©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
*, a UV-inducible Shorter Form of the Subunit of DNA Polymerase III of Escherichia coli
II. OVERPRODUCTION, PURIFICATION, AND ACTIVITY AS A POLYMERASE PROCESSIVITY CLAMP (*)

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

Rami Skaliter Moshe Bergstein Zvi Livneh (§)

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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Control elements located inside the coding sequence of dnaN, the gene encoding the beta subunit of DNA polymerase III holoenzyme, direct the synthesis of a shorter and UV-inducible form of the beta subunit (Skaliter, R., Paz-Elizur, T., and Livneh, Z.(1996) J. Biol. Chem. 271, 2278-2281, and Paz-Elizur, T., Skaliter, R., Blumenstein, S., and Livneh, Z.(1996) J. Biol. Chem. 271, 2282-2290). The protein, termed beta*, was overproduced using the phage T7 expression system, leading to its accumulation as inclusion bodies at 5-10% of the total cellular proteins. beta* was purified in denatured form, followed by refolding to yield a preparation >95% pure. Denatured beta* had a molecular mass of 26 kDa and contained two isoforms when analyzed by two-dimensional gel electrophoresis. The major isoform had a pI of 5.45, and comigrated with cellular beta*. Size exclusion high performance liquid chromatography under nondenaturing conditions and chemical cross-linking experiments indicate that beta* is a homotrimer. DNA synthesis by DNA polymerase III* was stimulated up to 10-fold by beta*, primarily due to an increase in the processivity of polymerization. It is suggested that beta* functions as an alternative sliding DNA clamp in a process associated with DNA synthesis in UV-irradiated cells.


INTRODUCTION

DNA-damaging agents in general, and UV radiation in particular, affect dramatically the physiology of Escherichia coli(1) . These changes are regulated at the molecular level by several stress regulons, most notably the SOS and heat shock responses(2, 3, 4) . The immediate response is a transient arrest of DNA replication(5, 6, 7) which provides time for the repair of the damaged DNA. DNA replication than recovers in a process that requires SOS-inducible proteins(5, 6, 7) , but its mechanism is largely unknown. During the post-UV recovery period mutations are formed, primarily at sites of DNA damage. This mutagenesis pathway is believed to occur by polymerization through DNA lesions, a process termed bypass synthesis(7, 8, 9) .

A detailed biochemical analysis of in vitro replication of UV-irradiated or depurinated DNA with purified DNA polymerase III (Pol III) (^1)holoenzyme (10, 11, 12, 13) led us to concentrate on the beta subunit of the polymerase and to conclude that it modulates UV mutagenesis in vivo(14) and bypass of UV lesions in vitro(15) . The beta subunit is the major processivity factor of Pol III holoenzyme(16) . It is a homodimer that forms a ring structure(17) , and once loaded on DNA by the complex it functions as a sliding DNA clamp that tethers the polymerase to the DNA, thus endowing it with high processivity(18, 19, 20) . In companion studies (39, 40) we reported that in UV-irradiated E. coli cells a shorter form of the beta subunit, termed beta*, is produced, which corresponds to the C-terminal two-thirds of the beta subunit. This study describes the overproduction, purification, and characterization of beta* and its activity as an alternative processivity clamp for DNA polymerase III.


MATERIALS AND METHODS

Plasmids

Plasmid pSK11 that overproduces beta* was constructed as follows. The source for the dnaN* gene was plasmid pUN222, which is similar to pUN234 (14) except that the dnaN gene was cloned opposite to lacP. It was constructed by cloning the SspI(1516)-StyI (3031) DNA fragment carrying the entire dnaN gene from plasmid pSJS9 (21) into the SmaI site of plasmid pUC18. Plasmid pUN222 was digested with restriction nucleases EcoRI and AvaI, to yield a 1125-base pair fragment, that was further digested with HgaI. This resulted a 860-base pair fragment which carries the entire dnaN* gene, with its ATG codon at the 5`-terminus of the fragment. The ends of the fragment and of NdeI-cleaved plasmid pET3a (22) were filled-in with the Klenow fragment of DNA polymerase I and then the two DNA fragments were ligated to form plasmid pSK11. Plasmid pKT11 was derived from pSK11 by deleting a 134-base pair segment from the SacII site(2940) located downstream to the dnaN* terminator down to the BamHI site in the polylinker. This removed the recF sequence downstream to the dnaN* gene.

Proteins and Chemicals

The beta subunit was purified from JC13576 cells harboring plasmid pSJS9 as described(21) . DNA polymerase III* and single-strand DNA binding protein were purified as described by Lasken and Kornberg (23) and Weiner et al.(24) , respectively. Affinity-purified antibodies against the beta subunit and against beta* were described in companion studies(39, 40) . Restriction nucleases were purchased from Pharmacia Biotech Inc. and from New England Biolabs. T4 DNA ligase was the product of Stratagene, DNA polymerase I (Klenow fragment) and calf alkaline phosphatase were from U. S. Biochemical Corp., and chicken egg lysozyme was from Sigma. The sources for materials used are as follows: sodium deoxycholate, ethyl dimethyl carboimidate, and N-hydroxysuccinimide, Sigma; nucleotides and ampholines, Pharmacia; radiolabeled nucleotides, Amersham Corp.; dimethyl suberimidate, Pierce.

Purification of beta* and Inclusion Bodies

E. coli BL21(DE3) cells harboring plasmid pKT11 were grown at 37 °C in a 12-liter fermentor with constant stirring and aeration, to 100 Klett units. The cells were treated with 0.5 mM IPTG for 2 h to induce the synthesis of beta*. The cells (18 g) were collected, resuspended in an equal volume of a buffer containing 50 mM TrisbulletHCl, pH 7, 15% sucrose, and frozen in liquid nitrogen. The cells suspension was thawed at 10 °C, and its volume was increased to 100 ml with the same buffer. Cells were then disrupted by a 5-min sonication period in a Soniprep MSE sonicator equipped with a medium tip at 3/4 power, and centrifuged at 25,000 rpm in a Beckman Ti45 rotor for 1 h. The supernatant was discarded and the pellet was homogenized in water, spun at 15,000 rpm in a Ti45 rotor for 0.5 h, and washed again with water. The pellet was homogenized and treated with chicken egg lysozyme (0.3 mg/ml) for 1 h at 22 °C, followed by 2% sodium deoxycholate for 1 h at 22 °C. Following centrifugation in a Beckman Ti45 rotor at 15,000 rpm for 0.5 h, the pellet was washed once with a solution of 4 M urea in water for 0.5 h and twice with water. The washed inclusion bodies (final yield 200 mg) contained >90% pure beta* as estimated by Coomassie Blue staining.

Denaturation

The inclusion bodies were denatured with a buffer containing 8 M urea, 50 mM TrisbulletHCl, pH 7.4, 10 mM dithiothreitol, and 1 mM EDTA (buffer A). After 2 h of incubation at 4 °C, the solution was spun at 40,000 rpm in a Beckman Ti45 rotor at 4 °C to precipitate the insoluble material. The solution of denatured protein was incubated for 18 h at 4 °C with constant stirring. The solution was then cleared at 40,000 rpm in a Ti45 rotor at 4 °C for 2 h.

Q-Sepharose Chromatography

Denatured beta* were loaded on a 15-ml Q-Sepharose (Pharmacia) column equilibrated with 7 M urea, 50 mM Tris, pH 7.4, 2 mM dithiothreitol, and 0.5 mM EDTA (buffer B) at a rate of 1 column volume/h. The column was washed with 2 column volumes of buffer B, and the proteins were eluted with 10 volumes of a linear gradient of 0-500 mM KCl in buffer B. beta* eluted at 150 mM KCl.

Phosphocellulose Chromatography

The peak fractions from the Q-Sepharose column were combined and fractionated on a phosphocellulose column (Whatman, P11) in buffer B. beta* eluted in the flow-through. This column was used in order to remove DNA-binding proteins such as nucleases that interfere with replication assays.

Refolding of beta*

The fractions containing beta* (5 mg) were diluted to a concentration of 0.1 mg/ml protein and dialyzed against a refolding buffer containing 0.2 M KCl, 50 mM TrisbulletHCl, 0.5 mM EDTA, 10 mM beta-mercaptoethanol, and 10% glycerol (buffer C). After dialysis was completed, the solution was cleared with a 2-h spin at 200,000 times g. The supernatant contained the soluble beta*. About 40% of the beta* (2 mg) remained soluble. The purity was greater than 95% as estimated by Coomassie Blue staining. beta* was concentrated using Centriprep 10 (Amicon) and by dialysis against buffer C with 50% glycerol. Refolding at concentrations higher than 0.1 mg/ml led to precipitation of beta*.

Size Exclusion Chromatography

beta* was analyzed by HPLC size exclusion chromatography on a TSK3000SW column (Tosohass) equilibrated with a buffer containing 50 mM TrisbulletHCl, pH 7.4 and 0.1 M KCl at a flow rate of 0.7 ml/min. The elution profile was monitored in parallel with a UV detector at 214 nm and by Western blot analysis, using anti-beta antibodies. The column was calibrated with HPLC standards (Boehringer Mannheim).

Chemical Cross-linking of Proteins

beta* at a concentration of 10 µg/ml was incubated with 1 mg/ml dimethyl suberimidate (25) in a buffer containing 100 mM TrisbulletHCl pH 8.5 for 4 h at room temperature, after which it was fractionated by SDS-PAGE on an 8% gel. The cross-linked proteins were detected by Western blot analysis with anti-beta antibodies. The beta subunit (30 µg/ml in 50 mM TrisbulletHCl, pH 7.4) was cross-linked with 66 mM ethyl dimethyl carboimidate and 13 mMN-hydroxysuccinimide for 4 h at room temperature.

Purification of beta* from a Denaturing Polyacrylamide Gel

One mg of denatured beta* was fractionated by SDS-PAGE (12.5% gel). The edges of the gel were cut out and stained with Coomassie Blue in order to localize beta*. The gel region containing beta* was cut out, and dialyzed against PAGE running buffer containing 10% glycerol. beta* was electroeluted overnight at 70 V at 4 °C. At the end a reverse voltage of 100 V was applied for 30 min order to detached the protein from the wall of the dialysis bag. The solution containing beta* was cleared by centrifugation at 200,000 times g and dialyzed again overnight at 4 °C against a renaturation buffer containing 50 mM TrisbulletHCl, 10% glycerol, 100 mM NaCl, 0.5 mM EDTA, and 1 mM dithiothreitol. The final dialysis was against the same buffer except that the glycerol was raised to 50%. The final concentration of beta* was 0.1 mg/ml.

Standard Replication Assays

The synthetic oligonucleotide primer 5`-GAAACCATCGATAGC-3`, complementary to nucleotides 2524-2538 in M13mp8 ssDNA, was annealed at a molar ratio of 30:1 to SSB-coated M13mp8 circular ssDNA (0.83 µg of SSB and 35 fmol of circles). Annealing was carried out for 10 min at 30 °C in a final volume of 25 µl containing 20 mM TrisbulletHCl, pH 7.5, 80 µg/ml bovine serum albumin, 5 mM dithiothreitol, 4% glycerol, 8 mM MgCl(2), and 0.1 mM EDTA. The standard replication mixture contained the annealing mixture supplemented with 0.5 mM ATP, 40 mM KCl, 50 µM dGTP, 50 µM dCTP, 0.15 pmol of DNA polymerase III*, 35 fmol (as circles) of primed M13mp8 ssDNA in a volume of 25 µl. beta* (as a trimer) and the beta subunit (as a dimer) were added when required, usually at a final concentration of 200 nM. After a 10-min preincubation the reaction was started by the addition of 50 µM dATP and 10 µM [alpha-P]dTTP. The reaction was carried out at 30 °C. Incorporation of [alphaP]dTTP into acid insoluble material was determined by trichloroacetic acid precipitation, and replication products were analyzed by alkaline agarose gel electrophoresis as described previously(26) .

Processivity of DNA Polymerase III*

This was assayed under standard conditions as described above except that an excess of primed M13mp8 ssDNA (2.8 pmol) over the polymerase (0.05 pmol) was used, and SSB was omitted.


RESULTS

Overproduction of beta*

Due to the low abundance of beta*, we decided to overproduce it before attempting purification. We chose the phage T7 expression system(22) , and cloned the coding sequence of the dnaN* gene by precise tailoring of the ATG initiation codon of dnaN* to the Shine-Dalgarno and promoter sequences of the phage T7 10 gene in plasmid pET3a to yield plasmids pSK11 and pKT11. Pulse labeling with [S]methionine of cells harboring plasmid pKT11 followed by SDS-PAGE and autoradiography revealed that, 30 min after addition of IPTG, beta* was synthesized almost exclusively (Fig. 1). Based on densitometric scanning of Coomassie Blue-strained gels beta* comprised up to 8% of the total protein (e.g.Fig. 2, lane 1). Immunoblots of extracts of overproducing cells were probed with polyclonal antibodies against the beta subunit of Pol III, assuming that at least some antigenic determinants are common to beta* and the beta subunit. Indeed, the overproduced protein reacted with the anti-beta antibodies (Fig. 1).


Figure 1: Overproduction of beta*. E. coli BL21(DE3) cells harboring plasmid pKT11 were induced by IPTG treatment, and the synthesis of beta* was assayed by Western blot analysis (left panel) or by pulse labeling with [S]methionine (right panel). Cells were collected at the indicated time points after addition of IPTG (0.5 mM), fractionated on 15% SDS-PAGE, and electroblotted onto a nitrocellulose membrane. The membranes were probed with anti-beta subunit antibodies, and reactive proteins were detected by the enhanced chemiluminescence method (ECL, Amersham Corp.; left panel). In parallel, 1-ml samples were withdrawn and labeled with 50 µCi of [S]methionine for 10 min at 37 °C. The cells were then collected, and total cellular proteins were separated by 15% SDS-PAGE. The gel was dried and fluorographed (right panel). C, control cells harboring the vector pET3a.




Figure 2: Purification of beta*. A Coomassie Blue-stained gel summarizing the purification process of beta*. 1, total proteins from beta* overproducing cells; 2, purified inclusion bodies; 3, the peak fraction eluted from a Q-Sepharose column; 4, refolded beta* after phosphocellulose chromatography. 5, protein size standards.



Purification of beta*

The overproduced beta* protein was present in the cells as insoluble inclusion bodies. Those were washed extensively, solubilized in 8 M urea, purified by ion exchange chromatography on Q-Sepharose and phosphocellulose, and refolded (Fig. 2). The critical step in the purification procedure was the refolding step, and successful renaturation required that beta* be present at a concentration of <0.1 mg/ml. Under these conditions, beta* remained soluble and active (Fig. 2; see below). N-terminal protein sequence analysis revealed that the first 10 amino acids of the purified protein were Met Lys Arg Leu Ile Glu Ala Thr Gln Phe, as expected for beta*(40) . The presence of small amounts of the beta subunit in the preparation of beta* may interfere with the assay for beta* activity. We examined the presence of contaminations of the beta subunit in the purified beta* preparation by Western blot analysis using anti-beta subunit antibodies. Even at high amounts of beta* no traces of the beta subunit were detected (Fig. 3; detection limit was 1 ng of the beta subunit).


Figure 3: Analysis of the beta* preparation for the presence of the beta subunit. beta* in increasing amounts was fractionated by 10% SDS-PAGE. The protein was then transferred to a nitrocellulose membrane and probed with anti-beta antibodies. Lanes 1, 2, 3, and 4 contained 1, 2, 5, and 10 µg of beta*, respectively. Lane 4 contained 0.4 µg of the beta subunit.



Analysis of beta* by Two-dimensional Gel Electrophoresis

The purified beta* was analyzed by two-dimensional electrophoresis (isoelectric focusing and SDS-PAGE) according to O'Farrell(27) . After separation the proteins were blotted onto a nitrocellulose membrane and probed with anti-beta subunit antibodies. As can be seen in Fig. 4, beta* had two major isoforms. The major form had a pI of 5.45 in agreement with the calculated pI value of 5.46. The significance of the two isoforms is not known yet. The relative abundance of the two isoforms varied in different preparations, most likely due to variations in the overproduction. The beta subunit which was fractionated in the same gel as an internal marker had a pI of 5.15.


Figure 4: Immunoblot of a two-dimensional fractionation of beta*. Purified beta* was separated by two-dimensional gel electrophoresis, isoelectric focusing in the first dimension, and 12.5% SDS-PAGE in the second dimension. The beta subunit was added as an internal marker. Purified beta subunit and beta* were fractionated in the second dimension as markers.



Determination of the Molecular Mass of Native beta*

When analyzed by SDS-PAGE beta* had a molecular mass of 26 kDa as expected from the size of the open reading frame in the dnaN* gene (Fig. 2). In order to estimate the molecular mass of native beta*, we analyzed it by HPLC size exclusion chromatography, using a TSK3000SW column. The elution profile was monitored with a UV detector at 214 nm, and the fractions were tested for the presence of beta* by Western blot analysis. Fig. 5shows the profile of elution from the HPLC column and the Western blot analysis of the protein peak (retention time of 27.4 min). Based on calibration with HPLC protein standards native beta* has an apparent molecular mass of 75 kDa, representing most likely a trimeric form of beta* (calculated molecular mass, 78 kDa).


Figure 5: Size exclusion chromatography of beta* under native conditions. beta* was analyzed on a 60-cm long TSK3000SW size exclusion column, as described under ``Materials and Methods.'' The protein was detected by a Western blot analysis (Panel A) or by absorbance at 214 nm (Panel B). The left lane in Panel A contains a marker of beta*.



In order to further support its suggested trimeric structure, the beta subunit and the HPLC peak of beta* were each subjected to chemical cross-linking. These proteins proved to be remarkably resistant to cross-linking attempts by a variety of chemical agents. Successful cross-linking of beta* was achieved with dimethyl suberimidate(25) . The cross-linked products were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with anti-beta antibodies. Despite considerable efforts to reduce smearing, the treated proteins migrated as smeared bands; still the major result is clearly seen. The cross-linking of beta* produced two forms in addition to the monomeric beta*: a beta* dimer formed most likely due to partial cross-linking, and a putative beta* trimer representing fully cross-linked beta* (Fig. 6, lane 1). The apparent molecular mass of the putative beta* trimer was 107 kDa in this gel, higher than the calculated molecular mass of 78 kDa. To examine whether this discrepancy is due to an aberrant migration of the cross-linked protein in the gel we used cross-linked dimeric beta subunit as a marker. The beta subunit was cross-linked with ethyl dimethyl carboimidate and N-hydroxysuccinimide since dimethyl suberimidate was ineffective. As can be seen in Fig. 6(lane 2) the only band in addition to the monomeric beta subunit, presumably the beta subunit dimer, had an apparent molecular mass of 107 kDa, identical to that of the putative beta* trimer. Taken together with the HPLC data these result suggests that the native beta* is a trimer.


Figure 6: Chemical cross-linking of beta*. beta* was cross-linked with dimethylsuberimidate, and the beta subunit was cross-linked with ethyl dimethyl carboimidate and N-hydroxysuccinimide as described under ``Materials and Methods.'' The cross-linked proteins were fractionated by 8% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with anti-beta antibodies. Lane 1, cross-linked beta*; lane 2, cross-linked beta subunit.



beta* Stimulates DNA Polymerase III*

Pol III*, a subassembly of Pol III holoenzyme that lacks the beta subunit, is 50-100-fold less active than the holoenzyme(15, 23) . This is primarily due to the lower processivity of Pol III*, 190 nucleotides per binding event, as compared to a processivity of >8000 nucleotides per binding event of the holoenzyme(16, 28, 29) . Does beta* have a DNA clamp activity similar to the beta subunit, and can it stimulate Pol III*? As can be seen in Fig. 7, beta* stimulated DNA synthesis by Pol III* up to 10-fold. The kinetics of this DNA synthesis was slow relative to that of Pol III holoenzyme(10) , suggesting a slow initiation step and/or slow elongation. We next examined replication products by alkaline agarose gel electrophoresis (Fig. 8). In order to detect early replication products we preincubated all of the components of the reaction, except for dATP and dTTP, and then initiated polymerization by the addition of the two missing dNTPs. This allows the formation of polymerase-DNA complexes such that polymerization commences immediately upon addition of the dNTPs. Under these conditions, Pol III* synthesized DNA products not longer than several hundred nucleotides in length, which are hardly seen at the bottom of the autoradiogram. The longer products seen are most likely due to contaminating traces of the beta subunit in the Pol III* preparation. They are visible here since the experiment was conducted with an excess of enzyme. Upon addition of beta*, the amount and size of DNA products increased dramatically. The kinetics of elongation was fast, and full-length products were observed by 15 s. Thus, the addition of beta* to Pol III* formed a polymerizing complex which behaved similarly to Pol III holoenzyme.


Figure 7: beta* stimulates DNA synthesis by DNA polymerase III*. The effect of beta* on DNA synthesis by Pol III* was tested using oligonucleotide-primed M13mp8 ssDNA as described under ``Materials and Methods.'' DNA synthesis was measured in the absence (open circles) or presence (closed circles) of beta* by scintillation counting of radiolabeled dTTP that was incorporated into acid-insoluble replication products.




Figure 8: Analysis of DNA products synthesized by DNA polymerase III* in the presence of beta*. A time course of the replication of SSB-coated and primed M13mp8 ssDNA with Pol III* (12 nM) and beta* (200 nM). The reaction mixture was preincubated at 30 °C for 10 min in the absence of dATP and [P]dTTP, after which DNA synthesis was initiated by the addition of the dNTPs. Replication products were separated on a 1% alkaline agarose gel and visualized by autoradiography. The details are presented under ``Materials and Methods.''



Can the activity observed be due to a contamination of the beta subunit in our beta* preparation? As shown in Fig. 3no beta subunit could be identified in immunoblots of beta* preparations. However, in order to rule out completely this possibility, we fractionated the purified beta* on an SDS-polyacrylamide gel, eluted the beta* band and renatured it. This preparation of beta*, which was resolved on the gel from any contamination of the beta subunit, stimulated Pol III* (Fig. 9, lane 2 compared to lane 1 at each time point), although to a lesser extent than the original beta* preparation (Fig. 9, lane 3 compared to lane 2 at each time point). Presumably the harsh treatment led to a considerable decrease in the activity of beta*. Still, the results clearly show that the stimulation of Pol III* was caused by beta*.


Figure 9: Gel-purified beta* stimulates Pol III*. beta* that was purified by SDS-PAGE followed by renaturation was examined for its effect on Pol III* as described under ``Materials and Methods.'' The template used was primed M13mp8 ssDNA in the absence of SSB. Lanes marked 1 contained Pol III* alone; lanes marked 2 contained Pol III* and 100 nM beta* (purified by SDS-PAGE); lanes 3 and 4 contained 100 nM and 300 nM beta*, respectively, purified by the standard procedure.



The Effect of beta* on DNA Polymerase III* Is ATP Dependent

DNA synthesis by Pol III holoenzyme requires ATP (or dATP) for assembling a polymerase-DNA initiation complex(30) . Does the effect of beta* on Pol III* depend on ATP? To examine this issue we omitted ATP from the reaction mixture, and replacing dATP by dATPalphaS. dATPalphaS is incorporated into DNA by the polymerase, but in contrast to dATP it does not support the formation of a stable initiation complex between the primed DNA and the polymerase(13) . As can be seen in Fig. 10, stimulation of Pol III* by either the beta subunit or by beta* was completely dependent on the presence of ATP. Notice that DNA synthesis in the presence of beta* was 10-fold lower than in the presence of the beta subunit.


Figure 10: ATP is required for stimulation of DNA polymerase III* by beta*. SSB-coated and oligonucleotide-primed M13mp8 ssDNA was replicated with Pol III* (12 nM), and with beta* (200 nM) or the beta subunit (200 nM) as described under ``Materials and Methods,'' with dATPalphaS instead of dATP. The traces of long radiolabeled products synthesized by Pol III* in the absence of added beta* or beta subunit were most likely due to trace contamination of beta subunit in the Pol III* preparation.



beta* Increases the Processivity of DNA Polymerase III*

In order to examine whether the stimulation of Pol III* by beta* occurred via an increase in the processivity of the polymerase, replication assays were performed with the template present at a 60-fold excess over the polymerase. This enabled to monitor the size of products that were replicated by the polymerase following a single binding event. Under these conditions, the length distribution of replication products remains unchanged with time, but their amounts increase. If processivity is affected, a change in the length of the replication products is expected. Under these conditions, Pol III* alone synthesized products of no longer than several hundred nucleotides in length, that were obscured by the unincorporated radiolabeled dNTP at the bottom of the autoradiogram (Fig. 11). In contrast, in the presence of beta*, the polymerase synthesized long products, up to fully replicated DNA. Two major products were observed. The fully replicated DNA (7.2 kilobase pairs) and a shorter product of approximately 4 kilobase pairs, the result of a major pause site in the ssDNA template (Fig. 11). This was caused most likely by a secondary structure in the ssDNA, formed in the absence of SSB, that impeded the progression of the polymerase. Thus, similarly to the beta subunit, beta* conferred high processivity on Pol III*; however, the efficiency of the formation of processive polymerase complexes was lower.


Figure 11: beta* increases the processivity of DNA polymerase III*. DNA replication was performed with a 55-fold excess of primed M13mp8 ssDNA over the polymerase, in the absence of SSB. Aliquots were withdrawn at the indicated time points and the replication products were fractionated on a denaturing alkaline agarose gel. An autoradiogram of radiolabeled products is shown. The details are described under ``Materials and Methods.''




DISCUSSION

Our results suggest that beta* can act as an alternative polymerase clamp, and confer high processivity on Pol III* (Fig. 11). This activity requires ATP and is thus likely to involve loading of a Pol III*bulletbeta(3) complex on the primer template, similar to the case of Pol III holoenzyme(18, 19) . The overall slow synthesis activity of this polymerase (Fig. 7) is likely to be due to a slower initiation stage, e.g. a slow loading process of beta(3) on the DNA by the complex which is part of Pol III*(18, 19) . Consistent with this possibility, when a period of preincubation was allowed, a rapid phase of polymerization was observed, similar to that of Pol III*bulletbeta(2) ( Fig. 8and Fig. 10). A possible explanation is that the preincubation period allows the assembly of Pol III*bulletbeta(3) complexes, and thus upon addition of the dNTPs polymerization resumes immediately. According to this model the assembly of a Pol III*bulletbeta(3) complex on the primer-template is less efficient than the assembly of a Pol III*bulletbeta(2) initiation complex. However, once a Pol III*bulletbeta(3) complex is assembled on the DNA, it has a high processivity similar to that of Pol III holoenzyme.

It was recently shown by x-ray crystallography that the beta subunit is composed of three structurally similar domains, and it dimerizes to form a hexagon-like ring(17) . beta* contains precisely two of the three domains of the beta subunit(40) . The fact that beta* appears as a trimer suggests that it may from a beta(2)-like ring structure, composed of three two-domain proteins, forming an alternative DNA clamp. The three repeating domains of the beta subunit are structurally very similar, although there is no significant homology at the amino acid sequence level(17) . Although the overall structure of beta(3) may be similar to that of beta(2), it is expected to show significant differences in activity as compared to beta(2), since it is lacking the N-terminal domain of the beta subunit.

Polymerase clamps composed of homotrimers are in fact more common than dimeric clamps. The gp45 gene of bacteriophage T4, which serves as an auxiliary subunit of T4 DNA polymerase(31) , is an homologue of the beta subunit(17) . The gp45 protein has a molecular mass of 25 kDa, and based on its amino acid sequence it is a homologue of two of the three repeating domains of the beta subunit(17) . Indeed, gp45 forms a trimer in solution (32, 33) and is likely to form a beta(2)-like sliding clamp. In eukaryotes, proliferating cell nuclear antigen (PCNA) serves as a processivity factor of DNA polymerase , which is involved in DNA replication and repair(34, 35, 36) . PCNA has usually a size of 29 kDa, and it forms a trimeric hexagonal ring-shaped structure very similar to that of the beta subunit(37) . Interestingly, it was recently reported that two PCNA genes were found in carrot, encoding putative PCNA molecules of 29 and 40 kDa(38) . Based on its size, its trimeric structure and its activity as a processivity factor, beta* could thus be viewed as an analog of the T4 gp45 and the eukaryotic PCNAs, with a possible evolutionary link.

The two major DNA synthesis processes in UV-irradiated cells are repair synthesis of short ssDNA gaps(1) , and the resumption of DNA replication, that involves both reactivation of replication forks stalled at UV lesions and the activity of new origins of replication (7) . In addition, trans-lesion synthesis occurs and gives rise to mutations. The involvement of beta* in these processes, and its effects on each of the three E. coli DNA polymerases are currently under investigation. However, its activity as a processivity clamp, and the increase in cell UV survival caused by its overproduction (39) suggest that it is involved in a process related to DNA synthesis in the UV-irradiated cell.


FOOTNOTES

*
This research was supported by grants from the Dorot Science Fellowship Foundation and the Scheuer Research Foundation of the Israel Academy of Sciences, and from 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.: 972-8-9343203; Fax: 972-8-9344169; :BCLIVNEH{at}WEIZMANN.WEIZMANN.AC.IL.

(^1)
The abbreviations used are: Pol III, DNA polymerase III; HPLC, high performance liquid chromatography; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; dATPalphaS, deoxyadenosine-5`-O-(1-thiotriphosphate); PAGE, polyacrylamide gel electrophoresis; ss, single-stranded; PCNA, proliferating cell nuclear antigen; SSB, single-strand binding protein.


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