Abstract
The mechanisms that control the fidelity of DNA replication are being investigated by a number of approaches, including detailed kinetic and structural studies. Important tools in these studies are mutant versions of DNA polymerases that affect the fidelity of DNA replication. It has been suggested that proper interactions within the core of DNA polymerase III (Pol III) of Escherichia coli could be essential for maintaining the optimal fidelity of DNA replication (H. Maki and A. Kornberg, Proc. Natl. Acad. Sci. USA 84:4389–4392, 1987). We have been particularly interested in elucidating the physiological role of the interactions between the DnaE (α subunit [possessing DNA polymerase activity]) and DnaQ (ɛ subunit [possessing 3′→5′ exonucleolytic proofreading activity]) proteins. In an attempt to achieve this goal, we have used the Saccharomyces cerevisiae two-hybrid system to analyze specific in vivo protein interactions. In this report, we demonstrate interactions between the DnaE and DnaQ proteins and between the DnaQ and HolE (θ subunit) proteins. We also tested the interactions of the wild-type DnaE and HolE proteins with three well-known mutant forms of DnaQ (MutD5, DnaQ926, and DnaQ49), each of which leads to a strong mutator phenotype. Our results show that the mutD5 and dnaQ926 mutations do not affect the ɛ subunit-α subunit and ɛ subunit-θ subunit interactions. However, the dnaQ49 mutation greatly reduces the strength of interaction of the ɛ subunit with both the α and the θ subunits. Thus, the mutator phenotype of dnaQ49 may be the result of an altered conformation of the ɛ protein, which leads to altered interactions within the Pol III core.
Replication of the Escherichia coli chromosome is performed by the DNA polymerase III holoenzyme (Pol III HE) (15, 16, 21). Pol III HE is an asymmetric, dimeric complex containing a total of 18 subunits (10 distinct), which are capable of coordinately synthesizing leading and lagging strands. The complex contains two polymerase cores, one for each strand, composed of α, ɛ, and θ subunits (22).
With regard to the fidelity of the replication process, the α and ɛ subunits of the Pol III core are of particular importance. The α subunit (dnaE gene product) is the DNA polymerase, which selects the correct nucleotides during template-directed DNA synthesis (18). The ɛ subunit (dnaQ gene product) performs the 3′→5′ exonucleolytic proofreading activity, which preferentially removes incorrect bases inserted by the polymerase (27). The function of the third subunit, the θ subunit, has yet to be identified. The ɛ subunit of the Pol III HE plays a complex role in DNA replication. Besides its proofreading activity, it stabilizes the core by tightly binding to both the α and θ subunits (22, 29). Interestingly, the α and ɛ subunits are each less active individually than when bound together in the Pol III core (19). One may hypothesize that the fidelity of DNA replication depends not only on the intrinsic accuracy of the polymerase and the strength of the 3′→5′ exonuclease activity but also on the appropriate interactions between the subunits within the Pol III core. Thus, the decreased fidelity of DNA replication observed in E. coli strains carrying mutations within the dnaQ gene could be due either to the defective catalytic properties of the ɛ subunit or to aberrant subunit interactions within the Pol III core.
Several mutators which carry mutations in the dnaQ gene have been isolated, e.g., dnaQ49, mutD5, and dnaQ926 strains. Two of these mutations, mutD5 and dnaQ49, have been extensively studied. mutD5 is a particularly strong mutator allele, leading to mutation rates of up to 105-fold above the wild-type level (3, 4). This is due not only to the proofreading defect but also to the concomitant impairment (by saturation) of postreplicative mismatch repair (25, 26). dnaQ49 strains differ from mutD5 strains in several respects. First, dnaQ49 is a temperature-sensitive mutator, possessing modest mutator activity below 30°C but strongly enhanced activity at 37°C (7, 10, 11). Second, dnaQ49 strains are unable to grow at 44.5°C in salt-free rich medium because of the inhibition of DNA synthesis (10). Third, the dnaQ49 allele is recessive with respect to the wild-type gene, while mutD5 is dominant (3, 20). The dnaQ49 and mutD5 alleles result from different missense mutations within the dnaQ gene (9, 30; see also Table 1). On the basis of genetic data, a model in which the DnaQ49 protein has a reduced ability to bind to the α subunit has been proposed (30). The third allele, dnaQ926, is the strongest known mutator of E. coli (9). It was constructed by site-specific mutagenesis by changing the codons for two conserved amino acid residues in the ExoI motif of the ɛ subunit (Table 1) known to be essential for the catalytic activity of other polymerase-associated proofreading exonucleases (1). When residing on a plasmid, dnaQ926 confers a strong, dominant mutator phenotype, suggesting that the protein, although deficient in exonuclease activity, may still efficiently bind to the α subunit. When dnaQ926 was transferred to the chromosome, replacing the wild-type gene, the cells were essentially inviable. dnaQ926 strains survived well, however, when carrying a dnaE antimutator mutation (6, 8) or a multicopy plasmid containing the E. coli mutL+ gene. Thus, the poor viability of dnaQ926 strains was proposed to result from excessively high mutation rates due, as in the case of mutD5 strains, both to the proofreading defect and to the collapse of the mismatch repair system (error catastrophe).
TABLE 1.
dnaQ mutants tested in this work
A mechanism coordinating DNA polymerization and DNA excision, relying on structural and functional communication between the different subunits of Pol III HE, may play an important role in maintaining optimal fidelity of DNA replication. We have been particularly interested in elucidating the physiological role of interactions between the α and ɛ subunits. In an attempt to achieve this goal, we used the Saccharomyces cerevisiae two-hybrid system to investigate the in vivo interactions between mutant and wild-type DnaQ protein with the wild-type DnaE and HolE proteins.
MATERIALS AND METHODS
Bacterial and yeast strains and media.
E. coli DH5α [supE44 ΔlacU169 (φ80lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1] was the transformation recipient strain for all plasmid constructions. All two-hybrid system experiments were done with S. cerevisiae Y187 (MATα gal4Δ gal80Δ his3 trp1-901 ade2-101 ura3-52 leu2-3,112 met URA3::GAL1-lacZ). Strain Y187 was kindly provided by S. Elledge (Baylor College of Medicine, Houston, Tex.). Yeast extract-peptone-dextrose medium and synthetic medium (SMM) were prepared as described previously (23). For drug selection, Luria broth plates were supplemented with ampicillin (100 μg/ml).
Methods.
Manipulations and sequencing of DNA were carried out by standard procedures (24). The S. cerevisiae Y187 strain was transformed simultaneously with a pGBT9-derived plasmid (e.g., pGBT9dnaE) and a pGAD424-derived plasmid (e.g., pGAD424-2dnaQ) by the method of Chen et al. (2).
β-Galactosidase assay.
For quantitative studies, yeast strains were grown at 25 or 28°C to stationary phase in synthetic medium (SMM plus 3% glucose) lacking leucine and tryptophan, diluted 10 times in SMM plus 2% ethanol (lacking leucine and tryptophan), and then incubated at 25 or 28°C for 48 h. The β-galactosidase activity was determined as described previously (23).
Construction of GAL4 protein fusion plasmids.
Plasmids for the GAL4 two-hybrid fusion assay were prepared by cloning PCR-amplified fragments into pGBT9 (Clontech) or pGBT9-2 (13) (both containing amino acids 1 to 147 of the DNA-binding domain of GAL4) and pGAD424 (Clontech) or pGAD424-2 (13) (both containing amino acids 768 to 881 from the trans-activation domain of GAL4). The dnaQ coding region was obtained from plasmid pIP1 (12), the dnaE coding region was obtained from plasmid pMWE103 (kindly provided by C. McHenry [University of Colorado Health Sciences Center, Denver]), and the holE coding region was obtained from the chromosome of E. coli DH5α. In all cases these were obtained via PCR amplification with the following forward and reverse primers, respectively: dnaQ, 5′-ATGAGCACTGCAATTACACGC-3′ and 5′-TTTTTAGCGCCTTCACAGG-3′; dnaE, 5′-ATGTCTGAACCACGTTTCGTA-3′ and 5′-AATCAAGGAAATTCAGACTCA-3′; and holE, 5′-ATGCTGAAGAATCTGGCTA-3′ and 5′-CAGGCGTTATGTAAGAAAG-3′. The PCR conditions and cloning of PCR amplification products were as described previously (13). To confirm the presence of the in-frame junction of the genes with the respective GAL4 domains, recombinant plasmids pGBT9-2dnaQ+, pGBT9-2holE+, and pGBT9dnaE+ were sequenced with the primer GAL4bd (5′-GAAGAGAGTAGTAACAAAGG-3′). The entire DNA sequences of the PCR-derived holE and dnaQ inserts were verified by dideoxy sequencing. The dnaE sequence was verified by sequencing pGBT9dnaE+ from the ATG codon (position 796) to the HpaI site (position 1035) and from the SmaI site (position 4022) to the 3′ end of the gene (numbering system as in reference 31). Then the region in pGBT9dnaE+ between the HpaI site (position 1035) and the SmaI site (position 4022) was removed and replaced by the HpaI-SmaI fragment (2,987 bp) from pMWE103 carrying the wild-type dnaE gene.
Plasmids pGBT9-2dnaQ49, pGBT9-2mutD5, and pGBT9-2dnaQ926 were constructed as follows. pGBT9-2dnaQ+ has unique BamHI and MluI sites (at positions 839 and 375 of the dnaQ gene, respectively) (numbering according to the method of Maki et al. [17]). The 464-bp BamHI-MluI fragment was cut out and replaced by the corresponding fragment of plasmid pIP21, which carries the dnaQ49 gene (14). The presence of the dnaQ49 mutation in the resulting plasmid, pGBT9-2dnaQ49, was confirmed by DNA sequencing. As sources of mutD5 and dnaQ926 DNA for PCR amplification we used plasmids pIF45 and pIF44, respectively (9). After the PCR fragments were cloned into pGBT9-2, the presence of the mutD5 and dnaQ926 mutations in the resulting plasmids, pGBT9-2mutD5 and pGBT9-2dnaQ926, was confirmed by sequencing.
RESULTS AND DISCUSSION
Interactions within the Pol III core.
It has been shown that the αɛθ heterotrimer can be formed in vitro (22, 29). Structural studies showed the α subunit to bind to the ɛ subunit and the θ subunit to bind to the ɛ subunit but not to the α subunit, indicating a linear arrangement of the α, ɛ, and θ subunits in the Pol III core. To evaluate the usefulness of the two-hybrid system for studying interactions within the Pol III core, we have examined interactions between the DnaE (α subunit), DnaQ (ɛ subunit), and HolE (θ subunit) fusion proteins. We cloned the complete DNA coding sequences of the dnaE, dnaQ, and holE genes, each from the first ATG codon, into both pGBT9 and pGAD424. All pairwise combinations of pGBT9, pGBT9dnaE+, pGBT9-2dnaQ, or pGBT9-2holE+ and pGAD424, pGAD424dnaE+, pGAD424-2dnaQ+, or pGAD424-2holE+ were introduced into the yeast reporter strain Y187. After selection, cotransformants were screened for their ability to produce β-galactosidase by filter assay (data not shown) and by quantitative measurement of β-galactosidase activity (Table 2). The results in Table 2 indicate that the DnaE fusion protein is able to interact specifically with the DnaQ fusion protein. Also, the DnaQ fusion protein binds tightly to the HolE fusion protein. The HolE fusion protein does not interact with DnaE fusion protein. The observed efficiency of (hetero)dimer formation, measured by the activity of the lacZ reporter gene, followed the order DnaQ-HolE ≫ DnaQ-DnaE. This does not necessarily reflect the relative strengths of the α subunit-ɛ subunit and ɛ subunit-θ subunit interactions in E. coli cells, as the two-hybrid system results are additionally determined by the levels of expression of various fusion proteins and by any effects of the GAL4 fusion domains on the binding efficiencies. None of the three tested proteins formed homodimers. We noticed stronger interactions of the DnaE and HolE fusion proteins with the DnaQ fusion protein when dnaQ was cloned into pGBT9 than when cloned into pGAD424. We interpret these differences to reflect the need for appropriate tertiary structures of the respective proteins in order to permit the specific interactions. The presence of the additional 113 amino acids of the GAL4 trans-activation domain fused at the N terminus of DnaQ in the case of pGAD424 may interfere with the optimal folding of the protein and, consequently, affect its interaction with the other proteins. Our results on the specificities and strengths of the α subunit-ɛ subunit and ɛ subunit-θ subunit interactions are in good agreement with those obtained from in vitro experiments using purified subunits (29). The present data as well as previous data demonstrating specific interactions between the UmuC and UmuD′ or UmuD and UmuD′ proteins (13) indicate that the yeast two-hybrid system can be used successfully for investigating protein-protein interactions of E. coli proteins.
TABLE 2.
Interaction of DnaQ, HolE, and DnaE fusion proteins: quantitative assay of β-galactosidase activity
| Plasmidsa | β-Galactosidase activityb |
|---|---|
| pGBT9-2holE+ + pGAD424 | <2 |
| pGBT9-2dnaQ+ + pGAD424 | <2 |
| pGBT9dnaE+ + pGAD424 | <2 |
| pGBT9 + pGAD424-2holE+ | <2 |
| pGBT9 + pGAD424-2dnaQ+ | <2 |
| pGBT9 + pGAD424dnaE+ | <2 |
| pGBT9-2holE+ + pGAD424-2holE+ | <2 |
| pGBT9-2dnaQ+ + pGAD424-2dnaQ+ | <2 |
| pGBT9dnaE+ + pGAD424dnaE+ | <2 |
| pGBT9-2holE+ + pGAD424-2dnaQ+ | 665 ± 113 |
| pGBT9-2dnaQ+ + pGAD424-2holE+ | 2,709 ± 571 |
| pGBT9-2holE+ + pGAD424dnaE+ | <2 |
| pGBT9-2dnaE+ + pGAD424-2holE+ | <2 |
| pGBT9-2dnaQ+ + pGAD424dnaE+ | 108 ± 32 |
| pGBT9dnaE+ + pGAD424-2dnaQ+ | 34 ± 11 |
The reporter strain Y187 was transformed with the indicated plasmids.
β-Galactosidase specific activities were calculated as nanomoles of O-nitrophenyl galactoside hydrolyzed per minute per milligram of protein (22). The values are averages for the four transformants, each assayed in triplicate, ± standard deviations.
Interactions of the HolE and DnaE fusion proteins with mutant DnaQ proteins.
To investigate the importance of proper DnaE-DnaQ interactions in maintaining the high fidelity of DNA replication, we examined the ability of the DnaE fusion protein to interact with several DnaQ mutant proteins. E. coli strains carrying the dnaQ49, mutD5, and dnaQ926 mutations were originally isolated as strong mutators (3, 9, 10, 20). Amino acid substitutions responsible for the mutator phenotype of each mutant allele (Table 1) have been identified (9, 30). If the proper interaction of DnaE with DnaQ has biological significance in maintaining the high fidelity of DNA replication, one may expect that at least some mutations in the dnaQ gene affect the ability of DnaQ to interact with DnaE.
To test this hypothesis, we cloned dnaQ49, mutD5, and dnaQ926 mutant DNA sequences into the pGBT9-2 plasmid (see Materials and Methods) and assessed the interactions of the mutant proteins with DnaE and HolE fusion proteins according to their ability to trans-activate the lacZ reporter construct. The experiment with the dnaQ49 allele was performed at 25°C in addition to the normal temperature of 28°C, because this strain has a temperature-dependent mutator activity (and hence, presumably, a temperature-dependent proofreading deficiency). Unfortunately, the yeast strain Y187 used for the two-hybrid system experiment grew very poorly above 30°C and experiments could not be performed at higher temperatures. Our data (Table 3) indicate that the MutD5 and DnaQ926 mutant proteins exhibit the same strengths of interaction with the DnaE and HolE fusion proteins as the wild-type DnaQ fusion protein. In contrast, the DnaQ49 fusion protein showed a sixfold-weaker interaction with DnaE than did wild-type DnaQ. Unexpectedly, the DnaQ49 fusion protein also exhibited a 10-fold-weaker interaction with the HolE fusion protein at 28°C, although little or no effect was observed at 25°C (Table 3).
TABLE 3.
Interaction of HolE and DnaE fusion proteins with wild-type and mutant DnaQ proteins: quantitative assay of β-galactosidase activity
| Plasmids | β-Galactosidase activitya
|
|
|---|---|---|
| 25°C | 28°C | |
| pGBT9-2dnaQ+ + pGAD424dnaE+ | 67 ± 4 | 108 ± 32 |
| pGBT9-2mutD5 + pGAD424dnaE+ | ND | 95 ± 9 |
| pGBT9-2dnaQ926 + pGAD424dnaE+ | ND | 110 ± 5 |
| pGBT9-2dnaQ49 + pGAD424dnaE+ | 10 ± 2 | 17 ± 4 |
| pGBT9-2dnaQ+ + pGAD424-2holE+ | 2,157 ± 310 | 2,709 ± 571 |
| pGBT9-2mutD5 + pGAD424-2holE+ | ND | 2,778 ± 526 |
| pGBT9-2dnaQ926 + pGAD424-2holE+ | ND | 2,452 ± 271 |
| pGBT9-2dnaQ49 + pGAD424-2holE+ | 1,859 ± 210 | 280 ± 64 |
Determined as described in Table 2, footnote b. ND, not determined.
One objective of this work was to correlate the phenotypes of the dnaQ mutators with the altered subunit interactions within the DNA Pol III core. Our results strongly suggest that neither mutD5 nor dnaQ926 affects the ɛ subunit-α subunit and ɛ subunit-θ subunit interactions. These data are consistent with the localization of the mutations within the dnaQ gene. Both mutations are located in the ExoI region presumed responsible for the catalytic activity of the ɛ subunit (9). Thus, it is reasonable to assume that the mutator phenotype of these mutations directly reflects the decreased 3′→5′ exonuclease activity (5) without affecting the α subunit-ɛ subunit interaction. In contrast, our data indicate that the dnaQ49 mutation results in decreased strength of the ɛ subunit-α subunit interaction. This result is consistent with the genetic analysis of dnaQ49 by Takano et al. (30), who, based on the recessive nature of the dnaQ49 mutation (in contrast to the dominant mutD5 allele), suggested that the dnaQ49 ɛ subunit is impaired in its binding to the α subunit. Such a binding defect could result from a specific affinity loss if the responsible mutation in the ɛ subunit were to reside at or near the site of interaction with the α subunit or, alternatively, from a global loss of protein structure. Such a global loss would also likely result in a loss of binding to the θ subunit, as is indeed observed at 28°C. However, since binding of DnaQ49 to the θ subunit appears to be normal at 25°C, but its interaction with the α subunit is severely impaired at this temperature, a specific local defect in binding to the α subunit may be involved, which at higher temperatures could expand to a global effect. It should be noted that even at 25°C the dnaQ49 mutant exhibits a significant mutator phenotype as shown by Fijalkowska et al. (7). At this temperature, the frequency of Rif mutations in E. coli cells bearing dnaQ49 is about 50-fold higher than that in wild-type cells. It is tempting to speculate that the mutator phenotype observed in dnaQ49 strains at 25°C may reflect poor communication between the α and ɛ subunits.
Our results indicating that the DnaQ49 protein also shows decreased strength of interaction with the HolE (θ subunit) protein raise the question of whether the temperature-dependent loss of ɛ subunit-θ subunit binding is related to the temperature-dependent dnaQ49 mutator effect. The function of the θ subunit is not yet clear. A strain carrying a holE null mutation is viable and shows no detectable mutant phenotype (28). Biochemical analysis of the θ subunit indicated that it has no effect on the polymerase activity of the α subunit or the α subunit-ɛ subunit complex (29). However, purified θ subunit was shown to stimulate (about threefold) the 3′→5′ exonuclease activity of the ɛ subunit on a substrate carrying a 3′-terminal G:T mismatch (29). Since the θ subunit interacts only with the ɛ subunit, this may indicate that the θ subunit is involved, either directly or indirectly, in the fidelity of DNA replication by modulating the activity of the ɛ subunit and/or by acting as a protein that ensures communication between the α and ɛ subunits; loss of interaction with the θ subunit could in principle cause a mutator effect. However, the lack of a mutator effect with the ΔholE strain (28) is not consistent with such a hypothesis. Furthermore, the observed reduction in α subunit-ɛ subunit binding at 25°C, at which temperature ɛ subunit-θ subunit binding appears to be normal, suggests that the primary defect of dnaQ49 lies at the level of α subunit-ɛ subunit interaction. Obviously, loss of the ɛ subunit from the replication will be highly mutagenic.
We believe that the usefulness of the yeast two-hybrid system for testing E. coli mutant proteins will facilitate further in vivo studies of the subunit-subunit interactions within the Pol III HE.
ACKNOWLEDGMENTS
We thank members of our research groups for many helpful discussions, C. S. McHenry for providing plasmid pMWE102, and S. Elledge for providing yeast strain Y187.
This work was supported by grants from KBN (6P04 A 043 09 to P.J. and I.J.F. and 6P04 A 015 09 to A.N. and Z.C.) and from the Polish-U.S.A. M. Sklodowska-Curie Foundation (FMKS/KP-96-739 to I.J.F. and R.M.S.).
REFERENCES
- 1.Blanco L, Bernard A, Salas M. Evidence favouring the hypothesis of a conserved 3′-5′ exonuclease active site in DNA-dependent DNA polymerases. Gene. 1992;112:139–144. doi: 10.1016/0378-1119(92)90316-h. [DOI] [PubMed] [Google Scholar]
- 2.Chen D, Yang B, Kuo T. One-step transformation of yeast in stationary phase. Curr Genet. 1992;21:83–84. doi: 10.1007/BF00318659. [DOI] [PubMed] [Google Scholar]
- 3.Cox E C, Horner D L. Dominant mutators in Escherichia coli. Genetics. 1982;100:7–18. doi: 10.1093/genetics/100.1.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Degnen G E, Cox E C. Conditional mutator gene in Escherichia coli: isolation, mapping, and effector studies. J Bacteriol. 1974;117:477–487. doi: 10.1128/jb.117.2.477-487.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Echols H, Lu C, Burgers P M J. Mutator strains of Escherichia coli, mutD and dnaQ, with defective exonucleolytic editing by DNA polymerase III holoenzyme. Proc Natl Acad Sci USA. 1983;80:2189–2192. doi: 10.1073/pnas.80.8.2189. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Fijalkowska I J, Dunn R L, Schaaper R M. Mutants of Escherichia coli with increased fidelity of DNA replication. Genetics. 1993;134:1023–1030. doi: 10.1093/genetics/134.4.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Fijalkowska I J, Dunn R L, Schaaper R M. Genetic requirements and mutational specificity of the Escherichia coli SOS mutator activity. J Bacteriol. 1997;179:7435–7445. doi: 10.1128/jb.179.23.7435-7445.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Fijalkowska I J, Schaaper R M. Effects of Escherichia coli dnaE antimutator alleles in a proofreading-deficient mutD5 strain. J Bacteriol. 1995;177:5979–5986. doi: 10.1128/jb.177.20.5979-5986.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Fijalkowska I J, Schaaper R M. Mutants in the Exo I motif of Escherichia coli dnaQ: defective proofreading and inviability due to error catastrophe. Proc Natl Acad Sci USA. 1996;93:2856–2861. doi: 10.1073/pnas.93.7.2856. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Horiuchi T, Maki H, Sekiguchi M. A new conditional lethal mutator (dnaQ49) in Escherichia coli K12. Mol Gen Genet. 1978;163:277–283. doi: 10.1007/BF00271956. [DOI] [PubMed] [Google Scholar]
- 11.Isbell R J, Fowler R G. Temperature-dependent mutational specificity of an Escherichia coli mutator, dnaQ49, defective in 3′-5′ exonuclease (proofreading) activity. Mutat Res. 1989;213:149–156. doi: 10.1016/0027-5107(89)90146-2. [DOI] [PubMed] [Google Scholar]
- 12.Jonczyk P, Fijalkowska I J, Ciesla Z. Overproduction of the ɛ subunit of DNA polymerase III counteracts the SOS mutagenic response of Escherichia coli. Proc Natl Acad Sci USA. 1988;85:9124–9127. doi: 10.1073/pnas.85.23.9124. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Jonczyk P, Nowicka A. Specific in vivo protein-protein interactions between Escherichia coli SOS mutagenesis proteins. J Bacteriol. 1996;178:2580–2585. doi: 10.1128/jb.178.9.2580-2585.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Kanabus M, Nowicka A, Sledziewska-Gojska E, Jonczyk P, Ciesla Z. The antimutagenic effect of a truncated ɛ subunit of DNA polymerase III in Escherichia coli cells irradiated with UV light. Mol Gen Genet. 1995;247:216–221. doi: 10.1007/BF00705652. [DOI] [PubMed] [Google Scholar]
- 15.Kelman Z, O’Donnell M. DNA polymerase III holoenzyme: structure and function of a chromosomal replicating machine. Annu Rev Biochem. 1995;64:171–200. doi: 10.1146/annurev.bi.64.070195.001131. [DOI] [PubMed] [Google Scholar]
- 16.Kornberg A, Baker T. DNA replication. W. H. New York, N.Y: Freeman; 1992. [Google Scholar]
- 17.Maki H, Horiuchi T, Sekiguchi M. Structure and expression of the dnaQ mutator and the RNase H genes of Escherichia coli: overlap of the promoter region. Proc Natl Acad Sci USA. 1983;80:7137–7141. doi: 10.1073/pnas.80.23.7137. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Maki H, Kornberg A. The polymerase subunit of DNA polymerase III of Escherichia coli. J Biol Chem. 1985;260:12987–12992. [PubMed] [Google Scholar]
- 19.Maki H, Kornberg A. Proofreading by DNA polymerase III of Escherichia coli depends on cooperative interaction of the polymerase and exonuclease subunits. Proc Natl Acad Sci USA. 1987;84:4389–4392. doi: 10.1073/pnas.84.13.4389. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Maruyama M, Horiuchi H, Maki H, Sekiguchi M. A dominant (mutD5) and a recessive (dnaQ49) mutator of Escherichia coli. J Mol Biol. 1983;167:757–771. doi: 10.1016/s0022-2836(83)80109-0. [DOI] [PubMed] [Google Scholar]
- 21.McHenry C S. DNA polymerase III holoenzyme of Escherichia coli. Annu Rev Biochem. 1988;57:519–550. doi: 10.1146/annurev.bi.57.070188.002511. [DOI] [PubMed] [Google Scholar]
- 22.McHenry C S, Crow W. DNA polymerase III of E. coli: purification and identification of subunits. J Biol Chem. 1979;254:1748–1753. [PubMed] [Google Scholar]
- 23.Rose M D, Winston F, Hieter P. Methods in yeast genetics. A laboratory course manual. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 1990. [Google Scholar]
- 24.Sambrook J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 1989. [Google Scholar]
- 25.Schaaper R M. Mechanisms of mutagenesis in the Escherichia coli mutator mutD5: role of DNA mismatch repair. Proc Natl Acad Sci USA. 1988;85:8126–8130. doi: 10.1073/pnas.85.21.8126. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Schaaper R M, Radman M. The extreme mutator effect of Escherichia coli mutD5 results from saturation of mismatch repair by excessive DNA replication errors. EMBO J. 1989;8:3511–3516. doi: 10.1002/j.1460-2075.1989.tb08516.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Scheuermann R H, Echols H. A separate editing exonuclease for DNA replication: the ɛ subunit of Escherichia coli DNA polymerase III holoenzyme. Proc Natl Acad Sci USA. 1984;81:7747–7751. doi: 10.1073/pnas.81.24.7747. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Slater S C, Lifsics M R, O’Donnell M, Maurer R. holE, the gene coding for the θ subunit of DNA polymerase III of Escherichia coli: characterization of a holE mutant and comparison with a dnaQ (ɛ-subunit) mutant. J Bacteriol. 1994;176:815–821. doi: 10.1128/jb.176.3.815-821.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Studwell-Vaughan P S, O’Donnell M. DNA polymerase III accessory proteins. V. θ encoded by holE. J Biol Chem. 1993;268:11758–11791. [PubMed] [Google Scholar]
- 30.Takano K, Nakabeppu Y, Maki H, Horiuchi T, Sekiguchi M. Structure and function of dnaQ and mutD mutators of Escherichia coli. Mol Gen Genet. 1986;205:9–13. doi: 10.1007/BF02428026. [DOI] [PubMed] [Google Scholar]
- 31.Tomasiewicz H G, McHenry C S. Sequence analysis of the Escherichia coli dnaE gene. J Bacteriol. 1987;169:5735–5744. doi: 10.1128/jb.169.12.5735-5744.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]