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Journal of Bacteriology, May 2004, p. 2774-2780, Vol. 186, No. 9
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.9.2774-2780.2004

The Subunit of Escherichia coli DNA Polymerase III: a Role in Stabilizing the Proofreading Subunit

Sharon A. Taft-Benz and Roel M. Schaaper^*

Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Received 18 December 2003/ Accepted 16 January 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
The function of the subunit of Escherichia coli DNA polymerase III holoenzyme is not well established. is a tightly bound component of the DNA polymerase III core, which contains the subunit (polymerase), the subunit (3'5' exonuclease), and the subunit, in the linear order --. Previous studies have shown that the subunit is not essential, as strains carrying a deletion of the holE gene (which encodes ) proved fully viable. No significant phenotypic effects of the holE deletion could be detected, as the strain displayed normal cell health, morphology, and mutation rates. On the other hand, in vitro experiments have indicated the efficiency of the 3'-exonuclease activity of to be modestly enhanced by the presence of . Here, we report a series of genetic experiments that suggest that has a stabilizing role for the proofreading subunit. The observations include (i) defined holE mutator effects in mismatch-repair-defective mutL backgrounds, (ii) strong holE mutator effects in certain proofreading-impaired dnaQ strains, and (iii) yeast two- and three-hybrid experiments demonstrating enhancement of - interactions by the presence of . appears conserved among gram-negative organisms which have an exonuclease subunit that exists as a separate protein (i.e., not part of the polymerase polypeptide), and the presence of might be uniquely beneficial in those instances where the proofreading 3'-exonuclease is not part of the polymerase polypeptide.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The DNA polymerase III (Pol III) holoenzyme (HE) is the major chromosomal replication enzyme in Escherichia coli (19, 22, 30, 31). The enzyme is composed of 17 subunits, 10 of which are distinct (19, 31). HE contains two polymerase core molecules, each consisting of an , , and subunit arranged in the linear order --. The two cores are connected through a dimer of the subunit, establishing the basic arrangement of a dimeric polymerase that simultaneously replicates the leading and lagging strands (14). In addition, the HE contains, for each core, a ß-clamp (ß₂) tethering the polymerase to the DNA to ensure high processivity and the five-subunit -complex (') responsible for loading and unloading the ß-clamp. The precise functioning of the HE complex in chromosomal replication is under active investigation (5, 24, 26, 31).

Within the Pol III core, (the dnaE gene product) is the DNA polymerase, while (the dnaQ gene product) is the 3'5' proofreading exonuclease. The function of (the holE gene product), which is tightly bound to , is unclear. Genetic analysis of the Pol III core constituents has provided insight into the role of its constituents. For example, dnaE(Ts) mutants, encoding temperature-sensitive polymerase subunits, are conditionally lethal, as expected in view of the essential nature of the Pol III replication function. Several dnaE mutants display mutator or antimutator effects (9, 28), indicating the important fidelity role of this enzyme. Many dnaQ mutants exhibit strong mutator phenotypes, indicating the importance of the 3'-exonuclease activity for replication fidelity (45). Deletion mutants of dnaQ (23, 27) or mutants lacking the domain necessary for interaction with the polymerase (46) have been generated, but these mutants proved essentially inviable unless accompanied by a suppressing mutation in dnaE. Based on these studies, is assigned at least two functions: a fidelity function through its 3'-exonuclease activity and a structural function based on its tight, and presumably stabilizing, interaction with the polymerase (23, 27, 45, 46).

In contrast, the role of the subunit of the Pol III core is unknown. Loss of (holE) results in healthy cells with no morphology changes and little or no change in mutant frequencies (42). Based on these studies, it was suggested that is not necessary or important for proper functioning of the Pol III core. does not affect DNA synthesis by or - (43); however, gel filtration (44), coexpression (1), and yeast two-hybrid experiments (18) have demonstrated a tight interaction between and , but none between and . Interestingly, was shown to moderately affect the 3'5' proofreading exonuclease activity, as addition of to an exonuclease assay measuring the removal of a G · T mispair increased -mediated excision of the terminal T residue by about 2.5-fold (44).

The above findings suggest that , while not essential, could play a role in DNA replication and its fidelity, presumably indirectly through its interaction with the subunit. The precise nature of this interaction is being pursued structurally by both nuclear magnetic resonance (NMR) and crystallography studies. Structures of both and have been reported (6, 15, 20), as well as the - interaction surface on (7). Here, we report on a series of genetic experiments on the - interaction. Specifically, we have studied (i) in greater detail, the possible mutator effect resulting from a holE deletion, (ii) the effect of the holE deletion on dnaQ mutator mutants, and (iii) the effect of on the - interaction as measured by yeast two- and three-hybrid assays. The results suggest that may be a stabilizing factor for the subunit, which has been shown to be intrinsically unstable (11).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Media. Luria-Bertani (LB) medium and minimal glucose medium (MM) were standard recipes (38). MMLac plates for determination of the lacZ reversion mutant frequency contained 0.2% lactose instead of glucose. Antibiotics were tetracycline (15 µg/ml), rifampin (100 µg/ml), kanamycin (50 µg/ml), ampicillin (100 µg/ml), and chloramphenicol (20 µg/ml). For the yeast two-hybrid assay, yeast (Saccharomyces cerevisiae) strain Y187 (16) was grown in yeast-peptone-dextrose medium (standard recipe). Yeast strain Y190 (Y153, but cyh^r) (American Type Culture Collection, Manassas, Va.) was grown in yeast-peptone-dextrose for use in the yeast three-hybrid assay. For yeast transformation, yeast synthetic minimal medium was prepared as described previously (34).

Strains. The E. coli strains used are listed in Table 1. Constructions were generally performed by P1 transductions using P1virA. The holE201::cat allele was introduced from RM4030 (42) using chloramphenicol resistance as the selectable marker. The mutL211::Tn5 allele was transferred from ES1293 (40) using kanamycin resistance. The dnaQ49 allele was transferred from NR9695 (37) with the tetracycline resistance conferred by the zae-502::Tn10 transposon, followed by testing for mutator phenotype (40% linkage). The mutD5 mutator allele was transferred from NR9458 (37) with the tetracycline resistance conferred by zaf-13::Tn10, followed by testing for mutator phenotype (90% linkage). The mutagenesis experiments shown below in Table 2 were performed with strains NR9559 (mutL) and NR11853 (mutL holE) into which the F'(pro-lacIZ) from strains CC101 through CC111 (3, 4) had been transferred by conjugation (F' transfer). The experiment shown below in Table 3 was performed using dnaQ and dnaQ holE derivatives of the mismatch-repair-defective strains NR9606 or NR9501. The complete genotype of NR9606 is hisG4(Oc) trp-3(Oc) metE46 thi-1 fhuA1 ara-9 tsx-3 supE44 galK2 ^– rfbD1? rpsL8 or rpsL9 malA1(^r), mtl-1 lacZ118(Oc) mutL::Tn5, as described by Oller et al. (32). NR9501 is identical to NR9606 except that it contains lacZ75(Fs) instead of lacZ118(Oc) (J.-Y. Mo and R. M. Schaaper, unpublished data). This distinction is not relevant to the experiments presented here. Into these strains, we first introduced the series of dnaQ mutator alleles described by Taft-Benz and Schaaper (45), followed by introduction of holE. In the case of the very strong mutD5 and dnaQ49 alleles, holE was inserted first, followed by mutD5 or dnaQ49.

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TABLE 1. List of E. coli strains used

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TABLE 2. lac reversion frequencies in mismatch-repair-defective hol+ and holE strainsa

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TABLE 3. Rifr mutant frequencies in dnaQ mutators with or without the subunita

Plasmids. The plasmids used in the yeast two-hybrid assay were described previously (18, 46). The dnaE and holE genes were contained in plasmid pGAD424, while the various dnaQ alleles were contained in plasmid pGBT9-2 (18). Insertion of the dnaQ gene from the various dnaQ mutator strains was performed as described by Jonczyk et al. (18).

For the three-hybrid assay, vector pBridge (Clontech) was restricted with EcoRI and PstI and treated with calf intestinal alkaline phosphatase (CIAP). The dnaQ and dnaQ49 genes from plasmids pGBT9-2dnaQ or pGBT9-2dnaQ49 (18) were excised using EcoRI and PstI, and the dnaQ fragments were ligated into the EcoRI-PstI-CIAP-treated pBridge to create pBridge/dnaQ and pBridge/dnaQ49, respectively. The recombinant plasmids were sequenced to verify in-frame insertion and correctness of the inserted sequences. To insert the holE gene, pBridge/dnaQ and pBridge/dnaQ49 were restricted with NotI and BglII and treated with CIAP. The entire holE gene (start to stop codon) was PCR amplified from genomic DNA prepared from strain MG1655 (41) using primers containing NotI (forward primer) or BglII (reverse primer) restriction sites. The forward primer was 5'-AGTGTGGCGGCCGCAATGCTGAAGAATCTGGCTAAA-3' (the ATG start codon is underlined), and the reverse primer was 5'-GCGTGTGCGAGATCTCAGGCGTTATGTAAGAAAGTG-3'. The PCR product was restricted with NotI and BglII, gel purified, and ligated into NotI-BglII-CIAP-treated pBridge/dnaQ and pBridge/dnaQ49. The new plasmids, sequenced to verify correct in-frame sequence of the holE gene, were designated pBridge/dnaQholE and pBridge/dnaQ49holE, respectively. The dnaE plasmid used in this assay was pGAD424dnaE, as in the two-hybrid assay.

Mutant frequency determinations. In each experiment, mutant frequencies were determined from 8 to 15 independent cultures from each strain grown overnight in 1 ml of LB medium with agitation at the indicated temperature. Aliquots of appropriate dilutions were plated on LB-rifampin or MMLac plates to determine the number of Rif^r or Lac⁺ mutants, respectively. The total number of cells was determined by plating aliquots of appropriate dilutions on LB or MM plates, respectively. Mutant frequencies were calculated by dividing the median number of mutant colonies by the total number of cells.

Yeast two- and three-hybrid assays. Yeast strains were grown as previously described (18). For the two-hybrid assays, yeast strain Y187 was transformed (2) simultaneously with pGBT9-2dnaQ and pGAD424dnaE to permit assaying of the - interaction, or simultaneously with pGBT9-2dnaQ and pGAD424holE to permit assaying of the - interaction. The dnaQ gene was either the wild type or any of the mutant dnaQ alleles listed below in Table 4. The assays were performed at 30°C as described before (46). For the three-hybrid assay, strain Y190 was transformed simultaneously with pGAD424dnaE and either pBridge/dnaQholE or pBridge/dnaQ49holE. The assay was performed as for the two-hybrid assay, except that 1 mM L-methionine was included in half of the cultures. ß-Galactosidase activity was determined as described by Taft-Benz and Schaaper (46).

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TABLE 4. Yeast two-hybrid analysis of - and - interactions for dnaQ mutator allelesa

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mutator effect of holE deletion. Slater et al. (42) measured the effect of a holE deletion on spontaneous mutant frequencies and observed no significant effect on the rate of nalidixic acid-resistant (Nal^r) mutants and only a slight increase in the rate of rifampin-resistant (Rif^r) mutants. On this basis, it was concluded that plays no significant role in the fidelity of DNA replication. In order to reevaluate this question, we again measured mutant frequencies in holE strains, using two modifications. First, we used mismatch-repair-defective (mutL) strains. In these strains, replication errors are not corrected and, hence, such strains provide a more direct view of replication errors. Second, we used a series of lacZ alleles that revert via defined base-pair substitution or frameshift pathways (3, 4). Such reversion systems might demonstrate effects on replication fidelity that would be obscured in forward systems such Rif^r or Nal^r, in which multiple sites are scored simultaneously. We measured the number of lac⁺ revertants in mutL and mutL holE strains carrying the lacZ alleles from strains CC101 through -111 (Table 2). We noted that mutant frequencies were consistently increased for several, although not all, of the lac alleles. The CC101 allele (A · TC · G) proved quite variable in three independent experiments. We have observed significant variability with this allele in related experiments, presumably due to the variable production of 8-oxo-dGTP, which is believed responsible for the measured A · TC · G transversion (12). More consistent results were obtained for the CC104 (G · CT · A) allele (3.8-fold average increase), the CC105 (A · TT · A) allele (3.5-fold average increase), and the CC110 (+A frameshift) allele (4.5-fold increase). Smaller increases were observed for strains containing the CC106 (A · TG · C) (2.7-fold increase) and CC111 (–A frameshift) (1.8-fold increase) alleles. These increases in both base-substitution and frameshift mutant frequencies in holE strains suggest that does play a role in DNA replication fidelity.

The holE201::cat construct contains a partial deletion of holE, resulting in deletion of the C-terminal 53 amino acids of (42). Although it is unlikely that the remaining 23 N-terminal amino acids have any residual activity, we also constructed a complete deletion of holE (residues 1 to 76) and again measured the above reversion frequencies. The data showed, likewise, modest but consistent mutator effects similar to those for strains carrying the holE201::cat construct (data not shown).

Effect of the holE deletion on the dnaQ49(Ts) allele. The effect of on lac mutant frequencies, as described above, is presumably due to its effect on the proofreading subunit with which it tightly interacts (1, 33, 44). To further investigate this possibility, we studied the effect of the deletion on a recessive dnaQ(Ts) allele, dnaQ49. The DnaQ49 protein contains an amino acid substitution (V96G) (47) that has been proposed to result in temperature-dependent destabilization of the protein. Based on the recessive nature of this allele (29), it was suggested that this destabilization leads to progressive loss of binding to the subunit, resulting in the observed temperature-dependent mutator phenotype (17, 29). Indeed, Jonczyk et al. (18), using a yeast two-hybrid assay, showed that the DnaQ49 protein had decreased binding to the subunit. Based on these findings, we reasoned that the unstable DnaQ49 protein might display increased sensitivity to the loss of the subunit.

We constructed dnaQ49 holE⁺ and dnaQ49 holE201::cat strains and measured their respective Rif^r mutant frequencies at various temperatures. The control dnaQ⁺ strains showed slightly elevated mutant frequencies at 37°C compared to 30°C (Fig. 1), as also noted by others (13). The dnaQ49 strain, as expected, exhibited a large temperature-dependent increase in mutant frequency: at 25°C there was a modest 10-fold mutator effect and at 30°C the effect was about 100-fold, while at 37°C the effect was greater than 1,000-fold. Interestingly, the mutant frequency of the dnaQ49 holE strain was consistently elevated to the highest level, regardless of temperature. At 25°C, the strain showed a 10,000-fold mutator activity, more than 1,000-fold higher than the corresponding dnaQ49 hol⁺ strain. The mutant frequency reached, greater than 100 x 10^–6, is essentially at the level observed in strains completely defective in proofreading (10). The dnaQ49 holE strain also suffered from viability problems, as evidenced by lower viable titers in overnight cultures and the production of significantly smaller and more heterogeneous colonies on plates, as seen previously for near-completely proofreading-defective strains (10). These data suggest that plays an important role in stabilizing the intrinsically unstable DnaQ49 protein.

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FIG. 1. Effect of holE on the Rifr mutant frequencies of dnaQ49 strains. Mutant frequencies were determined as described in Materials and Methods from 15 independent cultures grown at the indicated temperatures. The strains used were KA796 (wild type) and NR11852 (holE) and their respective dnaQ49 transductants, constructed as indicated in Materials and Methods. Note the log scale on the y axis. The frequencies for the two dnaQ+ strains (hol+ and holE) were similar in this experiment, and the data points overlap (line indicated by dnaQ+).

Effect of holE on other dnaQ alleles. Since the DnaQ49 (V96G) protein is thought to be structurally compromised, we were interested to see if would exert a similar stabilizing effect on other, potentially less compromised mutant DnaQ proteins. Previously, we characterized in some detail a large number of new dnaQ mutator mutants (45). Ten of these displayed relatively moderate increases in mutant frequencies at 37°C compared to the dnaQ49 strain, suggesting that these mutant dnaQ alleles encode proofreading subunits with less severe defects (45). For these 10 dnaQ alleles, we determined the Rif^r mutant frequency in both holE⁺ and holE backgrounds at 37°C. We also included the dnaQ49 and mutD5 alleles. mutD5 (T15I) is a strong, dominant, and temperature-independent mutator allele whose defect is thought to be catalytic with little effect on the protein structure (10, 29, 45). The strains used in this experiment were also mismatch repair defective (mutL). The use of the mutL background avoids complications brought about by saturation of mismatch repair that can occur in proofreading-defective strains (10, 35, 36, 39), and the effect of on the proofreading ability can be more effectively evaluated in this background. The results described in Table 3 indicate that four of the dnaQ alleles showed -related changes in mutant frequency similar to those of the dnaQ49 strain: dnaQ920 (R56W), dnaQ923 (H66Y), dnaQ924 (L171F), and dnaQ928 (G17S). A 3.2- to 13-fold increase in mutant frequency at 37°C was observed upon loss of . In contrast, no effect was observed for the mutD5 mutant. These data suggest that is generally capable of stabilizing a certain class of impaired subunits. It is likely that these mutants, like dnaQ49, carry defects that bear a structural component.

- interactions as measured by yeast two-hybrid assays. Whether the dnaQ alleles that proved sensitive to the absence of suffer, like dnaQ49, from impaired interactions with the polymerase subunit was investigated using the yeast two-hybrid system. This system was used previously to evaluate both - and - interactions (18, 46). Consistent with the genetic data, the DnaQ49 protein, but not the MutD5 protein, was shown to be impaired in its binding to the subunit (18). Here, we inserted the various mutant dnaQ genes in the pGBT9-2dnaQ fusion vector (see Materials and Methods) and measured the relative strength of the - and - interactions. The data in Table 4 indicate that among the tested dnaQ alleles, in addition to dnaQ49, several alleles (dnaQ920, dnaQ923, dnaQ924, dnaQ928, dnaQ930, and dnaQ932) suffered from significantly reduced - interaction. The interaction of with was not affected significantly for any of the alleles except for dnaQ49. The - deficiency of dnaQ49 at 30°C (but not at 25°C) has been described elsewhere (18, 46).

The reduced - interaction for the dnaQ920, dnaQ923, dnaQ924, dnaQ928, dnaQ930, and dnaQ932 gene products suggests that for these alleles a reduced ability to bind the polymerase provides at least part of the explanation for their proofreading defect. The dnaQ932 allele contains a termination codon in the C terminus (Table 3) that defines a polymerase-binding domain (residues 187 to 243) (33, 46), and impairment in this domain is a sufficient explanation for this two-hybrid result. The remaining six alleles carry missense mutations in the N-terminal catalytic domain (residues 1 to 186), and we therefore conclude that their reduced - interaction most likely results from a structural impairment, as in the case of dnaQ49. Five of these are also ones described in Table 3 for which the mutator phenotype was significantly enhanced by loss of . Thus, a good correlation exists between presumed structural defects in (as measured in the two-hybrid assay) and sensitivity to loss of (as measured by mutant frequencies). This correlation strengthens our suggestion that is important in maintaining in its proper conformation.

Yeast three-hybrid analysis. To show more directly that affects the - interaction, we utilized the yeast three-hybrid assay using plasmid pBridge. This assay is similar to the two-hybrid assay but involves additional expression of a third protein, whose effect on the interaction of the two fusion proteins is studied. In our version of the system, we again expressed and as interacting fusion proteins and assayed their interaction in the presence or absence of . We constructed plasmids pBridge/dnaQholE and pBridge/dnaQ49holE as described in Materials and Methods. From these plasmids, dnaQ⁺ or dnaQ49 is expressed as a fusion protein, while holE is expressed as a free protein from the Pmet₂₅ promoter, which is repressible by the presence of 1 mM methionine. Thus, this assay allows, in principle, controlled expression of by manipulating the medium formulation. In our hands, we found the Pmet₂₅ promoter to be leaky and not completely repressible even by the presence of 1.5 mM methionine, a concentration at which the yeast cells were exhibiting growth delays. The leakiness of the Pmet₂₅ promoter on a multicopy plasmid was also noted previously (21).

In Table 5, we compare the strength of the - interaction for both the wild-type and DnaQ49 proteins under conditions promoting or inhibiting the expression of holE. The interaction of with wild-type was relatively unchanged by the production of , although we observed a two- to fourfold decrease in the interaction when was expressed. This decrease could have been caused by intrinsic expression differences between plasmids with or without the holE gene or by small changes in plasmid copy number. In contrast, the -DnaQ49 interaction was dramatically increased (up to 100-fold) when was expressed. In fact, the strength of this interaction in the presence of was near that of the wild-type - interaction. These results are in strong support of the conclusion that is an important stabilizing factor for the subunit.

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TABLE 5. Effect of on the - interaction in a yeast three-hybrid assay

Top Abstract Introduction Materials and Methods Results Discussion References

 
The data presented here indicate an important role for the subunit of DNA Pol III in stabilizing the proofreading subunit. This effect of was demonstrated both indirectly, through the effect of on bacterial mutation frequencies, and directly, using the yeast three-hybrid assay.

The beneficial effect of was noted in particular for dnaQ alleles that were impaired in their interaction with the polymerase subunit, as determined from the two-hybrid assay. As the underlying dnaQ mutations were located in the N-terminal catalytic domain of , we suggest that these proteins suffer from a structural impairment (see also reference 45). This structural defect in the catalytic domain likely affects the stability of the full-length protein and its ability to interact effectively with . This is likely the case, even though the C-terminal domain of was shown to contain the primary (but perhaps not only) determinant for binding to (46). In addition to impaired interaction with , the structural impairment of the catalytic domain could also have direct consequences for the exonuclease reaction itself. Thus, the proofreading impairment of these alleles could also have a catalytic component, as also suggested previously by analysis of the NMR model structure of (6). We noted that among -sensitive dnaQ alleles, dnaQ928 (G17S) contains a mutation that altered residues in or near the catalytically important exonuclease I motif (45). Regardless of the precise mechanism of the proofreading defect, appears capable of stabilizing the structure, leading to improved proofreading ability and reduced errors. This proposed role of in stabilizing is consistent with the in vitro observation of stimulated terminal mismatch removal by purified in the presence of (44).

While large effects of were observed in the case of mutant DnaQ proteins, smaller, but significant effects were also observed with the dnaQ⁺ wild-type gene. Here, we demonstrated that the loss of is correlated with increased mutant frequencies for several mutational markers, including transitions, transversions, and frameshifts. This antimutagenic effect of suggests that the stability of can also be rate limiting to proofreading during normal DNA replication. One could speculate that this is relevant as to why E. coli and other gram-negative bacteria contain the subunit as part of their replication complex. As mutation rates are under genetic control (8), the modest effect of in reducing error rates could be significant on an evolutionary time scale.

It is interesting that the replication complex of gram-negative bacteria is characterized by both a subunit and a separate proofreading subunit (i.e., not part of the polymerase polypeptide). In other organisms where the proofreading exonuclease is part of the same polypeptide as the polymerase, no subunit is found, nor is any -like domain in the polymerase or other replication protein. This correlation of with a free proofreading exonuclease is certainly consistent with the idea of as being a specific, protective (or even chaperone-like) subunit for the benefit of .

A BLAST search revealed a holE homolog in the following gram-negative bacteria: E. coli, Shigella flexneri, Salmonella enterica, Yersinia pestis, Wigglesworthia brevipalpis, and Photorhabdus luminescens. Most interestingly, a holE homolog is also found on two plasmids (Proteus vulgaris Rts1 and S. enterica serovar Typhimurium pSLT) and on one phage genome (bacteriophage P1 hot gene). The function of these extrachromosomal holE homologs remains to be determined, but their presence is a further indication that must play a meaningful role.

Foster and Marinus (11), using dnaQ-lacZ fusion proteins, found evidence that is a rather unstable protein, as its cellular level is reduced in strains lacking DnaK, DnaJ, or GrpE heat shock proteins. Apparently, the chaperone function of these proteins is important in maintaining in the proper conformation and helping it to avoid rapid proteolysis. Interestingly, in vitro experiments with purified have also indicated it to be a protein of limited stability, including facile proteolysis (or hydrolysis) (33) and its relatively unstructured behavior during NMR experiments (6).

In addition to stabilizing within the Pol III core, another potentially important role of that needs to be considered is sequestration of free in the cell by capturing it in the - complex. In this complex, is significantly more stable than the free protein (7, 25). The order in which the Pol III core is assembled from its constituents is not known, and the complexation of with could be a first step designed to ensure sufficient supply of the intrinsically unstable for interaction with to form the Pol III core. In this model, performs essentially a chaperone function.

 
We thank Sean Moore and Jeffrey Wu for performing the lacZ mutation experiments and Piotr Jonczyk for providing the yeast two-hybrid vectors containing the dnaQ, dnaE, and holE genes. We thank R. London and S. Holmes of the National Institute of Environmental Health Sciences for a careful review of this paper.

 
* Corresponding author. Mailing address: NIEHS, Laboratory of Molecular Genetics, MD E3-01, P.O. Box 12233, 111 T. W. Alexander Dr., Research Triangle Park, NC 27709. Phone: (919) 541-4250. Fax: (919) 541-7613. E-mail: schaaper{at}niehs.nih.gov.

Present address: Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, May 2004, p. 2774-2780, Vol. 186, No. 9
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.9.2774-2780.2004

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