment of pSU19 containing the cat gene and a 0.2-kb smtA fragment produced using primers VI (5'-CGCGGATCCTCATGACCTCAACA-3') and VII (5'-CCGGAATTCGGATTAGCAGGGAAACAGT-3') (Fig. 1) with pJHNR49 as a template. To confirm co-integration of the dnaG(C597S) mutation, the carboxyl-terminal region of dnaG was amplified using primers VIII (5'-TCAATGCAAATGTTCAAAGG-3') and IX (5'-CATGTATCAAGTCGAGCG-3') (Fig. 1) with genomic DNA as a template, ligated to pGEM-T, and sequenced.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Isolation and Analysis of dnaG Adjacent to the MT Divergon-- Sequences upstream of the MT divergon, smt, have been determined (GenBankTM accession no. X94247). These contain an open reading frame encoding a protein with similarity to known bacterial DNA primases, which has therefore been designated dnaG. Immediately upstream of dnaG is an open reading frame encoding a protein with sequence similarity to PheT, a subunit of phenylalanyl-tRNA synthetase (Fig. 2). Reverse transcriptase-PCR revealed co-transcription of pheT and dnaG in Synechococcus PCC 7942 (data not shown). In E. coli, and many other bacteria (33), dnaG is located within the MMS operon (34, 35). The MMS operon contains the genes rpsU-dnaG-rpoD whose products (S21, DNA primase, sigma 70 subunit of RNA polymerase) are required to initiate protein, DNA, and RNA synthesis. Fig. 2A illustrates the arrangement, and transcription, of genes in the region of dnaG in Synechococcus PCC 7942 and compares this with the more typical gene architecture exemplified by the MMS operon of E. coli (Fig. 2C).


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Fig. 2.   Physical map of genes in the region of dnaG. Panel A, the pheT and dnaG genes from Synechococcus PCC 7942 are shown (shaded rectangles coincide with open reading frames) upstream of the MT divergon smt. A predicted transcriptional terminator is shown as a circle (standard free energy = -23.7 kJ mol-1, calculated using "FoldRNA"). Panel B, diagrammatic representation of the predicted Cys2/His2-type zinc-finger at the carboxyl terminus of Synechococcus PCC 7942 DNA primase. The Trp residue excited by fluorescence spectroscopy is shown (underlined). Panel C, the MMS operon from E. coli. Panel D, diagrammatic representation of the E. coli DNA primase carboxyl terminus. Residues involved in DNA primase-DnaB interaction (8) are shown (underlined).

The p10.5 Polypeptide Comprising the Carboxyl-terminal Region of DNA Primase Binds Zn2+ and Cd2+-- The carboxyl-terminal region of DNA primase from Synechococcus PCC 7942 is predicted (using the "motifs" program) to form a eukaryotic type Cys2/His2 zinc-finger (Fig. 2B). Such a carboxyl-terminal zinc-finger motif is not found in other primases (Fig. 2D) and therefore recombinant polypeptides were generated to investigate proposed metal-association with this region. Recombinant p10.5 was extracted from E. coli (JM101) cells containing the plasmid pGEXAB1.1. Purified recombinant p10.5 was incubated for 2 h with 65Zn before fractionation on Sephadex G-25. Protein and 65Zn co-eluted in the void volume (fractions 7-11) with no free 65Zn detected in the total volume (fractions 12-25), the elution volume for the latter being identified by subsequent resolution of an aliquot of 65Zn (Fig. 3).


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Fig. 3.   65Zn binding to p10.5 in vitro. Purified p10.5 was incubated for 2 h with 65Zn and then fractionated on Sephadex G-25. Fractions were analyzed for protein (squares) and 65Zn (circles). A second elution profile of 65Zn alone has been superimposed (diamonds).

Upon Cys modification of p10.5 an increase in A500 (Delta A500) was observed to 2 mol equivalents of PMPS (Fig. 4). By using calibration curves, reacting known amounts of Zn2+ with PAR, it was estimated that only 0.04 (± 0.01) g atom of Zn2+ was released mol-1 of p10.5 upon addition of PMPS (Table I). The analysis was repeated using p10.5 extracted from cells supplemented with 200 µM Zn2+. While the p10.5 yield declined, the stoichiometry increased (Fig. 4B), but the latter remained substantially less than 1:1. It was initially hypothesized that this could be due to relative insolubility of the metallated-, compared with apo-, p10.5 since insolubility of a Zn2+-saturated prokaryotic Zn2+-binding protein expressed in E. coli has been reported (36). In previous studies of a metal-saturated prokaryotic protein containing a Cys4-type Zn2+-binding domain, such insolubility problems were overcome by using Cd2+ as an analogue of Zn2+ (36). Cys2/His2 zinc-fingers can bind one equivalent of Cd2+ (37, 38) and there are precedents for the metal ions being introduced either by in vitro exchange and/or following growth of E. coli cells in Cd2+ supplemented medium (39).


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Fig. 4.   Release of metal ions from p10.5 or p7.5 by titration with the mercurial reagent PMPS. p10.5 (squares) or p7.5 (circles) were extracted from cells grown with A, no metal supplement; B, 200 µM Zn2+; C, 250 µM Cd2+; or D, 250 µM Cd2+ and incubated with a 2-fold molar excess of Cd2+ in vitro, were titrated with PMPS, and metal ion release was detected via the increase in A500 following reaction with PAR.

                              
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Table I
Metal contents of p10.5 and p7.5
The total amount of metal displaced from p10.5, or p7.5, by saturating amounts of PMPS was determined for independent protein preparations. Means and standard deviations of the sets of triplicate estimations of metal contents of p10.5 are shown in parentheses.

There was a greater than 4-fold reduction in p10.5 yield following exposure of E. coli to 250 µM Cd2+ (Table I), but titration with PMPS released a greater amount of metal (Fig. 4C). The mean estimated stoichiometry was 0.95 (±0.10), a value close to 1:1 (Table I). An aliquot of the recombinant protein purified from cells exposed to 250 µM Cd2+ was also incubated in vitro with a 2-fold molar excess of Cd2+ and again titrated with PMPS (Fig. 4D) to obtain an estimated stoichiometry of 0.87 (Table I). Equivalent analyses were performed with p7.5, which is truncated and does not contain the zinc-finger motif. Only trace amounts of Cd2+, 0.03 g atom mol-1 (Table I), were displaced from p7.5 (Fig. 4C).

It was noted that an immediate color change (prior to addition of PMPS) observed when p10.5 isolated from Zn2+ exposed cells was incubated with PAR, was not apparent with p10.5 (or p7.5) from Cd2+-exposed cells. Thus an alternative explanation for the low estimated Zn2+ stoichiometry is that a proportion of the Zn2+ (but not Cd2+) associated with the purified p10.5 is accessible to PAR prior to the addition of PMPS. The amount of Zn2+ reacting with PAR prior to the addition of PMPS was therefore quantified. Two separate preparations gave 0.86 and 0.81 g atom Zn2+ mol-1 p10.5, with a further 0.10 and 0.12 g atom, respectively, detected after addition of saturating amounts of PMPS. This suggests Zn2+ stoichiometries of 0.96:1 and 0.93:1 (mean value of 0.95:1).

Incubation with Zn2+ Increases Structural Complexity in the Carboxyl-terminal Region of p10.5-- Use of radiotracer, and PMPS titrations, revealed that Zn2+ (or Cd2+) associate with p10.5, and furthermore this involves the carboxyl-terminal 24 residues and at least two of the residues (the Cys residues) predicted to form a zinc-finger. Protein fluorescence was therefore used to test whether Zn2+ association induces formation of protein structure in the region of the zinc-finger motif. The p10.5 polypeptide is amenable to fluorescence studies due to the presence of a single Trp residue at the tip of the predicted loop with none elsewhere in p10.5 (Fig. 2B). An increase in intensity combined with a reduction in the wavelength (blue shift) of fluorescence was always observed when p10.5 was titrated with Zn2+ (Fig. 5). Subsequent addition of EDTA always resulted in a diminution in intensity and an increase in the wavelength of fluorescence. Fig. 5 shows an initial spectrum and final spectra after titration of p10.5 with saturating amounts of Zn2+ followed by saturating amounts of EDTA. Calculating the difference between the baricentric means of the corrected emission spectra gave an overall shift of 2.48 (±0.90) nm. This value represents the difference between the minimum (after titration with Zn2+) and maximum baricentric means for three preparations of p10.5, noting that in one preparation the magnitude of the Zn2+-induced blue shift exceeded the subsequent EDTA-induced red shift and for this sample the initial mean gave the maximum wavelength.


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Fig. 5.   Fluorescence spectra of p10.5. The corrected emission spectra of ~25 µg of p10.5 in factor Xa cleavage buffer (50 mM Tris, 150 mM NaCl, 1 mM CaCl2, pH 7.5) (squares), following incubation with saturating levels of Zn2+ (diamonds) and subsequently EDTA (circles) are shown.

Merodiploids in Which Some Copies of dnaG Are Inactive Have a Reduced Number of Chromosomes-- It was speculated that a phenotype associated with impaired action of dnaG in this organism could be a reduced number of chromosomes. To test this hypothesis mutants were first generated in which dnaG was inactivated on a proportion of chromosomes. Construct pABNR1.1 was generated containing Synechococcus PCC 7942 chromosomal sequences interrupted by neo. The Synechococcus PCC 7942 sequences included in pABNR1.1 are separated by 256 bp in the chromosome, within the 3' end of dnaG. These 256 bp are therefore absent from pABNR1.1. Synechococcus PCC 7942 was transformed to kanamycin resistance with a fragment of DNA released from pABNR1.1, and transformants were repeatedly restreaked on selective media. Two transformants were grown in liquid culture, and their genotypes were examined (Fig. 6).


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Fig. 6.   Southern analysis demonstrating integration of neo into dnaG and nonsegregation of mutant chromosomes. PstI-digested DNA from two kanamycin-resistant mutants of Synechococcus PCC 7942 resulting from introduction of a 4.3-kb ApaI/SacI fragment of pABNR1.1 (lanes 1 and 2) and wild-type Synechococcus PCC 7942 (lane 3) was electrophoresed on a 0.8% agarose gel, transferred to a membrane, and probed with neo (A); the 256 bp "diagnostic"-deleted region (B); retained sequences to the 3' of the deleted region (C); and retained sequences to the 5' of the deleted region (D).

Integration of neo by homologous recombination will delete a part of dnaG (256 bp) containing a PstI site leading to the loss of a 0.86-kb PstI fragment and the creation of a diagnostic, novel, 3.1-kb PstI fragment. Fig. 6A shows that a ~3.1-kb PstI fragment that hybridizes to neo is present in DNA from the kanamycin-resistant mutants and, of course, absent in DNA from wild-type cells. This confirms integration of neo within dnaG. However, hybridization to a ~0.86-kb PstI fragment (and weakly to a smaller fragment) was also detected using a probe containing the 256-bp "deleted" region, in DNA from both wild-type and mutant cells (Fig. 6B). This region must therefore be retained on a proportion of the chromosomes in the mutants. Using DNA from both wild-type and mutant cells a ~0.94-kb PstI fragment hybridized to a probe corresponding to sequences downstream of the site of insertion of neo. An additional ~3.1-kb fragment was also detected in DNA from the mutants (Fig. 6C). A ~0.86- and ~3.1-kb fragment were similarly detected upon probing with sequences upstream of the site of insertion of neo (Fig. 6D). The mutants are thus merodiploids containing some wild-type chromosomes that retain dnaG and others that contain neo within dnaG. The same four probes were also hybridized to SalI-digested DNA from wild-type and mutant cells (data not shown), and these results also confirmed that the mutants are merodiploids. Additional uncharacterised recombination events were detected in one of the mutants (Fig. 6, A, C, and D, lane 2), hence the alternative mutant line, designated Synechococcus PCC 7942(dnaG), was used in subsequent analyses. The fact that both of the analyzed mutants are merodiploids (Fig. 6) is consistent with this region of DNA, and hence dnaG, being essential. Previous attempts to inactivate essential genes in cyanobacteria have also led to the generation of merodiploids with the subsequent introduction of an homologous E. coli gene on an autonomously replicating plasmid, allowing the interrupted mutant chromosomes to segregate to fixation (40).

Synechococcus PCC 7942(dnaG) was subcultured in liquid media at 2-day intervals to maintain cells in continuous logarithmic growth. These cells contained fewer chromosomes than cells in which all copies of dnaG were intact (Fig. 7A). Assays were performed in triplicate, and similar data were also obtained when the entire experiment was repeated on two further occasions (nine replicates in total). Differences in chromosome copy number did not correlate with altered growth rates, since Synechococcus PCC 7942(dnaG) did not grow any more slowly than wild-type cells (Fig. 7B). The mean estimated number of chromosomes in Synechococcus PCC 7942(dnaG) was ~2.5. Therefore, many of these merodiploids must contain only a single copy of each type of chromosome.


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Fig. 7.   DNA contents of logarithmically growing (at equivalent rates) Synechococcus PCC 7942 and dnaG mutants. Panel A, DNA contents of (from left to right) Synechococcus PCC 7942(dnaG) grown in the presence of kanamycin; Synechococcus PCC 7942(dnaG) grown in the absence of kanamycin; wild-type Synechococcus PCC 7942 grown in the absence of kanamycin; and wild-type Synechococcus PCC 7942 containing pUC105 with neo grown in the presence of kanamycin. Panel B, maintenance of logarithmically growing cultures of Synechococcus PCC 7942(dnaG) grown with (shaded square) and without (open square) kanamycin in the final culture medium, wild-type Synechococcus PCC 7942 (open circle) grown without kanamycin, and wild-type Synechococcus PCC 7942 containing pUC105 with neo (closed circle) grown with kanamycin. Growth was monitored by measuring the optical density at 595 nm. Cultures were diluted to an optical density (at 595 nm) of 0.03 at 48-h intervals.

Generation of Mutants Containing a Single Nucleotide Substitution within dnaG-- R2-PIM8(smt) is a Zn2+-sensitive variant of Synechococcus PCC 7942 in which we have previously insertionally inactivated the smt divergon (18) that lies adjacent to dnaG in this organism. A linear fragment of DNA, containing the smt divergon and mutant dnaG, with a nucleotide substitution converting codon C597S from TGC to TCC (Fig. 1), was released from pABNR2.2 and used to transform R2-PIM8(smt) to Zn2+ resistance. Zn2+-resistant (and chloramphenicol-sensitive) transformants were selected on medium containing 15 µM Zn2+. Two Zn2+-resistant colonies were grown in liquid culture, and Southern analysis confirmed the site of integration of smt (Fig. 8). A ~7.5-kb fragment of SalI digested DNA from R2-PIM8(smt) hybridized to cat, and this corresponds to the known size of the SalI fragment containing disrupted smt (18) (Fig. 8A). No hybridizing fragments were detected in either of the two mutants confirming that cat had been lost and hence that smt had (i) reintegrated at the homologous site and (ii) this had been followed by complete segregation of all chromosome copies. The Southern blot was also probed with smtA and a ~5.5-kb hybridizing fragment was observed in both of the mutants further confirming integration of smt into the genome at the homologous site (Fig. 8B). A ~7.5-kb hybridizing fragment, faintly visible using DNA from R2-PIM8(smt), corresponds to hybridization to a small region of smtA which is retained in this genome.


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Fig. 8.   Southern analysis of dnaG(C597S). SalI-digested DNA from R2-PIM8(smt) (lane 1) and two Zn2+-tolerant mutants resulting from introduction of a 5.5-kb SalI fragment of pABNR2.2 (lanes 2 and 3), was electrophoresed on a 1.0% agarose gel, transferred to a membrane, and probed with cat (A) and smtA (B).

To determine whether or not the homologous recombination event had also introduced the C597S codon substitution into dnaG, a region encoding the carboxyl terminus of DNA primase was amplified using template DNA recovered from the Zn2+-resistant cells. The PCR product was cloned and sequenced to reveal that the mutation had been introduced into the genome. These cells were therefore designated dnaG(C597S).

Chromosome Copy Number Is Reduced in dnaG Zinc-finger Mutants-- Analysis of Synechococcus PCC 7942(dnaG) has shown that reduced chromosome copy number can be associated with impaired activity of dnaG in this organism (Fig. 7A). To investigate the functional importance of the zinc-finger region we have estimated chromosome copy number in dnaG(C597S). Fig. 9 reveals that these cells contain less chromosomes than the equivalent wild-type cells. In two further experiments (data not shown) similar results were obtained (nine replicates in total). Wild-type and dnaG(C597S) cells grew at the same rates (data not shown).


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Fig. 9.   DNA content of dnaG(C597S) mutants. DNA contents were determined in rapidly growing cultures of Synechococcus PCC 7942 and dnaG(C597S), standardized cell-1 following microscopic cell counts. All experiments were performed with triplicate cultures and on at least three separate occasions.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Our results show that in Synechococcus PCC 7942 dnaG is co-transcribed with pheT and the gene is not flanked by rpsU and rpoD (Fig. 2). It has been suggested that in other bacteria MMS operon structure allows some coordination of the overall levels of synthesis of the three major informational macromolecules (33). This may provide some control over the ratios of total protein:DNA:RNA even when rates of cell proliferation change, or at cell division. The absence of a typical MMS operon in Synechococcus PCC 7942 suggests that any co-ordination of synthesis of informational macromolecules which is afforded by normal MMS operons in many other bacteria, must be either absent or mediated by alternative mechanisms.

The amino-terminal two-thirds of DNA primases tend to be the more highly conserved in sequence (33, 41). In common with other bacterial primases, the amino-terminal end of the deduced DNA primase from Synechococcus PCC 7942 contains a conserved Zn2+-binding region. Several studies have demonstrated that this region does indeed bind Zn2+ in other DNA primases (10-12, 42). However, a second predicted Zn2+-binding region, a Cys2/His2 type zinc-finger motif, is located at the extreme carboxyl terminus of the deduced Synechococcus PCC 7942 DNA primase (Fig. 2B). Our results demonstrate that a recombinant protein, p10.5, containing the carboxyl-terminal 91 residues of Synechococcus PCC 7942 DNA primase does bind Zn2+ and also Cd2+ (used as an analogue of Zn2+). Several lines of evidence imply that all (or at least a proportion) of these ions were associated with the deduced Cys2/His2 type zinc-finger in preparations of the recombinant polypeptide.

First, p10.5 binds 65Zn (Fig. 3). Second, Cys modification of p10.5 with PMPS displaced metal ions (Zn2+ and Cd2+) with 2-mol equivalents of the mercurial reagent representing saturating amounts (Fig. 4). There are only two Cys residues in p10.5, both are located within the zinc-finger motif, and the requirement for 2 mol equivalents of PMPS to displace the metal ions indicates that Hg2+-mercaptide bond formation must occur between both Cys residues and PMPS (20) in order to release all of the co-ordinated Zn2+ or Cd2+ ions.

The displacement of ~1 g atom of Cd2+ mol-1 p10.5 by saturating amounts of PMPS (Table I) is consistent with metal coordination to a single specific site on each molecule of p10.5. Estimates of the amount of Zn2+ displaced by PMPS from p10.5 were low, and also variable with an error greater than 20% (Table I). However, PAR (a metal-chelator) can access Zn2+ associated with some proteins without any requirement for the addition of PMPS (44). Stoichiometries close to 1:1 were obtained for p10.5 if the Zn2+ reacting with PAR prior to the addition of PMPS was also included in the estimates. Thus, only a proportion (10 and 13%) of the Zn2+ associated with preparations of p10.5 was inaccessible to PAR prior to addition of 2 equivalents of PMPS, implying that only the coordination of these ions involved the two Cys residues. Thus each purified polypeptide can "either" bind a Zn2+ ion in a conformation which involves the two Cys, "or" some other conformation in which the Zn2+ is immediately accessible to PAR. The second conformation may have formed in vitro, possibly due to oxidation of Cys. However, this site could also be important in vivo, for example as an intermediate in Zn2+ acquisition. The second site could involve other residues in the region of the zinc-finger motif, for example, two additional His residues which are arranged to form CXXXCXH and HXXXHXH motifs (where X represents an amino acid other than Cys or His, and the underlined residues are those identified by the motifs program). It is notable that the coordination of all Cd2+ ions involves both Cys in preparations of p10.5. Either (i) the coordination of Cd2+ to Cys is in conjunction with different residues to those coordinating Zn2+ and/or (ii) the site is more stable when associated with Cd2+ or (iii) the alternative, PAR-accessible, site is capable of coordinating Zn2+ but incapable of coordinating Cd2+.

Only an estimated 0.03 g atom Cd2+ mol-1 was displaced from p7.5 (Table I). This again indicates that the metal ions are coordinated to the zinc-finger motif contained within the carboxyl-terminal 24 residues of p10.5, but absent from p7.5.

An increase in the intensity, combined with a reduction in the wavelength, of fluorescence emitted from a Trp residue located at the tip of the zinc-finger, was observed upon titration of p10.5 with Zn2+ (Fig. 5). An enhanced intensity of fluorescence from a single aromatic amino acid located within a Cys3/His-type Zn2+-binding motif was previously observed in (i) the murine leukemia virus Ncp10 protein (45), and (ii) the "amino-terminal" Zn2+-binding motif of E. coli DNA primase (13) coincident with a Zn2+-induced conformational change. The increase in intensity and decrease in wavelength of fluorescence indicate that Zn2+ encourages the formation of greater structure in the region of the Trp residue, the latter becoming less solvent-accessible upon addition of the metal ion. Addition of EDTA confers the opposite responses, a reduction in intensity combined with an increase in the wavelength of fluorescence, consistent with metal removal by the chelating agent making this region less structured, causing the Trp residue to become more solvent-accessible.

Due to the presence of multiple, and a variable number of, chromosomes in Synechococcus PCC 7942 (2, 3), it was anticipated that an inhibition of DNA synthesis could be manifest in a change in DNA content in this organism. Such a phenotype was indeed observed (Fig. 7) in merodiploids in which dnaG had been insertionally inactivated on a proportion of chromosomes (Fig. 6).

It has been possible to introduce a single nucleotide substitution into dnaG within the genome of Synechococcus PCC 7942 by using the adjacent smt divergon as a marker to select for Zn2+-resistant recombinants (Fig. 8). These dnaG(C597S) mutants will thus be otherwise genetically identical to wild-type Synechococcus PCC 7942. This technical approach could be used to address further questions concerning the structure and function of DNA primase via introduction of other substitutions. The analysis of the dnaG(C597S) mutants has revealed that the exclusive substitution of Cys597 with Ser in DNA primase reduces the DNA content of Synechococcus PCC 7942 (Fig. 9). It is proposed that the abolition of the metal-mercaptide bond formation at Cys597 "impairs" formation of the zinc-finger. Clearly the thiol group of Cys597 is required for optimum chromosome replication.

A carboxyl-terminal zinc-finger in DNA primase from Synechococcus PCC 7942 is likely to be involved in protein-protein or protein-DNA interactions during DNA-replication. There are precedents for "lone" Zn2+-binding domains of the Cys4-type (46, 47), Cys3/His-type (48), and Cys2/His2-type (49) being involved in protein-protein interactions. Clearly in Synechococcus PCC 7942, the interactions which are known to be mediated by the extreme carboxyl terminus of E. coli DNA primase (6-9) must involve either a different region of the protein or this zinc-finger motif. In E. coli, mutations within the extreme carboxyl terminus of DNA primase impair interaction with helicase and thereby modify rates of DNA synthesis (6-8). There is no direct evidence that a carboxyl-terminal zinc-finger in DNA primase from Synechococcus PCC 7942 interacts with helicase, but this is a most obvious hypothesis.

There is circumstantial evidence suggesting that some eukaryotic MTs donate/remove Zn2+ to/from some zinc-finger proteins (43, 50-54). Due to the proximity of dnaG to smtA in Synechococcus PCC 7942 it is tempting to speculate that SmtA may act as a chaperone donating Zn2+ to, or removing Zn2+ from, DNA primase with implications for the control of DNA synthesis. The focus of this study has been the novel and additional carboxyl-terminal zinc-finger, although it is also plausible that SmtA could influence Zn2+ occupancy of the amino-terminal region. This is the subject of ongoing studies.

    FOOTNOTES

* This work was supported by a research grant from the United Kingdom Biotechnology and Biological Sciences Research Council (BBSRC) Genes and Developmental Biology Committee.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X94247.

Dagger These authors have contributed equally to this work.

§ Supported by a research studentship from the Biotechnology and Biological Sciences Research Council.

To whom correspondence should be addressed: Tel.: (0)191 222 7695; Fax: (0)191 222 7424; E-mail: n.j.robinson{at}newcastle.ac.uk.

The abbreviations used are: MMS, macromolecular synthesis; bp, base pair(s); kb, kilobase pair(s); MT, metallothionein; PAR, 4-(2-pyridylazo)resorcinol; PMPS, p-(hydroxymercuri)phenylsulfonatePCR, polymerase chain reaction.
    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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