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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1998 Nov 24;95(24):14106–14111. doi: 10.1073/pnas.95.24.14106

The β subunit sliding DNA clamp is responsible for unassisted mutagenic translesion replication by DNA polymerase III holoenzyme

Guy Tomer 1, Nina Bacher Reuven 1, Zvi Livneh 1,†,‡
PMCID: PMC24334  PMID: 9826661

Abstract

The replication of damaged nucleotides that have escaped DNA repair leads to the formation of mutations caused by misincorporation opposite the lesion. In Escherichia coli, this process is under tight regulation of the SOS stress response and is carried out by DNA polymerase III in a process that involves also the RecA, UmuD′ and UmuC proteins. We have shown that DNA polymerase III holoenzyme is able to replicate, unassisted, through a synthetic abasic site in a gapped duplex plasmid. Here, we show that DNA polymerase III*, a subassembly of DNA polymerase III holoenzyme lacking the β subunit, is blocked very effectively by the synthetic abasic site in the same DNA substrate. Addition of the β subunit caused a dramatic increase of at least 28-fold in the ability of the polymerase to perform translesion replication, reaching 52% bypass in 5 min. When the ssDNA region in the gapped plasmid was extended from 22 nucleotides to 350 nucleotides, translesion replication still depended on the β subunit, but it was reduced by 80%. DNA sequence analysis of translesion replication products revealed mostly −1 frameshifts. This mutation type is changed to base substitution by the addition of UmuD′, UmuC, and RecA, as demonstrated in a reconstituted SOS translesion replication reaction. These results indicate that the β subunit sliding DNA clamp is the major determinant in the ability of DNA polymerase III holoenzyme to perform unassisted translesion replication and that this unassisted bypass produces primarily frameshifts.

Numerous lesions are formed continuously in DNA (1). Most of these lesions are removed effectively via DNA repair, thus restoring the structural integrity of DNA (1). When DNA lesions that have escaped repair are replicated, they often give rise to mutations caused by the tendency of DNA polymerase to insert an incorrect nucleotide opposite the lesion. This process was termed translesion replication, bypass synthesis, or error-prone repair and is responsible for induced mutagenesis in prokaryotes and in eukaryotes (1–3).

In Escherichia coli, replication-blocking lesions, such as the UV light-induced cyclobutyl pyrimidine dimers (2), or abasic sites (4) give rise to mutations in a process that depends on the SOS stress response and requires DNA polymerase III (pol III) and the SOS-regulated proteins RecA, UmuD′, and UmuC (1, 2, 5). This led to the hypothesis that pol III alone cannot replicate the “blocking lesions”; only with the assistance of RecA, UmuD′, and UmuC can pol III perform translesion replication through these lesions and give rise to mutations. Studies on the ability of purified pol III holoenzyme to replicate blocking lesions unassisted yielded conflicting results (6–11). Recently, it has been shown that this controversy resulted from the usage of different DNA substrates for the translesion replication assay. Indeed, when a native-like gapped plasmid DNA was used as a substrate, pol III holoenzyme could replicate through a synthetic abasic site (unpublished data). Here, we report that the ability of pol III holoenzyme to perform translesion replication depends on its β subunit sliding DNA clamp. It is noteworthy that the proliferating cell nuclear antigen, the eukaryotic homologue of the β subunit, is a key target for regulating cellular responses to DNA damage (12).

MATERIALS AND METHODS

Materials.

The sources of materials used were as follows: nucleotides, DTT, BSA, and proteinase K, Boehringer Mannheim; [γ-32P]ATP, NEN; NA45 DEAE membranes, Schleicher & Schuell; and dSpacer CE phosphoramidite, a synthetic abasic site building block used in oligonucleotides synthesis, Glen Research, Sterling, VA.

Proteins.

Pol III holoenzyme (0.03 mg/ml) (13), pol III* (0.07 mg/ml) (14), the β subunit of pol III (1 mg/ml; >98% pure) (15), and ssDNA-binding protein (0.6 mg/ml; >95% pure) (16) were purified as described. Pol III core (2.45 mg/ml), the γ complex of pol III (3 mg/ml), and the purified α (0.65 mg/ml), ɛ (1.7 mg/ml), θ (4.1 mg/ml), and τ (0.77 mg/ml) subunits were generous gifts from M. O’Donnell (The Rockefeller University, New York). Restriction nucleases, T4 DNA ligase, and T4 polynucleotide kinase were from New England Biolabs. S1 Nuclease was from Promega, and T7 gp6 exonuclease was from Amersham.

DNA Substrates.

The preparation of the gapped plasmid carrying a site-specific lesion has been described (unpublished work). In brief, plasmid pSKSL (3361 bp), a derivative of plasmid pBluescript II SK(+) (Stratagene), was cleaved with restriction nucleases BstXI and BsaI and was ligated to a synthetic gapped duplex oligonucleotide whose termini were complementary to those of the cleaved plasmid. Two substrates were used that differed only in the DNA sequence context in the vicinity of the lesion (a site-specific synthetic abasic site, marked X) (12). Substrate GP21 was prepared by annealing the oligonucleotides 5′-GGAATCACTTCGTTG-3′, 5′-*CTGGTTCAAGTAGCC-3′ and 5′-ACCGCAACGAAGTGATTCCCGTCGTGACTGXGAAAACCCTGGGCTACTTGAACCAGACCG-3′. Substrate GP31 was prepared by annealing the oligonucleotides 5′-GGAATCACTTCGTTG-3′, 5′-*CTGGTTCAAGTAGCC-3′, and 5′-ACCGCAACGAAGTGATTCCTGGCGTTACCCXACTTAATCGCGGCTACTTGAACCAGACCG-3′ (The asterisks mark 5′-32Pradiolabeled phosphate groups, and the XmnI cleavage site is in bold).

The desired gapped plasmids were gel-purified and were used for translesion replication assays. To obtain plasmids with longer gaps, substrates GP21 or GP31 (16 ng/μl) were incubated with 2.5 units/μl of T7 gp6 exonuclease in a total volume of 300 μl containing 40 mM Tris⋅HCl (pH 7.5), 20 mM MgCl2, and 150 mM NaCl for 30 min at 16°C. The enzyme then was heat-inactivated (80°C, 10 min), and the DNA was extracted with phenol/chloroform, and ethanol was precipitated.

All oligonucleotides were synthesized and purified by the Synthesis Unit of the Biological Services Department in our Institute. Oligonucleotides containing a synthetic abasic site were synthesized similarly by using dSpacer CE phosphoramidite as a building block. The synthetic abasic site analog is a modified tetrahydrofuran moiety that is a stable analog of 2′-deoxyribose in the abasic site. It has a hydrogen instead of a hydroxyl residue on the 1′ carbon of the deoxyribose ring (17).

Translesion Replication Assay.

The translesion replication reaction mixture (25 μl) contained buffer B (20 mM Tris⋅HCl, pH 7.5/8 μg/ml BSA/5 mM DTT/0.1 mM EDTA/4% glycerol), 1 mM ATP, 10 mM MgCl2, 0.5 mM each of dATP, dGTP, dTTP, and dCTP, and 0.1 μg (2 nM) gapped plasmid. The DNA polymerase and accessory subunits were added at the following concentrations: Pol III holoenzyme, 1 nM; Pol III*, 0.3–6 nM; the β subunit, 20–500 nM; and reconstituted pol III holoenzyme, 10 nM. Reactions were carried out at 37°C for 1–30 min, after which they were terminated by adding SDS to 0.2% and EDTA to 20 mM and were heat-inactivated at 65°C for 10 min. The proteins were digested with 0.4 mg/ml proteinase K at 37°C for 30 min, after which the DNA was extracted with phenol:chloroform (1:1) and was ethanol-precipitated. The DNA was digested with XmnI (3 units/tube) at 37°C for 1 hr. Then, 5 units of BstXI were added, and incubation continued at 55°C for another hour. The DNA was fractionated by electrophoresis on 15% polyacrylamide gels containing 8 M urea. Gels were run at 1,500–2,000 V for 2–3 hr, after which they were dried and were visualized and quantified by using a Fuji BAS 2000 phosphorimager. The extent of translesion replication was calculated by dividing the amount of bypass products (all products that extend beyond the lesion) by the amount of the extended primers.

DNA Sequence Analysis of Translesion Replication Products.

The analysis was performed as described (18). In brief, after translesion replication, all of the DNA products were isolated, were linearized with HindIII, and then were subjected to treatment with S1 nuclease to cleave plasmid molecules in which the gap was not fully filled in. The newly synthesized strand was specifically amplified by using linear PCR. The amplified ssDNA product was isolated, was amplified again by using a second round of regular PCR, and was subcloned into plasmid pUC18. After introduction of this DNA into E. coli cells, individual colonies were picked, and their plasmid contents were extracted and subjected to DNA sequence analysis. In this procedure, each translesion replication event was scored, and there was no selection for specific mutagenic events.

RESULTS

Recently, a new method has been developed for the preparative construction of gapped plasmids containing site-specific lesions in the single-stranded region (unpublished data). By using these gap/lesion plasmids, it was shown that DNA pol III holoenzyme replicates past a synthetic abasic site, unassisted by other proteins (unpublished data). To study the molecular basis underlying this bypass ability, we examined translesion replication with subassemblies of pol III holoenzyme. The assay involved replication of the gapped plasmid with the DNA polymerase, after which the DNA was restricted with BstXI, which cuts just upstream to the internal 32P present in the primer DNA strand, and with XmnI, which cuts downstream to the gap (Fig. 1). The DNA products then were fractionated by urea-PAGE and were visualized and quantified by phosphorimaging. With substrates GP21 and GP31, the unextended DNA is represented in the phosphorimage by 15-mer whereas replication arrest before or opposite the lesion is represented by 25-mer or 26-mer, respectively. Translesion replication is represented by oligonucleotides longer than 26-mer: A pause of the polymerase at the end of the gap will yield a 37-nt product whereas further elongation, which requires strand displacement, will form products longer than 37-mer. Replication past the XmnI site will be indicated by products 43 nucleotides long, dictated by the XmnI cleavage site (Fig. 1).

Figure 1.

Figure 1

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The translesion replication assay. The substrate for the translesion replication was a gapped plasmid, carrying a site-specific synthetic abasic site, and an internal radiolabeled phosphate. After replication, the DNA was restricted with restriction nucleases BstXI and XmnI, and the products were fractionated by urea-PAGE. Visualization and quantification was done by phosphorimaging. The bottom panel shows the types of replication products obtained. The unextended DNA is a 15-mer whereas replication arrest before or opposite the lesion forms a 25-mer or 26-mer, respectively. Translesion replication forms products longer than 26-mer: A pause of the polymerase at the end of the gap yields a 37-mer whereas further elongation, which requires strand displacement, forms products longer than 37-mer. Replication past the XmnI site will be indicated by products 43 nucleotides long, dictated by the XmnI cleavage site. If the polymerase skips the lesion, producing a −1 deletion, a 42-nt product is expected. Black rectangle, abasic site analog; black circle, radiolabeled phosphate; gray bar, newly synthesized DNA.

A previous study from this laboratory has found essentially no bypass by pol III core [composed of the α, ɛ, and θ subunits (unpublished data)], or by pol III′ [composed of pol III core plus the τ subunit (data not shown)]. Similarly, DNA polymerase III*, which contains 9 of the 10 subunits of pol III holoenzyme, lacking only the β subunit (19, 20), was blocked effectively by the synthetic abasic site (Fig. 2). Addition of purified β subunit caused a dramatic increase in the ability of the polymerase to replicate through the lesion. Thus, at a polymerase concentration of 1.5 nM, addition of 100 nM β subunit caused an increase of at least 28-fold, from <0.5 to 14%, in translesion replication (Fig. 2). At high concentrations of the β subunit, there was a weak 1.5-fold inhibition of bypass (Fig. 2). Bypass depended on the concentration of the polymerase, reaching nearly 53% within 5 min at a polymerase concentration of 6 nM (Fig. 2). Some replication products, 37–42 nucleotides long, were seen (Fig. 2), indicating replication pauses at the end of the gap (37-mer). The fact that most bypass products were full length indicates that the polymerase continued replication beyond the end of the gap while performing strand displacement.

Figure 2.

Figure 2

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The β subunit sliding DNA clamp is required for translesion replication by pol III holoenzyme. Translesion replication was performed as described in Materials and Methods with substrate GP21 and with pol III* in the absence or presence of the indicated concentrations of the β subunit. The reactions with 6 nM pol III* were carried out for 5 min whereas all other reactions were carried out for 2 min. The extents of translesion replication are indicated at the bottom of each lane.

A time course of the translesion replication with pol III* plus the β subunit reaction is shown in Fig. 3. Bypass increased with time, reaching remarkable extents of 46 and 86% at 8 and 30 min, respectively. This represents nearly complete bypass by pol III holoenzyme. Bypass by pol III* was <5% that of holoenzyme (data not shown). Notice that, when the reaction was performed under 7-fold excess of template (0.3 nM pol III), full bypass products were obtained when most of the primers were still unextended (Fig. 3; 2-, 4-, and 8-min time points). This indicates that translesion replication occurred via a processive reaction, with bypass occurring without dissociation of the polymerase from the DNA at the lesion. Once dissociated, binding would have been favored to unextended primers, which are present at high excess.

Figure 3.

Figure 3

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Kinetics of translesion replication by pol III holoenzyme reconstituted from pol III* and the β subunit. Translesion replication was performed as described in Materials and Methods with the indicated concentrations of pol III* and with 80 nM β subunit. The extents of bypass are indicated at the bottom of each lane.

The gapped plasmid that we have constructed contained a gap of 22 nucleotides. The sizes of ssDNA gaps in vivo are not well defined, but at least some are likely to be larger. In addition, larger gaps are needed to study the effects of DNA binding proteins such as ssDNA-binding protein, which stimulates pol III holoenzyme [a tetramer of ssDNA-binding protein binds 35 nucleotides (21)]. We thus have modified our methodology to enable the extension of the gap. This was done by using the phage T7 gp6 5′→3′ exonuclease. Conditions were optimized to allow controlled extension of the gap, as can be seen in Fig. 4. Treatment of the gapped plasmid led to an increase in the mobility of the DNA in agarose gel electrophoresis (Fig. 4). The average size of the gap was determined by digesting the ssDNA region with S1 nuclease and determining the size of the remaining dsDNA fragment. Based on this analysis, a treatment of 30 min under our conditions generated a gap with an average size of 350 nucleotides (Fig. 4A). Cleavage of the substrate containing the extended gap (termed GP21-L) with restriction nuclease DraIII, which cuts once in the plasmid (Fig. 1), generated a DNA fragment migrating slightly faster than the full length linear plasmid (Fig. 4B). This verifies that GP21-L has a circular structure, as expected (a linear construct would have yielded two shorter bands; see “vector” lanes in Fig. 4B).

Figure 4.

Figure 4

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Construction of a gap/lesion plasmid with an extended gap. Substrate GP21 was reacted with T7 exonuclease gp6 as described in Materials and Methods to generate GP21-L containing an extended gap of 350 nucleotides. A shows the substrate GP21 after exonuclease treatment and S1 treatment to determine the size of the ssDNA gap. B shows the analysis of the substrate by restriction with DraIII, which cuts once in the plasmid. The formation of a single full length DNA band indicates that substrates GP21 and GP21-L are circular. Restriction of linear control plasmid with DraIII generated two fragments each of ≈1.4 kbp, as expected.

Side-by-side translesion replication assay was performed by using gapped plasmids with 22- or 350-nt ssDNA gaps. As can be seen in Fig. 5A, the rate of translesion replication was reduced by 80% on the long ssDNA region in substrate GP21-L. A similarly lower bypass was observed with another substrate (GP31-L) in which the lesion was within a different DNA sequence context (Fig. 5B). Translesion replication on the substrate with the long gap also totally depended on the β subunit (Fig. 5B). The same was true also for pol III holoenzyme reconstituted from purified subunits (Fig. 5B; lanes marked “Pol III* R”).

Figure 5.

Figure 5

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Translesion replication is reduced in a large gap, but it requires the β subunit. (A) Translesion replication was performed with pol III holoenzyme on substrate GP21 (22-nt gap) side-by-side with substrate GP21-L (350-nt gap) as described in Materials and Methods. B shows translesion replication using a different gapped plasmid with a large gap (GP31-L), using pol III* (1.5 nM), or using reconstituted pol III* (10 nM; Pol III* R), in the absence or the presence of purified β subunit (40 nM). The extents of translesion replication are indicated at the bottom of each lane.

To determine the identity of the nucleotide inserted opposite the synthetic abasic site by pol III holoenzyme, we have amplified the newly synthesized DNA strand by using linear-amplification PCR followed by regular exponential amplification of the linear DNA obtained. Gapped plasmids in which the gap was not completely filled in were digested with S1 nuclease before the PCR reaction and thus were not amplified. The amplified DNA fragment was cloned into pUC18 and was introduced into E. coli. Bacterial cultures were grown from individual colonies, and their plasmid content was extracted and subjected to DNA sequence analysis. DNA sequence of 15 plasmids revealed exclusively small deletions: 13 (87%) −1 frameshifts, 1 −2 frameshift, and 1 −3 deletion. All mutations were located opposite the synthetic abasic site. Thus, pol III holoenzyme skipped over the abasic site in essentially each translesion replication reaction.

DISCUSSION

DNA lesions such as abasic sites or pyrimidine dimers are obstacles to DNA replication, and, when copied, they give rise to mutations caused by misincorporation opposite the lesions (1, 2, 22). It was shown previously by several laboratories, including our own, that pol I (17, 23–26), pol II (9, 27), and pol III core (27) can bypass in vitro “blocking” DNA lesions. Translesion replication was achieved by multiple bypass attempts, which involved binding-dissociation cycles at the lesion. This multiple-attempt bypass was facilitated greatly by increasing the concentrations of the polymerase and the DNA, as expected (27). All of these DNA polymerases have low processivities of 5–20 nucleotides per binding event (19), much lower than the processivity of pol III holoenzyme, estimated to be from 7,000 up to 100,000, depending on the assay system (19, 20, 28). However, it was difficult to measure bypass by pol III holoenzyme because of the difficulties in the construction of preparative amounts of long native-like DNA containing site-specific lesions. Although it has been reported that pol III holoenzyme was able to bypass UV lesions (6, 7, 29) and abasic sites (8) that were randomly distributed on circular ssDNA, other investigators have reported no bypass by pol III holoenzyme through a site-specific synthetic abasic site (9, 10) or acetylaminofluorene-guanine adduct (11). Recently, it has been shown that this controversy resulted from the usage of different DNA substrates for the translesion replication assay (unpublished data). Indeed, when a native-like gapped plasmid DNA was used as a substrate, pol III holoenzyme could replicate through a synthetic abasic site (unpublished data, and this work).

The results presented in this study clearly show that the ability of pol III holoenzyme to perform effective translesion replication depends on its β subunit. This subunit is a homodimer that forms a ring structure that can slide on DNA (30, 31). The polymerase is tethered to the DNA by virtue of protein–protein interactions with the β subunit. Thus, the activity of the β subunit as a sliding DNA clamp and the high affinity of the polymerase to a primer–template structure make pol III holoenzyme a highly processive DNA polymerase (20). Translesion replication in our assay occurred under conditions of excess template, and bypass was observed when most of the primers were still unextended. This suggests that bypass by pol III holoenzyme was processive, namely occurring without dissociation from DNA. If dissociation had occurred, the polymerase would have had a better chance to bind the excess of unextended primers rather than the primer terminus located at the lesion. The effective bypass via a processive mechanism can be explained by assuming that the high processivity of the polymerase, representing high affinity to the DNA, prevents rapid dissociation of the polymerase, increases its residence time at lesions, and thus increases the chances of performing the kinetically slow bypass steps.

The decrease in the rate of translesion replication on plasmids with large gaps does not seem to be caused by the tendency of ssDNA to form secondary structures because ssDNA-binding protein, which is known to melt out secondary structures, had little effect on bypass (data not shown). Another possibility is that the narrow gap enables the polymerase to bind the DNA on both sides of the gap, thus increasing it residence time on DNA and, as a consequence, also its bypass ability. A similar phenomenon was reported for DNA polymerase β, which exhibited translesion synthesis through O6-methylguanine only in short gaps (32).

A previous study from our laboratory has shown that overproduction of the β subunit from a plasmid reduced chromosomal UV mutagenesis (33) and that excess β subunit inhibited in vitro translesion replication though UV lesions (34). These results, of a negative effect of the β subunit on bypass, seem to be inconsistent with its requirement in translesion replication, as presented here. This apparent discrepancy can be explained by opposite effects that the β subunit has when present at normal versus overproduced concentrations. The in vivo experiments were done under conditions in which the β subunit was overproduced 50-fold (ref. 33; intracellular concentration of 10–15 μM). We have suggested that a β-rich form of pol III holoenzyme exists under such conditions, which rapidly dissociates at lesions, and is thus deficient in bypass (33). An alternative explanation is that, when present at high concentration, free β dimers may complete with DNA-bound β dimers for binding with pol III core pausing at the lesion (20). This is expected to facilitate dissociation of the polymerase from the lesion, thus favoring dissociation over bypass. Notice that at higher β2 concentration (0.5 μM), a slight inhibition of bypass was observed in the present study too (Fig. 2). Recently, it was found, by using the yeast two-hybrid system, that the β subunit interacts with UmuD′ and UmuC and their plasmidic homologues MucA′ and MucB (L. Sarov-Blat and Z.L., unpublished observation). This suggests an additional inhibitory mechanism for the overproduced β subunit: It may sequester the Umu proteins, thus preventing them from participating in SOS translesion replication. Consistent with such an explanation is the finding that expressing of UmuDC or MucAB from a plasmid alleviated part of the inhibition of UV mutagenesis caused by the overproduced β subunit (33).

Given the ability of pol III holoenzyme to bypass the synthetic abasic site unassisted, the question arises: Why does in vivo translesion replication require UmuD′, UmuC, and RecA? These proteins may be needed for at least four functions: (i) Stimulating translesion replication by pol III. This was shown by the recent reconstitution of SOS translesion replication (18, 35). (ii) Relieving inhibition of translesion replication imposed by intracellular DNA damage-binding proteins (36). (iii) Competing against error-free recombinational repair (37, 38). And (iv), changing the specificity of bypass. In vivo, most of the mutations caused by an abasic sites in SOS-induced cells were found to be base substitutions, and only 7–10% were −1 deletions (39–41). Mostly base substitutions were observed in vivo also with the synthetic abasic site that was used in the present study (42). The fact that the mutations produced in the in vitro translesion replication reaction were exclusively small deletions (primarily −1 frameshifts) suggested that SOS proteins change the specificity of translesion replication. Recently, we have reconstituted SOS translesion replication with purified components and have demonstrated directly such a change in specificity (18).

Acknowledgments

This research was supported by grants from The U.S.–Israel Binational Science Foundation (96-00448), the Israel Ministry of Science (6107), and the Forchheimer Center for Molecular Genetics.

ABBREVIATION

pol III

polymerase III

Footnotes

This paper was submitted directly (Track II) to the Proceedings Office.

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