©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
*, a UV-inducible Smaller Form of the Subunit Sliding Clamp of DNA Polymerase III of Escherichia coli
I. GENE EXPRESSION AND REGULATION (*)

(Received for publication, August 22, 1995; and in revised form, November 15, 1995 )

Tamar Paz-Elizur Rami Skaliter Sara Blumenstein Zvi Livneh (§)

From the Department of Biochemistry, Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The 40.6-kDa beta subunit of DNA polymerase III of Escherichia coli is a sliding DNA clamp responsible for tethering the polymerase to DNA and endowing it with high processivity (Stukenberg, P. T., Studwell-Vaughan, P. S., and O'Donnell, M.(1991) J. Biol. Chem. 266, 11328-11334). UV irradiation of E. coli induces a smaller 26-kDa form of the beta subunit, termed beta*, that, when overproduced from a plasmid, increases UV resistance of E. coli (Skaliter, R., Paz-Elizur, T., and Livneh, Z.(1996) J. Biol. Chem. 271, 2478-2481). Here we show that this protein is synthesized from a UV-inducible internal gene, termed dnaN*, that is located in-frame inside the coding region of dnaN, encoding the beta subunit. The initiation codon and the Shine-Dalgarno sequence of dnaN* were identified by site-directed mutagenesis. The dnaN* transcript was shown to be induced upon treatment with nalidixic acid, and transcriptional dnaN*-cat gene fusions were UV inducible, suggesting induction of dnaN* at the transcriptional level. Analysis of translational dnaN*-lacZ gene fusions revealed that UV induction was abolished in strains carrying the recA56, lexA3, or DeltarpoH mutations, indicating involvement of both SOS and heat shock stress responses in the induction process. Expression of dnaN* represents a strategy of producing several proteins with related functional domains from a single gene.


INTRODUCTION

UV irradiation of Escherichia coli cells leads to the formation of both mutagenic and inactivating DNA lesions(1) . The cells respond by an immediate arrest of DNA replication, followed by a period of extensive DNA repair, that operates to eliminate DNA damage in order to prevent replication obstacles(2) . These processes are controlled primarily by the SOS stress regulon, which involves more than 20 genes that are commonly regulated by the LexA repressor and the RecA activator(3, 4) . However, UV irradiation induces change also in heat shock genes (5) and other genes (6) which affect the post-UV physiology of the cell. We have previously found that the beta subunit of DNA polymerase III holoenzyme, the major replicase of the E. coli chromosome(7) , limits the ability of the purified polymerase to replicate UV-irradiated single-stranded DNA(8) . Consistent with this result, overproduction of the beta subunit from a plasmid caused a reduction in UV resistance and in UV mutagenesis of E. coli cells(9) .

This involvement of the beta subunit in UV irradiation effects prompted us to examine whether it may be present in a different form in UV-irradiated cells. We found that upon UV irradiation a smaller form of the beta subunit, termed beta*, was induced. When overproduced from a plasmid under the inducible lac promoter, beta* caused up to a 6-fold increase in UV resistance of E. coli cells, suggesting a role in recovery from UV damage, e.g. by involvement in DNA repair or reactivation of DNA replication(48) .

Smaller derivatives of proteins that are found in cells are frequently generated by proteolysis, as in the case of the mutagenesis protein UmuD` that is formed from UmuD by specific cleavage promoted by the RecA protein(10) . Alternatively, the protein can be translated from the overlapping mRNA by a de novo internal translational start, or it can be expressed from an internal in-frame gene. The present study shows that beta* is synthesized from a novel UV-inducible gene which is located in-frame inside the coding region of the dnaN gene, and it is subjected to indirect regulation by both the SOS and heat shock stress responses.


EXPERIMENTAL PROCEDURES

Materials

The sources for materials used in these studies were as follows: isopropyl beta-D-thiogalactopyranoside (IPTG), (^1)rifampicin, nalidixic acid, o-nitrophenyl-beta-D-galactoside, 5-bromo-4-chloro-3-indolyl-beta-D-galactoside, ampicillin, spermidine, sodium deoxycholate, Tween 20, Nonident P-40, and n-butyryl CoA, Sigma; radiolabeled nucleotides, Amersham Corp.; [^3H]chloramphenicol (30.7 Ci/mmol), DuPont NEN; urea, ICN; nitrocellulose membrane, Schleicher & Schuell; M9 medium, minimal A medium, and LB were prepared as described by Miller(11) .

Proteins

LexA repressor and the beta subunit of DNA polymerase III were purified as described by Little (12) and Johanson et al.(13) , respectively. beta* was purified from an overproducer strain that was constructed in our laboratory(49) . Anti-beta antibodies were affinity-purified on beta* immobilized on nitrocellulose as previously described(48) . RNA polymerase was a gift from R. Burgess (University of Wisconsin). DNA polymerase I, T4 DNA ligase, alkaline phosphatase, T7 RNA polymerase, E. coli RNA polymerase, RNase T(1) and RNase A, and bovine serum albumin were purchased from Boehringer Mannheim. Restriction endonucleases were purchased from New England Biolabs. Polynucleotide kinase was from U. S. Biochemical Corp. Proteinase K, chicken egg white lysozyme, and anti-beta-galactosidase antibody were obtained from Sigma.

Bacterial Strains and Bacteriophages

The bacterial strains used in this study are listed in Table 1. E. coli R40NL8 was obtained by removing the imm21 prophage from E. coli R40 (14) by superinfection with a heteroimmune derivative, b2 ( immunity). Phage b2 has a deletion at the att site so it cannot integrate into the chromosome; rather it enters the lytic pathway, and can supply the proteins needed for excision of the prophage from the chromosome. The reduced level of heat shock proteins (that are needed for the life cycle of ) slows down the lytic infection by b2. This raises the probability of obtaining colonies of -free E. coli R40 cells as a result of asymmetric segregation during cell division that has occurred after excision. E. coli R40NL8 was sensitive to all types of , as expected from a non-lysogen, and remained temperature-sensitive like the parental R40. Transformation of R40NL8 with plasmid pFN97, carrying the rpoH gene, yielded temperature-resistant colonies suggesting that the temperature sensitivity of the cells was indeed due to the DeltarpoH mutation.



Construction of Plasmids

The plasmids used in this study are presented in Table 2. Plasmid pRPHF11 that served as a template for the synthesis of the dnaN* riboprobe is a pBluescript SK derivative in which the HpaII-(1942)-FspI(2142) dnaN DNA fragment was cloned in an orientation opposite to the T7 RNA polymerase promoter. Plasmid pCAT was derived from plasmid pCM4, a pBR327 derivative containing the coding sequence of the cat gene downstream to the tet promoter (Fig. 1). The BamHI site located at the 3` end of the cat gene was eliminated by a partial digestion of plasmid pCM4 to full-length linear DNA, followed by filling-in of the termini and self-ligation to yield plasmid pCMB. The T(1)rrnB terminator of E. coli was isolated from plasmid pPS1 (15) by digesting with restriction endonucleases HindIII and MboII, to produce a 326-bp fragment, followed by further digestion with restriction endonuclease AhaII to generate the desired 247-bp HindIII-AhaII fragment. It was cloned into the AatII site of pCMB, to yield plasmid pCAT. Plasmid pCAT was the parent for the various cat transcriptional gene fusions. They were constructed by eliminating the tet promoter from pCAT by cleavage with restriction nucleases EcoRV and ClaI (Fig. 1) and ligating the cleaved vector to the appropriate dnaN* promoter fragment as follows. Plasmid pNCB17, BstUI-(1896-2228); pNCH6, BstUI-(1896)-HgaI-(2037); pNCS14, SfaNI-(1918-2090); pPC1, BstUI-(2228)-SfaNI-(2090), P(x) promoter; pRC5, NciI-(62-219), recA promoter; pNCH20, HgaI-(2037)-BstUI-(1896). Plasmids pTEN5, pNB3, and pSB6 are dnaN*-lacZ translational gene fusions. They were constructed by cloning dnaN* fragments into the SmaI site in plasmid pMC1403 (16) leading to the fusion of parts of beta* to the 8th amino acid of beta-galactosidase. The following dnaN* gene fragments were used. Plasmid pTEN5, SfaNI-(1918-2090); Plasmid pNB3, BstUI-(1896-2228); plasmid pSB6, AccI-(1965)-BstUI-(2228). The coordinates of the dnaN* gene are according to Ohmori et al.(17) . Control cI-lacZ and dnaA-lacZ translational gene fusions were constructed as follows. Plasmid pHSC6 (cI-lacZ fusion) was constructed by isolating from plasmid pUN121 (18) the 550-bp HincII DNA fragment containing the control region and the coding sequence for the first 168 amino acids of cI repressor gene of phage and cloning it into the SmaI site in plasmid pMC1403. Plasmid pHSA2 (dnaA-lacZ fusion) was constructed by isolating from plasmid pHB21 (17) the 450-bp EcoRI fragment carrying the control region and the coding sequence for the first 20 amino acids of the dnaA gene and cloning it into the EcoRI site in plasmid pMC1403 after filling-in the termini of both vector and insert in order to synchronize the reading frames of dnaA and lacZ. The in-frame fusion at the cloning junctions was verified by DNA sequence analysis. Plasmid pCM1 is a 6-kb pBR322-derivative that carries the rpoH gene and the cat gene, conferring resistance to chloramphenicol. It was constructed from plasmid pFN97 (19) that carries the rpoH and bla genes, by inserting the 1.6-kb NheI-HincII fragment carrying the cat gene from plasmid pACYC184 (20) into the ScaI site inside the bla gene.




Figure 1: Vectors used to construct cat gene fusions. Plasmid pCAT was derived from pCM4 by eliminating the BamHI site at the 3` side of cat, and by inserting the TrrnB transcription terminator into the AatII site upstream to cat. Transcriptional gene fusions to cat were constructed by replacing the tet promoter (P) located on the EcoRV-ClaI fragment with the promoter to be studied. See ``Experimental Procedures'' for details.



Preparation of dnaN* Mutants by Site-directed Mutagenesis

M13mp8beta is a 8.7-kb derivative of phage M13mp8 that caries the entire dnaN gene cloned opposite to the lac promoter. It was constructed by cloning the BanI-ScaI 1.4-kb fragment which carries the dnaN gene into the HincII site of M13mp8 RF. Site-directed mutations were introduced into the control region of dnaN* in single-stranded DNA from phage M13mp8beta using the Amersham dNTPalphaS kit. The oligonucleotides used were: Met1, 5`-CAG GCA ACG C*TG AAG CGT C-3`; Met2, 5`-CAG TTT TCT C*TG GCG CAT C-3`; SD-S, 5`-TAC CCT GCC A*CAA*G CAA CGA TG-3`; SD-M, 5`-TAC CCT GCC GCAA*A* CAA CGA TG-3` (asterisks mark mutated nucleotides). Screening for mutant plaques was done by dot blot hybridization using the radiolabeled mutant oligonucleotides as probes. The nucleotide sequences of the mutated M13mp8beta regions were verified by DNA sequence analysis. The wild-type and mutant dnaN genes were obtained on 1.5-kb fragments from the M13 clones by cleavage with SmaI and HindIII, and cloned into the SmaI site of plasmid pUC18 under the lac promoter. This yielded plasmids pCW3, pCM13, pCM2, pCRS, and pCRM that carried the wild-type, Met1, Met2, SD-S, and SD-M sequences, respectively. DNA fragments (EcoRV-(1871)-BanI-(2832); coordinates of the dnaN gene) carrying the mutated dnaN* genes were cut out from these plasmids and cloned into the EcoRV site in plasmid pBluescript SK in two orientations, yielding two series of plasmids: pBSW7 (wild-type), pBSM11 (Met1), pBSM21 (Met2), pBSRS1 (SD-S), and pBSRM1 (SD-M) contained the dnaN* fragments under the phage T7 promoter, whereas plasmids pBSOW1 (wild-type), pBSOM11 (Met1), pBSOM22 (Met2), pBSORS1 (SD-S), and pBSORM1 (SD-M) contained the same fragments in the opposite orientation expressed from the lac promoter. The BamHI-(719)-HaeII-(1031) segments carrying the lac promoter were deleted from plasmids pBSW7, pBSM11, pBSM21, pBSRS1, and pBSRM1, yielding plasmids pNLW1, pNLM11, pNLM21, pNLRS1, and pNLRM1, respectively.

Kinetics of beta* Synthesis from the Phage T7 Promoter

E. coli BL21(DE3) cells harboring plasmid pNLW1 (wild-type), pNLM11 (ATG CTG mutation, Met1) and pNLM21 (ATG CTG mutation, Met2) were grown to OD = 0.4 in minimal medium supplemented with ampicillin (20 µg/ml), MgSO(4) (1 mM) and glucose (0.4%). Expression of dnaN* was triggered by the addition of IPTG (0.5 mM). After 40 min of induction, rifampicin (200 µg/ml) was added, to inhibit transcription by the host RNA polymerase, and incubation was continued for 30 min. At this point 300 µCi (20 µl) of [S]methionine were added to a cell culture of 4 ml. At various time points 0.5-ml samples were withdrawn, and added to 0.1 ml of a solution of 0.3% sodium azide containing 120 µg of unlabeled L-methionine to chase the metabolic labeling. The culture was cooled for 10 s in a dry ice-ethanol bath, and then kept on ice until assayed. The cell extracts were fractionated by 15% SDS-PAGE after which the gel was dried and fluorographed using a Kodak XAR-5 x-ray film. The intensities of the bands were quantified by scanning with a Molecular Dynamics 300 A computing densitometer.

Kinetics of Degradation of beta*

Four ml of E. coli BL21(DE3) cells harboring plasmid pNLW1 (wild-type) were pulse-labeled for 5 min with 300 µCi of [S]methionine, and then chased with unlabeled 960 µg of L-methionine. At various time points after the addition of the unlabeled methionine, 0.5-ml samples were withdrawn and added to 0.1 ml of 0.3% sodium azide. Samples were heated at 100 °C for 5 min and analyzed by 15% SDS-PAGE. The gel was dried, fluorographed, and the bands were quantified by scanning with a Molecular Dynamics 300 A computing densitometer.

Immunoblot Analysis of beta*

E. coli AB1157XL harboring plasmids that carried mutated dnaN* gene fragments expressed from the lac promoter were grown in LB medium supplemented with ampicillin (100 µg/ml) and glucose (0.2%) at 30 °C to OD = 0.5. The cells were induced with IPTG (0.5 mM) for 1.5 h at 30 °C, after which they were precipitated, resuspended to OD = 20 in 10 mM TrisbulletHCl, pH 7.5, 0.15 M NaCl, and frozen in liquid nitrogen. Aliquots of 500 µl of the frozen cells were thawed, disrupted by sonication, and analyzed by Western blot analysis. Protein concentration was determined in 0.1 M NaOH according to Bradford(21) . Aliquots containing 5 µg of protein were separated by 10% SDS-PAGE, blotted to a nitrocellulose membrane, and probed with affinity-purified anti-beta antibodies as described by Skaliter et al.(48) using enhanced chemiluminescence (ECL, Amersham Corp.) for detection.

Nalidixic Acid Induction of the dnaN* Transcript

AB1157 cells were grown to OD = 0.4-0.5, at which nalidixic acid (40 µg/ml) was added. At the desired time interval, samples were withdrawn, and cells were precipitated and resuspended in a solution of 10 mM TrisbulletCl, pH 7.5, 1 mM EDTA, and 150 mM NaCl. They were frozen and kept in liquid nitrogen. Total RNA was isolated as described elsewhere (22) and further purified by isopicnic centrifugation in CsCl (1.799 g/ml) containing ethidium bromide, followed by isopropanol extraction. Riboprobes were prepared from plasmid pRPHF11, in which the control region of dnaN* was cloned in an orientation opposite to the T7 RNA promoter. The riboprobe was synthesized with T7 RNA polymerase in the presence of [alpha-P]UTP, after which it was purified on a 6% denaturing urea-polyacrylamide gel. Analysis of transcription initiation sites was performed by the RNase protection assay as described(22) . A hundred and fifty µg of RNA were hybridize with the radiolabeled riboprobe (2 times 10^6 cpm/reaction) in a final volume of 30 µl of a buffer containing 40 mM PIPES, pH 6.4, 1 mM EDTA, 0.4 M NaCl, and 80% formamide. The mixture was heated at 85 °C for 5 min, and then it was incubated at 45 °C for 12 h to allow annealing of the riboprobe to the specific RNA. The mixture was then cooled to room temperature, and 300 µl containing 0.3 M NaCl, 10 mM TrisbulletCl pH 7.4, 5 mM EDTA, 2 µg/ml RNase T(1), and 40 µg/ml RNase A were added. The mixture was incubated for 1 h at 30 °C, after which the RNases were digested with proteinase K (0.3 mg/ml) in the presence of SDS (0.6%) at 37 °C for 30 min. The RNA hybrid was extracted with phenol/chloroform and precipitated with ice-cold ethanol in the presence of 20 µg of tRNA as a carrier. The hybrid was fractionated on a 6% polyacrylamide gel containing urea.

Kinetics of UV Induction of the cat Gene Fusions

E. coli AB1157 cells harboring cat gene fusion plasmids, were grown in M9 medium supplemented with amino acids and glucose at 37 °C to OD = 0.1. The cultures were then divided into two 20-ml portions, one of which was UV-irradiated and returned for further growth at 37 °C. Usually a UV dose of 30 J m was used. Samples of 1.5-6 ml were withdrawn at various time points, sedimented, and resuspended in an extraction buffer (50 mM TrisbulletHCl, pH 7.5, 30 mM dithiothreitol). The cell suspension was then frozen in liquid nitrogen, thawed, and sonicated for 30 s. The mixture was centrifuged and the concentration of the soluble protein fraction was measured according Bradford(21) . CAT activity was assayed essentially as previously described(23) , based on the ability of CAT to transfer a butyryl residue from butyryl-CoA to one or two hydroxyl residues on the chloramphenicol molecule, thus causing its inactivation. Butyrylation of [^3H]chloramphenicol was followed by extracting the butyrylated chloramphenicol from the reaction mixture with an organic solvent. The reaction mixture (100 µl) contained 0.2 µCi of [^3H]chloramphenicol (preextracted twice with xylene), 25 µg of butyryl CoA, 20 mM TrisbulletHCl, pH 8, and the protein extract. The reaction mixtures were incubated for 1 h at 37 °C, after which they were extracted with 200 µl of 2:1 tetramethylpentadecane:xylene, and the amount of radioactivity extracted into the organic phase was determined by liquid scintillation counting.

Kinetics of UV Induction of beta-Galactosidase

E. coli cells transformed with plasmids carrying the lacZ gene fusions were grown at 37 °C to OD = 0.05-0.1, then divided into two 20-ml portions, of which one was UV-irradiated. The UV doses used, adjusted to give a survival of 30%, were: KY700 (wild-type), 60 J m; KY703 (lexA3), 5 J m; KY705 (recA56), 1 J m; KY706 (uvrA6), 5 J m; MC4100 (wild-type), 50 J m; R40NL8 (DeltarpoH), 25 or 50 J m. Culture growth was continued at 37 °C, and 1.5-ml samples were withdrawn at various time points for the determination of beta-galactosidase activity, as described elsewhere(11) .


RESULTS

Identification of the ATG Initiation Codon and the Shine-Dalgarno Sequence of dnaN*

The DNA sequence of the beginning of the dnaN* gene is shown in Fig. 2, including the putative control elements. Two ATG sequences were candidates for the initiation codon of dnaN*: ATG and ATG (Fig. 2). The sequence GCAGG, homologous to the Shine-Dalgarno sequence, was located 5 nucleotides upstream to ATG, whereas no such sequence was found at the appropriate distance upstream to ATG (Fig. 2), suggesting that ATG serves as the initiation codon of dnaN*. In order to examine this possibility we have mutated each of the ATG codons into a CTG codon by site-directed mutagenesis.


Figure 2: The 5`-region of the dnaN* gene. The dnaN* promoter, its Shine-Dalgarno sequence (SD), its ATG initiation codon, and codon ATG are indicated. In addition, the two recF promoters (P1 and P2) and the P promoter located inside dnaN/dnaN* and several cleavage sites of restriction nucleases are shown.



The mutated dnaN* genes were cloned under the strong phage T7 promoter in plasmid pBluescript SK. E. coli BL21(DE3) cells harboring these plasmids grew poorly, and they had variations in plasmid copy number. This was caused most likely by the induction of the T7 RNA polymerase due to titration of LacI by the lacP promoter present on the high copy number plasmid. Indeed, deletion of the lacP segment from the plasmids resolved the problem, and the synthesis of beta* could then be quantitatively monitored by metabolic labeling with [S]methionine, followed by SDS-PAGE and fluorography. To facilitate detection of beta*, transcription by the cellular RNA polymerase was inhibited by the addition of rifampicin, such that transcription was selectively initiated from the T7 promoter by the T7 RNA polymerase expressed from a prophage in the host cell. Indeed, as can be seen in Fig. 3, in the presence of rifampicin, the synthesis of beta* could be easily detected.


Figure 3: Kinetics of synthesis of beta* from mutant dnaN* plasmids assayed by [S]methionine labeling. Upper Panel,. E. coli BL21(DE3) cells harboring plasmid pNLW1 (wild-type dnaN*), pNLM11 (C*TG mutation, Met1), or pNLM21 (C*TG mutation, Met2) were grown to OD = 0.4 in minimal medium supplemented with ampicillin, MgSO(4) and glucose. Expression of dnaN* was turned-on by the addition of IPTG, followed by the addition of rifampicin to inhibit transcription by the host RNA polymerase. Newly synthesized proteins were pulse-labeled with [S]methionine and analyzed by SDS-PAGE followed by fluorography. Lower panel, Qualification of the fluorogram shown in A was done by scanning with a Molecular Dynamics 300A computing densitometer. Full squares, Wt; empty squares, Met2; circles, Met1.



The validity of measuring rates of protein synthesis by this procedure depends on the turnover of beta* in the cell. The half-life of beta* was determined by pulse-labeling of the protein with [S]methionine, followed by a chase with unlabeled methionine. From the decay in the amount of radiolabeled beta*, its half-life is approximately 40 min (Fig. 4), much higher than the time scale used to estimate metabolic rates of synthesis. Thus, beta* is relatively a stable protein, and its degradation is not expected to affect significantly the measurements of its synthesis.


Figure 4: Kinetics of degradation of beta*. E. coli BL21(DE3) cells harboring plasmid pNLW1 (wild-type) were grown and radiolabeled as described in the legend to Fig. 3. The kinetics of degradation was assayed by chasing these cells with unlabeled methionine and analyzing by SDS-PAGE and fluorography the amounts of beta* at the indicated time points after beginning the chase. The details are described under ``Experimental Procedures.'' The graph shows the results of tracing the fluorogram with a Molecular Dynamics 300A computing densitometer.



Mutating ATG (Met2) had essentially no effect on the rate of synthesis of beta* (Fig. 3). In contrast, mutating ATG (Met1) caused a 3-fold reduction in the rate of synthesis of beta* (Fig. 3), suggesting that ATG is the initiation codon of dnaN*.

To further support this conclusion, we have constructed another set of plasmids, in which the dnaN* gene was cloned under the lac promoter in plasmid pUC18. In this case we detected beta* by Western blot analysis of cell extracts, using affinity-purified anti-beta antibodies. As can be seen in Fig. 5(lanes 7-12), when the lac promoter was repressed by glucose, beta* was not produced. Upon induction by IPTG, the dnaN* plasmid yielded two products, a major product that comigrated with a sample of beta* purified from an overproducing cell and a minor product that migrated slightly faster (Fig. 5, lane 2). The major product comigrated with beta* synthesized in vivo from the chromosome(48) . As can be seen in Fig. 5(lane 3), mutating the ATG codon resulted in disappearance of the major beta* band, whereas the minor beta*-related band remained unchanged. On the other hand, mutating ATG (Fig. 5, lane 4) eliminated the minor band, and caused also a reduction in beta*. These results indicate that ATG is the initiation codon of dnaN*. The minor band seems to be the result of an alternative initiation from ATG when beta* was present on a plasmid.


Figure 5: The effects of site-directed mutations in the translation control elements of dnaN* on the synthesis of beta*. E. coli AB1157XL cells harboring mutated dnaN* genes cloned under the lac promoter in plasmid pBluescript SK were grown to mid-logarithmic phase and then treated with IPTG to induce the synthesis of beta*. Total cell lysates were fractionated by SDS-PAGE, blotted to a nitrocellulose membrane, and probed with affinity-purified anti-beta subunit antibodies using the enhanced chemiluminescence method for detection. The details are given under ``Experimental Procedures.'' Lanes 1-6 show IPTG-treated cells, whereas lanes 7-12 show controls with glucose repression. Lanes 1 and 7, cells harboring the vector pBluescript SK; lanes 2 and 8, cells harboring the wild-type dnaN* plasmid pBSOW1; lanes 3 and 9, cells harboring plasmid pBSOM11, with the ATG CTG dnaN* mutation (Met1); lanes 4 and 10, cells harboring plasmid pBSOM22 carrying the ATG CTG dnaN* mutation (Met2); lanes 5 and 11, cells with plasmid pBSORS1 carrying the GCAGG ACAAG mutation in the dnaN* Shine-Dalgarno sequence (SD-S); lanes 6 and 12, cells with plasmid pBSORM1 carrying the dnaN* mutation SD-M GCAGG GCAAA mutation in the dnaN* Shine-Dalgarno sequence (SD-M). Lane M contains purified beta subunit and beta* as markers.



The assignment of ATG as the initiation codon of dnaN* pointed to the GCAGG sequence as a likely Shine-Dalgarno sequence involved in ribosome binding. In order to examine this possibility we prepared two Shine-Dalgarno double mutants: GCA GG ACA AG (SD-S) and GCA GG GCA AA (SD-M). As can be seen in Fig. 5(lanes 5 and 6), both mutants exhibited reduced expression of dnaN*, consistent with the suggested role of the GCAGG sequence in ribosome binding.

The dnaN* Transcript Is Induced by Nalidixic Acid-Total RNA was isolated from E. coli cells, and the 5` termini of mRNAs initiating at the promoter region of dnaN* were analyzed using the RNase protection techniques. Several transcription initiation sites could be detected in the region analyzed, including the major recF transcript initiating at promoter P1, and a fully protected RNA probe, which represents the overlapping dnaN mRNA (Fig. 6, P(N)). In the dnaN* promoter region, a band of approximately 130 bases was detected, suggesting that transcription of dnaN* starts near position 2013 (Fig. 2).


Figure 6: Mapping of transcription initiation sites in the dnaN* control region by the RNase protection technique. Upper panel, cellular RNA was extracted from MC4100 wild-type cells at the indicated time points after induction of the SOS response by nalidixic acid (40 µg/ml). The RNA was purified and then hybridized to a uniformly labeled RNA probe transcribed from plasmid pRPHF11. The hybrids were digested with RNase A and RNase T(1), then treated with proteinase K, extracted with phenol, and separated on a denaturing 6% urea-polyacrylamide gel, after which the gel was dried and autoradiographed. P, P and P represent transcription initiation at the promoters of dnaN, dnaN*, and the major promoter of the recF gene, respectively. The weak second promoter of recF, P, is hardly seen under our conditions. The details are presented under ``Experimental Procedures.'' Lower panel, the riboprobe used in the assay shown in the upper panel and the predicted sizes of its protected regions that hybridize to mRNAs initiating inside the dnaN* gene.



The promoter region of the dnaN* gene contains the sequence 5`- CGCTGTCTACCCTGCCAGCG-3` (positions 1960-1979; Fig. 2), resembling the consensus sequence of the binding site of the LexA repressor, 5`-NNCTGTNTatNcaNNCAGNN-3`(3) . The most conserved 8 nucleotides are present in the dnaN* SOS box-like sequence, including the inverted repeat CTGNCAG, which in our case is part of a pentanucleotide inverted repeat, CGCTGNCAGCG. If indeed this sequence binds LexA, it is expected that the gene will be inducible by agents that induce the SOS regulon. In agreement with such a prediction, UV irradiation of E. coli cells was found to cause induction of beta*, the dnaN* gene product(48) . As can be seen in Fig. 6, treatment of cells with nalidixic acid, a potent inducer of the SOS and the heat shock responses, caused a 4-5-fold induction in the dnaN* transcript. Thus, induction of dnaN* expression is regulated, at least in part, at the transcriptional level.

In order to examine whether dnaN* is controlled directly by LexA, the global SOS repressor, we studied the binding of purified LexA repressor to the promoter region of dnaN*, using the gel mobility shift assay(22) . Binding of LexA to the promoter region of recA has been demonstrated by this technique(24) . Indeed, the LexA protein caused specific retardation of a 148-bp MspI restriction DNA fragment carrying the recA promoter which served as a positive control (data not shown). However, we could not to detect any specific binding of LexA to the dnaN* promoter region under a variety of condition (data not shown). This suggests that the inducibility of the dnaN* gene is not regulated directly by LexA.

The region of the recF promoters located inside the dnaN* gene contains an antisense promoter, termed P(x)(25) (Fig. 2). The transcript directed by this promoter is complementary the first 86 nucleotides of the dnaN* transcript. We have confirmed the activity of this promoter in our cells and found that its transcript was unaffected by treatment with nalidixic acid (data not shown). The role of this transcript is not clear, although it may function to regulate dnaN* and/or dnaN expression.

Transcriptional dnaN*-cat Gene Fusions Are UV-inducible

In order to examine by an independent method the induction of dnaN* at the transcriptional level we have utilized dnaN*-cat gene fusions. Since dnaN* has a weak promoter, it was necessary to use a vector with a low background of cat activity. Our vector, termed pCAT, carries the coding sequence of the cat gene under the control of the tet promoter (Fig. 1) and the independent T(1)rrnB terminator located upstream to cat. In order to examine the background CAT activity of a promoterless cat gene in plasmid pCAT we have deleted the 162-bp ClaI-EcoRV fragment(23-185), containing the tet promoter and inserted a spacer sequence between the terminator and the cat open reading frame. The spacer was the 141-bp HgaI-(2037)-BstUI-(1896) fragment from the dnaN gene, lacking any known promoter sequence (in the orientation opposite to dnaN*). Protein extracts prepared from cells harboring the resultant plasmid, termed pNCH20, had a background of 0.025 unit. UV irradiation of these cells did not affect CAT activity, as expected.

We have used the recA promoter, a classical SOS promoter, to serve as a positive control for a UV-inducible gene (Fig. 7). The CAT activity of cells harboring plasmid pRC5, containing the recA-cat fusion, was 4 units/mg of protein. UV irradiation at various doses led to the induction of CAT activity peaking at 30 min after irradiation. The extent of induction increased with increasing UV dose, up to an effect of 8-fold at 30 J m (Fig. 7). The increase in recA transcription as assayed by this recA-cat gene fusion is similar to the results obtained by assaying directly the level of recA mRNA, where a maximal 8-9-fold induction was found after 20 min(26) . Based on these results we used an inducing dose of 30 J m for examining the UV inducibility of dnaN*.


Figure 7: UV-dose dependence of the induction of CAT activity from a recA-cat fusion. AB1157 cells harboring the recA-cat fusion plasmid pRC5 were UV-irradiated at various doses and assayed for CAT activity at the indicated time points after irradiation as described under ``Experimental Procedures.'' The inducing UV doses were 0 (open circles); 2 J m (black squares); 5 J m (white triangles); 15 J m (black circles); 30 J m (white squares).



We have constructed three dnaN*-cat fusions plasmids, containing various portions of the dnaN* gene (Fig. 8). Plasmid pNCB17 contains the 332-bp BstUI (1896)-BstUI(2228) fragment of dnaN containing the two recF promoters, and 147 nucleotides upstream to the initiation codon of dnaN* including the SOS box-like sequence, the promoter, and the Shine-Dalgarno sequence. Plasmid pNCS14 contains the 172-bp SfaNI-(1918-2090) DNA fragment of dnaN, including the control region of dnaN*, but lacking the recF promoters. Plasmid pNCH6 contains the 141-bp BstUI-(1896)-HgaI-(2037) fragment, containing the SOS box-like sequence and the promoters of dnaN*, but no coding sequences. All three dnaN* gene fragments exhibited weak promoter activities as judged by the level of CAT activity (Fig. 9). The activity varied from 0.04 to 0.1 unit, which is 2-4-fold higher than the background activity of the control plasmid without a promoter (pNCH20). UV irradiation of cells harboring the dnaN*-cat gene fusion plasmids caused a 3-fold induction of CAT activity (Fig. 9), consistent with the dnaN* transcript analysis (Fig. 6). This included plasmid pNCS14 which did not contain the recF promoters. In order to analyze the P(x) promoter we have constructed a P(x)-cat fusion using the 138-bp BstUI-(2228)-SfaNI-(2090) DNA fragment (Fig. 8), containing the P(x) promoter. The resultant plasmid, termed pPC1, had a basal activity of 0.35 unit, which was 3-4-fold higher than the dnaN* gene fusion (Fig. 9). UV irradiation of cells harboring plasmid pPC1 did not affect CAT activity (Fig. 9). Thus, in contrast to the dnaN*-cat fusions, the antisense P(x)-cat fusion was not inducible by UV light, in agreement with the P(x) transcript analysis.


Figure 8: Structure of dnaN* gene fusions. Fragments containing various portions of dnaN* were fused to the cat gene in plasmid pCAT to form transcriptional gene fusions (above the dotted line) or to the 8th codon of the lacZ gene in plasmid pMC1403 to form translational gene fusions (below the dotted line). The constructions are described in detail under ``Experimental Procedures.'' The fused parts of the reporter genes are indicated by striped (cat) or gray (lacZ) bars. The arrows inside the bars indicate the direction of transcription of dnaN*. Thus, in plasmid pPC1 cat is transcribed from the antisense promoter P(x), and in the control plasmid pNCH20 there is no known promoter to transcribe cat.




Figure 9: UV-induction of CAT activity from plasmids carrying dnaN*-cat transcriptional gene fusions. E. coli AB1157 cells harboring the indicated dnaN*-cat gene fusion plasmids were UV-irradiated at a UV dose of 30 J m and assayed for CAT activity 75 min after irradiation as described under ``Experimental Procedures.'' Light bars, unirradiated cells; dark bars, UV-irradiated cells.



dnaN*-lacZ Translational Gene Fusions Are UV-inducible in a recA- and lexA-dependent Pathway

To further examine the UV inducibility of dnaN*, we used translational fusions to the lacZ gene (Fig. 8). Plasmid pTEN5 contains the 172-bp SfaNI DNA fragment(1918-2090) that contains the dnaN* promoter and the first 15 codons, plasmid pNB3 contains the BstUI DNA fragment(1896-2228) including the dnaN* promoter and the first 61 codons of dnaN* (a region that includes the two recF promoters), and pSB6, which contains a 263-bp AccI-(1965)-BstUI-(2228) DNA fragment, lacking the 5`-half of the SOS boxlike sequence ( Fig. 2and Fig. 8). Plasmid pTEN5 is the only dnaN* gene fusion plasmid that did not contain the two recF promoters that are located inside dnaN* ( Fig. 2and Fig. 8).

Plasmids pTEN5 and pNB3 gave rise to similar constitutive levels of beta-galactosidase activity, indicating that the recF promoters did not contribute to the expression of the gene fusion (Fig. 10, A and C). Upon UV irradiation of cells harboring these gene fusions, an increase of beta-galactosidase activity was observed. The level of induction was 5-10-fold, and was the same also for plasmid pSB6, in which the 5`-half of the SOS box-like sequence was deleted (Fig. 10D). The UV induction was completely abolished in isogenic mutant cells with either a lexA3 or recA56 mutation (Fig. 10). The lexA3 mutation renders the LexA repressor non-cleavable by activated RecA protein, whereas the recA56 mutation inactivates the RecA protein, thus the SOS response cannot be induced in cells carrying either of these mutations. The noninducibility of dnaN* in these strains suggested that its expression is under the control of the SOS stress regulon. The uvrA6 mutation, which inactivates nucleotide excision repair, and the umuC36 mutation, that inactivates UV mutagenesis, did not affect the UV inducibility of the dnaN*-lacZ fusions, indicating that the induction was not dependent on excision repair or UV mutagenesis (data not shown).


Figure 10: Kinetics of UV-induction of beta-galactosidase activity from dnaN*-lacZ translational gene fusions. Cells harboring plasmids with lacZ translational fusions were UV-irradiated and assayed for the amount of beta-galactosidase activity at the indicated time points after irradiation as described under ``Experimental Procedures.'' A, cells harboring plasmid pTEN5; B, cells harboring the control plasmids pHSA2 (dnaA-lacZ; circles and triangles), and pHSC6 (cI-lacZ; diamonds); C, cells with plasmid pNB3; D, cells with plasmid pSB6. Open symbols, unirradiated cells; closed symbols, UV-irradiated cells. Circles, E. coli KY700 (wild-type); squares, E. coli KY703 (lexA3); triangles, E. coli KY705 (recA56). The inducing UV doses were 60, 5 and 1 J m for strains KY700, KY703, and KY705, respectively.



The control fusion of the dnaA gene did show UV induction consistent with the report on the inducibility of dnaA by mitomycin C(27) . However, as can be seen in Fig. 10B, the UV induction was not dependent on the recA gene product, implying that the SOS response was not involved. The negative control for induction was the cI-lacZ fusion that was noninducible by UV irradiation (Fig. 10B).

The kinetics of induction of the dnaN*-lacZ fusions showed peak levels of beta-galactosidase activities at 3-5 h after irradiation (Fig. 10), whereas many SOS functions(26) , as well as the UV induction of beta* (48) peak an hour or less after irradiation. A similarly slow induction of SOS-inducible genes fused to lacZ was observed before(28) . This may be the result of the fact that the active structure of beta-galactosidase is a tetramer(29) , and that oligomerization of the fused beta-galactosidase molecules might be slow, particularly when their concentration is low. Indeed, when the induction of the beta*-beta-galactosidase protein was examined at the protein level, by Western blot analysis using polyclonal antibodies against beta-galactosidase, maximal induction of beta-galactosidase occurred approximately 60-90 min after UV irradiation (Fig. 11). This time period is close to the time of induction of the beta* protein(48) . This result shows that the UV-induced increase in the activity of beta-galactosidase was indeed due to an increase in the synthesis of the enzyme, and it is consistent with the suggestion that the slower kinetics of induction of the activity of the fused enzyme was due to the slow rate of assembly of the active tetrameric structure.


Figure 11: Kinetics of UV-induction of a beta*-beta-galactosidase fused protein assayed by immunoblot analysis. E. coli MC4100 cells harboring plasmid pTEN5 or the control plasmid pMC1403 were UV-irradiated at 50 J m and assayed for the induction of the beta*-beta-galactosidase protein by immunoblot analysis using anti beta-galactosidase antibodies and the enhanced chemiluminescence method for detection. Lane M contains a marker of beta-galactosidase.



dnaN* Is under the Control of the Heat Shock Response

UV irradiation and nalidixic acid induce in E. coli both the SOS and heat shock responses(5) . In order to test the involvement of the heat shock response in UV induction of dnaN we assayed a dnaN*-lacZ fused gene in cells which lack the heat shock subunit. E. coli R40NL8 is a derivative of E. coli MC4100 that has a deletion in rpoH, the gene encoding . Because is essential for growth at temperatures above 20 °C, R40NL8 (like its parent R40) (14) contains a suppressor mutation that causes overproduction of groE, and thus enables it to grow at 37 °C (but not at 42.5 °C). The kinetics of beta-galactosidase induction from the dnaN*-lacZ gene fusion was determined after UV-irradiation of the DeltarpoH30 mutant and its isogenic wild-type parent. As seen in Fig. 12B, there was no UV induction of beta-galactosidase activity from pTEN5 in cells which lack the heat shock subunit, whereas a 10-fold increase in activity was found in the isogenic wild-type cells (Fig. 12A). Interestingly, the basal level of beta-galactosidase from plasmid pTEN5 was the same in the wild-type and DeltarpoH30 strains. In order to establish that the loss of UV inducibility in strain R40NL8 was indeed due to the absence of the subunit, we supplied it in trans from plasmid pCM1. This plasmid did not affect the UV inducibility of the dnaN*-lacZ fusion from pTEN5 in the wild-type parent MC4100 (Fig. 12A). However, introduction of pCM1 into R40NL8 harboring pTEN5, rendered the dnaN*-lacZ fusion UV-inducible again (Fig. 12B). The extent of UV-induction of the dnaN*-lacZ fusion was 3-fold lower than in the wild-type strain. This may be due to the fact that the strains are not fully isogenic (i.e. R40NL8 but not MC4100 overproduces groE). In any case, it is clear that introducing the plasmid that expressed made the dnaN*-lacZ gene fusion UV-inducible again, suggesting that dnaN* is controlled by the heat shock activator, the RNA polymerase subunit.


Figure 12: Effect of the activator of the heat shock response on the induction of a dnaN*-lacZ translational fusion. Cells harboring plasmid pTEN5 were UV-irradiated at 50 J m and assayed for beta-galactosidase activity at the indicated time points after irradiation as described under ``Experimental Procedures.'' White symbols, unirradiated cells; black symbols, UV-irradiated cells. A, E. coli MC4100 (wild-type) cells harboring plasmid pTEN5 (triangles) or both plasmids pCM1 (carrying the rpoH gene) and pTEN5 (circles). B, E. coli R40NL8 (DeltarpoH) cells carrying plasmid pTEN5 (triangles) or both plasmids pCM1 and pTEN5 (circles).



We attempted transcribe dnaN* in vitro using purified RNA polymerase. We were unable to detect any in vitro initiation of transcription from the dnaN* promoter using either the regular RNA polymerase, or the heat shock-specific RNA polymerase, although the recF transcripts were observed (data not shown). Thus, it seems that transcription of dnaN* requires additional factors, or possibly another subunit. Possible candidates are , which is specific for some heat-induced genes(30, 31) , and ^s, which transcribes stationary phase genes (32, 33) . Consistent with such a possibility we found a higher amount of beta* in stationary phase cells(48) .


DISCUSSION

We have previously shown that a smaller form of the beta subunit of DNA polymerase III holoenzyme is induced in E. coli by UV irradiation(48) . Such a protein can be generated by proteolytic processing, like the mutagenesis protein UmuD`, that is formed from UmuD by specific proteolysis promoted by the RecA protein(10) . Alternatively, the protein can be translated from the dnaN mRNA by a de novo translational start, or it can be expressed from an internal in-frame gene.

The data presented here suggests that beta* is expressed from an internal in-frame gene termed dnaN*. This is based on the following observations. 1) The ATG initiation codon of dnaN* and its Shine-Dalgarno sequence were identified by site-directed mutagenesis. 2) A transcription initiation site was mapped inside dnaN, upstream to a Shine-Dalgarno sequence. 3) Plasmids carrying the dnaN* gene expressed beta*. 4) When cloned into a plasmid, the promoter region of dnaN* directed the expression of a promoter-less cat gene. 5) When the control region of dnaN*, including the beginning of its coding region, was fused in-frame to a portion of the lacZ gene lacking all transcriptional and translational control elements as well as its first 8 codons, it directed the synthesis of a fused beta*-beta-galactosidase protein.

The expression of dnaN* is complex and is likely to be regulated via several mechanisms. Transcription of dnaN* was not observed in vitro using either or RNA polymerase, suggesting that another transcription factor is required. Internal initiation of translation at the dnaN* ATG initiation codon on the intact dnaN mRNA seems to be very inefficient. This is indicated by the fact that overexpressing dnaN mRNA from the lac promoter on a plasmid did not yield any detectable beta*. Only after introducing a frameshift mutation into dnaN, upstream to dnaN*, that eliminated overproduction of the beta subunit, expression of beta* was observed from dnaN mRNA(48) . Thus, it seems that, under normal conditions, synthesis of beta* from dnaN mRNA is strongly inhibited, e.g. due to its engagement in translation of the beta subunit or due to direct inhibition by the beta subunit. The antisense transcript originating from P(x), may also be involved in the down-regulation of the expression of dnaN*.

UV induction of dnaN* is regulated at the transcriptional level, and subjected to control by both the SOS and heat shock responses, as indicated by the dependence of UV induction of dnaN*-lacZ gene fusions on recA, lexA, and rpoH. However, this dual regulation is indirect, since dnaN* did not bind LexA, and it was not transcribed by RNA polymerase. Thus, another factor(s) that is controlled by these major stress responses, is responsible for the UV induction of dnaN*. The role of the SOS box-like sequence in the promoter region of dnaN* is puzzling. It may represent a degenerated LexA binding site, or it may be a coincidental homology of no functional role, especially since its 5`-half was found to be dispensable for UV induction of dnaN*-lacZ gene fusions. It should be noted that if the sequence 5`-TACTGTATATATATACAGTA-3` is taken as the consensus LexA binding site, then based of the differences between it and the dnaN* SOS box-like sequence(34) , the latter is predicted to have no specific binding to LexA. Similar SOS box-like sequences, that did not bind LexA, were found in the phr gene, encoding DNA photolyase(35) , and in the uvrC gene, encoding a subunit of the UvrABC repair excinuclease(36) ; however, their significance remains unclear. In addition to dnaN* at least three other genes are inducible by DNA-damaging agents in a recA- and lexA-dependent pathway, but are not directly regulated by LexA: The phr gene mentioned above(35) , the dnaQ gene encoding the proofreading subunit of DNA polymerase III(37) , and the dnaN gene(37, 38) . The mechanism of this regulation is unknown yet, representing another layer of complexity of the SOS regulatory network. It may be performed by a factor which is by itself repressed directly by LexA.

Genes whose coding sequences overlap are not rare; however, extensively overlapping genes, or genes nested within other genes, are not common in the chromosome(39) . A well documented case is the phage T7 gene gp4, encoding a helicase-primase. The gene encodes two proteins of 63 and 56 kDa, the latter generated by an internal in-frame start site(40) . The dnaX gene encodes two subunits of DNA polymerase III holoenzyme: and . They both start at the same site, but is terminated before by a mechanism of ribosomal frameshifting, leading to the production of proteins of 47.5 and 71 kDa (7) .

The expression of the internal dnaN* gene produces a protein that lacks precisely one of the three repeating domains of the beta subunit. In this respect it belongs to a family of mechanisms such as alternative splicing, that produce from a single gene more than one protein, differing by a one or more defined functional domains. Such mechanisms generate a protein (or more) with a subset of the properties of the parental intact protein. They might be required to fulfill biochemically similar reactions under different conditions, or with conjunction with different counterpart proteins. Such are the cases of the dnaX gene and the T7 gp4 genes. The intact beta subunit forms a beta(2) ring-shaped sliding DNA clamp, that confers high processivity on DNA polymerase III holoenzyme by tethering it to the DNA(41, 42) . As shown in a companion study(49) , beta* forms an alternative DNA clamp for DNA polymerase III that may have a specialized function connected to DNA synthesis in the UV-irradiated cell. The increase in UV resistance caused by overproducing beta* is consistent with such a model(48) .


FOOTNOTES

*
This research was supported by grants from the the Dorot Science Fellowship Foundation and the Scheuer Research Foundation the Israel Academy of Sciences, and from the The Forchheimer Center for Molecular Genetics. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed. Tel.: 972-8-343203; Fax: 972-8-9344169; :BCLIVNEH{at}WEIZMANN.WEIZMANN.AC.IL.

(^1)
IPTG, isopropyl beta-D-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; CAT, chloramphenicol acetyltransferase; SD, Shine-Dalgarno; LB, Luria-Bertani medium; bp, base pair(s); kb, kilobase pair(s).


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