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Journal of Bacteriology, May 1999, p. 2963-2965, Vol. 181, No. 9
0021-9193/99/$04.00+0
The C-Terminal Domain of DnaQ Contains the
Polymerase Binding Site
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 22 September 1998/Accepted 22 February 1999
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ABSTRACT |
The Escherichia coli dnaQ gene encodes the 3'
5'
exonucleolytic proofreading (
) subunit of DNA polymerase III (Pol
III). Genetic analysis of dnaQ mutants has suggested that
might consist of two domains, an N-terminal domain containing the
exonuclease and a C-terminal domain essential for binding the
polymerase (
) subunit. We have created truncated forms of
dnaQ resulting in subunits that contain either the
N-terminal or the C-terminal domain. Using the yeast two-hybrid system,
we analyzed the interactions of the single-domain subunits with the
and 
subunits of the Pol III core. The DnaQ991 protein,
consisting of the N-terminal 186 amino acids, was defective in binding
to the subunit while retaining normal binding to the subunit.
In contrast, the N
186 protein, consisting of the C-terminal 57 amino
acids, exhibited normal binding to the subunit but was defective in
binding to the subunit. A strain carrying the dnaQ991
allele exhibited a strong, recessive mutator phenotype, as expected
from a defective binding mutant. The data are consistent with the
existence of two functional domains in , with the C-terminal domain
responsible for polymerase binding.
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TEXT |
The Escherichia coli DNA
polymerase III (Pol III) holoenzyme (HE) is responsible for the
efficient and accurate replication of the bacterial chromosome (for a
review, see references 9, 10, and
16). This multisubunit enzyme contains a core
polymerase with both polymerizing and exonucleolytic activities. Unlike
those of most other proofreading polymerases, these two activities of Pol III are contained in two separate polypeptides. The polymerase activity is contained in the subunit (dnaE gene
product), while the 3'5' exonuclease (Exo) activity, which serves as
a proofreader for replication errors, is contained in the subunit
(dnaQ gene product). The Pol III core also contains the subunit (holE gene product), but the function of this
subunit is unclear (4, 20, 21). The subunits are arranged in
a linear fashion, --, with binding both and (21). The precise interactions, both functionally and
structurally, between the core subunits and of the core subunits with
the additional accessory factors of the HE are the subject of active
current interest (2, 7, 14).
Previously, in an effort to better understand the roles of the subunit, we undertook a genetic analysis of a large collection of
dnaQ mutator mutants (23). These studies
confirmed and extended the importance of three conserved exonuclease
motifs in the catalytic activity of the DnaQ enzyme (see Fig. 1) and
also suggested that the C terminus of might be required for
interaction with the subunit. dnaQ932, a mutant
containing a stop codon 3 amino acids from the C-terminal end of ,
was recessive in the presence of a single copy of
dnaQ+, suggesting that the DnaQ932 protein does
not compete efficiently with the wild-type protein for binding to .
Furthermore, the region between residues 190 and 212 has been
identified as a so-called Q-linker (24). In certain
proteins, Q-linkers act as a hinge to link domains that have different
functions (22, 24). In , this linker may tether the
N-terminal exonuclease domain to the C-terminal polymerase-binding
domain (Fig. 1).
The modular structure of has been probed by limited proteolysis in
vitro (17). This study revealed a stable 186-amino-acid N-terminal fragment, created by proteolytic cleavage on the N-terminal side of the proposed Q-linker (Fig. 1).
The 186-amino-acid fragment, still comprising the three Exo motifs, has
been investigated biochemically and shown to be fully proficient in
exonuclease activity but to lack interaction with the subunit
(17). Here, we present a genetic analysis of the function of
the C terminus of . We used the yeast two-hybrid assay to study the
interactions of the proposed N- and C-terminal domains with the and
subunits. In addition, we analyzed the properties of an E. coli mutant carrying the truncated N-terminal 186-amino-acid as the sole source of DnaQ protein.
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FIG. 1.
Schematic drawing of E. coli DnaQ proteins.
Highlighted are the conserved Exo I, II, and III motifs (1,
3), the proposed Q-linker (24), and the C-terminal
domain. DnaQ991 is truncated at position 186, as indicated by the
arrowhead. The N186 protein starts at position 187, as indicated by
the arrowhead. Note that DnaQ903 lacks the Exo III motif in addition
to the putative C-terminal binding site.
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The yeast two-hybrid system was used previously to probe the
interactions of the , , and proteins (8). For
example, the subunit specified by the strong mutator allele
mutD5, carrying a Thr15Ile missense mutation in the Exo I
motif, was shown to be fully proficient in binding to both the and
subunits, consistent with its specific catalytic defect and
dominant mutator phenotype (5, 6, 18), while the subunit
specified by the recessive dnaQ49 mutant (15) was
defective in binding to (and, to various extents, also to ).
Our first experiments used dnaQ991, a dnaQ allele
generated by site-directed mutagenesis (Stratagene Quik Change kit) in
the two-hybrid vector pGBT9-2-dnaQ (8), by
converting codon 187 (Phe) into a TAA stop codon (TTTTAA), resulting
in a truncated containing only the N-terminal 186 amino acids (Fig.
1). pGBT9-2-dnaQ991 was combined in the same yeast cell with
pGAD424 containing either the dnaE+ gene or the
holE+ gene as described previously
(8), and the relative strengths of the - and -
interactions were assayed by 
-galactosidase activity. Table
1 shows that the subunits specified
by dnaQ991 and dnaQ49 have strongly reduced
- interactions compared to those encoded by
dnaQ+ or mutD5, their interaction
being essentially indistinguishable from the background level. Each
protein was also assessed for its ability to interact with . The
- interaction occurs more efficiently in the yeast two-hybrid
system, presumably due to the smaller size of (8 kDa) compared to
the subunit (130 kDa) (see also reference 8).
The data show (Table 1) that the DnaQ991 protein is normal in the
- interaction, in contrast to the DnaQ49 protein, which is very
defective in binding under these conditions. The specific loss of
binding of the DnaQ991 protein to the subunit is consistent with
the proposed role of the C-terminal domain of in binding to the
polymerase subunit. The normal binding of DnaQ991 to the subunit is
consistent with the 186-amino-acid fragment being properly folded, as
also suggested by the biochemical demonstration of exonucleolytic
proficiency (17).
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TABLE 1.
Interaction of mutant subunits with Pol III core
subunits and as determined in the yeast
two-hybrid assaya
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To more directly substantiate the role of the C terminus in the -
interaction, we amplified the region of dnaQ encoding residues 187 to 243, the last 57 C-terminal amino acids of , ligated
the PCR product into pGBT9-2 (as described in reference 8) to form plasmid pGBT9-2-N186, and tested the
resulting protein in the yeast two-hybrid system. Table 1 shows
proficient binding of the N186 fusion protein to the subunit.
This observation indicates that the C-terminal domain of DnaQ is not
only required, but is likely sufficient, for interaction with the polymerase.
The in vivo properties of the dnaQ991 allele were also
explored. As loss of the polymerase-binding domain would prevent from being incorporated into the Pol III core, strains carrying dnaQ991 are expected to mimic, in many respects, strains
that lack entirely. Deletion dnaQ strains have been
reported previously in both Salmonella typhimurium and
E. coli (11, 12, 20). The strains contain
(extensive) deletions from the 3' end of dnaQ, resulting in
truncated proteins lacking not only the C terminus but also part or
all of the exonuclease motifs (see DnaQ903 in Fig. 1). The strains
exhibit poor growth as well as highly elevated spontaneous mutation
rates. Cell health is improved considerably by spontaneously occurring
suppressor mutations, termed spq, that arise in the
dnaE gene. We inserted the 1.6-kb
SalI-SmaI dnaQ-rnh fragment from
pFF588 (20) into the SalI-SmaI
linearized allele exchange vector pKO3 (13) and
performed site-specific mutagenesis to create dnaQ991.
The pKO3-dnaQ991 plasmid was introduced into strain MG1655
(20), and a dnaQ+/dnaQ991
heterodiploid was created by plasmid integration at the dnaQ
locus at the nonpermissive temperature (13). Subsequent plating of the heterodiploids on Luria-Bertani (LB)-sucrose plates at
30°C yielded dnaQ991 and dnaQ+
haploid segregants. The dnaQ991 segregants were readily
distinguished by their small-colony morphology, similar to that
reported previously for dnaQ deletion derivatives (11,
20), and their strong mutator phenotype. Table
2 shows the mutant frequency for
rifampin-resistant mutants of the dnaQ991 haploids;
dnaQ deletion (dnaQ903::tet) strains (20) are shown for comparison. The
dnaQ991 mutation resulted in a mutant frequency that was
dramatically (>10,000-fold) increased over the background level,
similar to that seen for dnaQ903::tet.
A modest (~2-fold) but consistent increase in cell counts in
stationary cultures of the dnaQ991 haploid versus the dnaQ903::tet haploid was also observed
(Table 2), suggesting that the presence of active free 3' exonuclease
may be beneficial under the stressful conditions where the Pol III core
consists of only the single subunit. Like that of the
dnaQ903::tet strain, the health of
dnaQ991 haploids was significantly improved by the presence
of the spq-2 allele, a suppressor of the small-colony phenotype of dnaQ903::tet
(20), as evidenced by a much healthier colony morphology and
increased cell counts in the overnight cultures (Table 2). The
spq-2 allele did not affect the mutant frequency. Mutant
frequencies of dnaQ+/dnaQ991 and
dnaQ+/dnaQ903::tet
heterodiploids were also determined (Table
3). None of the heterodiploids exhibited
mutant frequencies significantly different from those of the parental
wild-type strain, indicating that dnaQ991 is fully
recessive. These mutational properties of dnaQ991 are
consistent with lack of binding of the DnaQ991 protein to the subunit and with the resulting absence of the subunit from the HE.
In summary, our data support the contention that the subunit is
composed of two distinct domains, the N-terminal domain, which contains
the exonuclease site (and also the -binding site), and the
C-terminal domain, which is necessary, and likely sufficient, for
interaction with the polymerase. Our results do not rule out the
existence of additional, secondary interactions between the polymerase
and the N terminus of that may be important for the proper
coordination of the two proteins. The discovery of a separate domain
within for binding to the subunit will aid in further understanding the precise nature of the - interaction, which is
likely important for efficient proofreading by the polymerase as well
as for the structural integrity of the replication complex.
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ACKNOWLEDGMENTS |
We thank Fred Perrino for sharing unpublished data, Piotr Jonczyk
and Iwona Fijalkowska for the yeast two-hybrid vectors and for help
with the assays, Russ Maurer for the
dnaQ+/dnaQ903 heterodiploid strain, and George
Church for plasmid pKO3. We also thank Dmitry Gordenin and Karin
Drotschman of NIEHS for careful review of the manuscript.
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FOOTNOTES |
*
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) 541-4043. Fax: (919) 541-7613. E-mail: schaaper{at}niehs.nih.gov.
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REFERENCES |
| 1.
|
Barnes, M. H.,
P. Spacciapoli,
D. H. Li, and N. C. Brown.
1995.
The 3'-5' exonuclease site of DNA polymerase III from gram-positive bacteria: definition of a novel motif structure.
Gene
165:45-50[Medline].
|
| 2.
|
Bertram, J. G.,
L. B. Bloom,
J. Turner,
M. O'Donnell,
J. M. Beechem, and M. F. Goodman.
1998.
Pre-steady state analysis of the assembly of wild type and mutant circular clamps of Escherichia coli DNA polymerase III onto DNA.
J. Biol. Chem.
273:24564-24574[Abstract/Free Full Text].
|
| 3.
|
Blanco, L.,
A. Bernad, and M. Salas.
1992.
Evidence favouring the hypothesis of a conserved 3'-5' exonuclease active site in DNA-dependent DNA polymerase.
Gene
112:139-144[Medline].
|
| 4.
|
Carter, J. R.,
M. A. Franden,
R. Aebersold,
D. R. Kim, and C. S. McHenry.
1993.
Isolation, sequencing and overexpression of the gene encoding the subunit of DNA polymerase III holoenzyme.
Nucleic Acids Res.
14:3281-3286.
|
| 5.
|
Echols, H.,
C. Liu, and P. M. J. Burgers.
1983.
Mutator strains of Escherichia coli, mutD and dnaQ, with defective exonucleolytic editing by DNA polymerase III holoenzyme.
Proc. Natl. Acad. Sci. USA
80:2189-2192[Abstract/Free Full Text].
|
| 6.
|
Fijalkowska, I., and R. M. Schaaper.
1996.
Mutants in the Exo I motif of Escherichia coli dnaQ: defective proofreading and inviability due to error catastrophe.
Proc. Natl. Acad. Sci. USA
93:2856-2861[Abstract/Free Full Text].
|
| 7.
|
Hingorani, M. M., and M. O'Donnell.
1998.
ATP binding to the Escherichia coli clamp loader powers opening of the ring-shaped clamp of DNA polymerase III holoenzyme.
J. Biol. Chem.
273:24550-24563[Abstract/Free Full Text].
|
| 8.
|
Jonczyk, P.,
A. Nowicka,
I. J. Fijalkowska,
R. M. Schaaper, and Z. Ciesla.
1998.
In vivo protein interactions within the Escherichia coli DNA polymerase III core.
J. Bacteriol.
180:1563-1566[Abstract/Free Full Text].
|
| 9.
|
Kelman, Z., and M. O'Donnell.
1995.
DNA polymerase III holoenzyme: structure and function of a chromosomal replicating machine.
Annu. Rev. Biochem.
64:171-200[Medline].
|
| 10.
|
Kornberg, A., and T. A. Baker.
1992.
DNA replication. W. H.
Freeman and Company, New York, N.Y.
|
| 11.
|
Lancy, E. D.,
M. R. Lifsics,
D. G. Kehres, and R. Maurer.
1989.
Isolation and characterization of mutants with deletions in dnaQ, the gene for the editing subunit of DNA polymerase III of Salmonella typhimurium.
J. Bacteriol.
171:5572-5580[Abstract/Free Full Text].
|
| 12.
|
Lifsics, M. R.,
E. D. Lancy, Jr., and R. Maurer.
1992.
DNA replication defect in Salmonella typhimurium mutants lacking the editing () subunit of DNA polymerase III.
J. Bacteriol.
174:6965-6973[Abstract/Free Full Text].
|
| 13.
|
Link, A. J.,
D. Phillips, and G. M. Church.
1997.
Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization.
J. Bacteriol.
179:6228-6237[Abstract/Free Full Text].
|
| 14.
|
Marians, K. J.,
H. Hiasa,
D. R. Kim, and C. S. McHenry.
1998.
Role of the core DNA polymerase III subunits at the replication fork. is the only subunit required for processive replication.
J. Biol. Chem.
273:2452-2457[Abstract/Free Full Text].
|
| 15.
|
Maruyama, M.,
T. Horiuchi,
H. Maki, and M. Sekiguchi.
1983.
A dominant (mutD5) and a recessive (dnaQ49) mutator of Escherichia coli.
J. Mol. Biol.
167:757-771[Medline].
|
| 16.
|
McHenry, C. S.
1991.
DNA polymerase III holoenzyme. Components, structure and mechanism of a true replicative complex.
J. Biol. Chem.
266:19127-19130[Free Full Text].
|
| 17.
| Perrino, F. W., S. Harvey, and S. M. McNeill. Personal communication.
|
| 18.
|
Pham, P. T.,
M. W. Olson,
C. S. McHenry, and R. M. Schaaper.
1998.
The base substitution and frameshift fidelity of Escherichia coli DNA polymerase III holoenzyme in vitro.
J. Biol. Chem.
273:23575-23584[Abstract/Free Full Text].
|
| 19.
|
Rose, M. D.,
F. Winston, and P. Hieter.
1990.
Methods in yeast genetics. A laboratory manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 20.
|
Slater, S. C.,
M. R. Lifsics,
M. O'Donnell, and R. Maurer.
1994.
holE, the gene coding for the subunit of DNA polymerase III of Escherichia coli: characterization of a holE mutant and comparison with a dnaQ (-subunit) mutant.
J. Bacteriol.
176:815-821[Abstract/Free Full Text].
|
| 21.
|
Studwell-Vaughan, P. S., and M. O'Donnell.
1993.
DNA polymerase III accessory proteins. V. encoded by holE.
J. Biol. Chem.
268:11785-11791[Abstract/Free Full Text].
|
| 22.
|
Sutrina, S. L.,
P. Reddy,
M. H. Saier, Jr., and J. Reizer.
1990.
The glucose permease of Bacillus subtilis is a single polypeptide chain that functions to energize the sucrose permease.
J. Biol. Chem.
265:18581-18589[Abstract/Free Full Text].
|
| 23.
|
Taft-Benz, S. A., and R. M. Schaaper.
1998.
Mutational analysis of the 3'5' proofreading exonuclease of Escherichia coli DNA polymerase III.
Nucleic Acids Res.
26:4005-4011[Abstract/Free Full Text].
|
| 24.
|
Wootton, J. C., and M. H. Drummond.
1989.
The Q-linker: a class of interdomain sequences found in bacterial multidomain regulatory proteins.
Protein Eng.
2:535-543[Abstract/Free Full Text].
|
Journal of Bacteriology, May 1999, p. 2963-2965, Vol. 181, No. 9
0021-9193/99/$04.00+0
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