(Received for publication, August 22, 1995; and in revised form, November 15, 1995 )
From the
The 40.6-kDa subunit of DNA polymerase III of Escherichia coli is a sliding DNA clamp responsible for
tethering the polymerase to DNA and endowing it with high processivity
(Stukenberg, P. T., Studwell-Vaughan, P. S., and O'Donnell,
M.(1991) J. Biol. Chem. 266, 11328-11334). UV
irradiation of E. coli induces a smaller 26-kDa form of the
subunit, termed
*, that, when overproduced from a plasmid,
increases UV resistance of E. coli (Skaliter, R., Paz-Elizur,
T., and Livneh, Z.(1996) J. Biol. Chem. 271, 2478-2481).
Here we show that this protein is synthesized from a UV-inducible
internal gene, termed dnaN*, that is located in-frame inside
the coding region of dnaN, encoding the
subunit. The
initiation codon and the Shine-Dalgarno sequence of dnaN* were
identified by site-directed mutagenesis. The dnaN* transcript
was shown to be induced upon treatment with nalidixic acid, and
transcriptional dnaN*-cat gene fusions were UV inducible,
suggesting induction of dnaN* at the transcriptional level.
Analysis of translational dnaN*-lacZ gene fusions revealed
that UV induction was abolished in strains carrying the recA56, lexA3, or
rpoH mutations,
indicating involvement of both SOS and heat shock stress responses in
the induction process. Expression of dnaN* represents a
strategy of producing several proteins with related functional domains
from a single gene.
UV irradiation of Escherichia coli cells leads to the
formation of both mutagenic and inactivating DNA lesions(1) .
The cells respond by an immediate arrest of DNA replication, followed
by a period of extensive DNA repair, that operates to eliminate DNA
damage in order to prevent replication obstacles(2) . These
processes are controlled primarily by the SOS stress regulon, which
involves more than 20 genes that are commonly regulated by the LexA
repressor and the RecA activator(3, 4) . However, UV
irradiation induces change also in heat shock genes (5) and
other genes (6) which affect the post-UV physiology of the
cell. We have previously found that the subunit of DNA polymerase
III holoenzyme, the major replicase of the E. coli chromosome(7) , limits the ability of the purified
polymerase to replicate UV-irradiated single-stranded DNA(8) .
Consistent with this result, overproduction of the
subunit from a
plasmid caused a reduction in UV resistance and in UV mutagenesis of E. coli cells(9) .
This involvement of the
subunit in UV irradiation effects prompted us to examine whether it may
be present in a different form in UV-irradiated cells. We found that
upon UV irradiation a smaller form of the
subunit, termed
*,
was induced. When overproduced from a plasmid under the inducible lac promoter,
* caused up to a 6-fold increase in UV
resistance of E. coli cells, suggesting a role in recovery
from UV damage, e.g. by involvement in DNA repair or
reactivation of DNA replication(48) .
Smaller derivatives of
proteins that are found in cells are frequently generated by
proteolysis, as in the case of the mutagenesis protein UmuD` that is
formed from UmuD by specific cleavage promoted by the RecA
protein(10) . Alternatively, the protein can be translated from
the overlapping mRNA by a de novo internal translational
start, or it can be expressed from an internal in-frame gene. The
present study shows that * is synthesized from a novel
UV-inducible gene which is located in-frame inside the coding region of
the dnaN gene, and it is subjected to indirect regulation by
both the SOS and heat shock stress responses.
Figure 1:
Vectors used to construct cat gene fusions. Plasmid pCAT was derived from pCM4 by eliminating
the BamHI site at the 3` side of cat, and by
inserting the TrrnB transcription terminator into the AatII site upstream to cat. Transcriptional gene fusions to cat were
constructed by replacing the tet promoter (P) located
on the EcoRV-ClaI fragment with the promoter to be
studied. See ``Experimental Procedures'' for
details.
Figure 2:
The
5`-region of the dnaN* gene. The dnaN* promoter, its
Shine-Dalgarno sequence (SD), its ATG
initiation codon, and codon
ATG are indicated. In
addition, the two recF promoters (P1 and P2)
and the P
promoter located inside dnaN/dnaN* and several cleavage sites of restriction
nucleases are shown.
The mutated dnaN* genes were
cloned under the strong phage T7 promoter in plasmid pBluescript
SK. E. coli BL21(
DE3) cells harboring
these plasmids grew poorly, and they had variations in plasmid copy
number. This was caused most likely by the induction of the T7 RNA
polymerase due to titration of LacI by the lacP promoter
present on the high copy number plasmid. Indeed, deletion of the lacP segment from the plasmids resolved the problem, and the
synthesis of
* could then be quantitatively monitored by metabolic
labeling with [
S]methionine, followed by
SDS-PAGE and fluorography. To facilitate detection of
*,
transcription by the cellular RNA polymerase was inhibited by the
addition of rifampicin, such that transcription was selectively
initiated from the T7 promoter by the T7 RNA polymerase expressed from
a
prophage in the host cell. Indeed, as can be seen in Fig. 3, in the presence of rifampicin, the synthesis of
*
could be easily detected.
Figure 3:
Kinetics of synthesis of * from
mutant dnaN* plasmids assayed by
[
S]methionine labeling. Upper Panel,. E. coli BL21(
DE3) cells harboring plasmid pNLW1
(wild-type dnaN*), pNLM11 (
C*TG mutation,
Met1), or pNLM21 (
C*TG mutation, Met2) were grown to
OD
= 0.4 in minimal medium supplemented with
ampicillin, MgSO
and glucose. Expression of dnaN*
was turned-on by the addition of IPTG, followed by the addition of
rifampicin to inhibit transcription by the host RNA polymerase. Newly
synthesized proteins were pulse-labeled with
[
S]methionine and analyzed by SDS-PAGE followed
by fluorography. Lower panel, Qualification of the fluorogram
shown in A was done by scanning with a Molecular Dynamics 300A
computing densitometer. Full squares, Wt; empty
squares, Met2; circles, Met1.
The validity of measuring rates of protein
synthesis by this procedure depends on the turnover of * in the
cell. The half-life of
* was determined by pulse-labeling of the
protein with [
S]methionine, followed by a chase
with unlabeled methionine. From the decay in the amount of radiolabeled
*, its half-life is approximately 40 min (Fig. 4), much
higher than the time scale used to estimate metabolic rates of
synthesis. Thus,
* is relatively a stable protein, and its
degradation is not expected to affect significantly the measurements of
its synthesis.
Figure 4:
Kinetics of degradation of *. E.
coli BL21(
DE3) cells harboring plasmid pNLW1 (wild-type) were
grown and radiolabeled as described in the legend to Fig. 3. The
kinetics of degradation was assayed by chasing these cells with
unlabeled methionine and analyzing by SDS-PAGE and fluorography the
amounts of
* at the indicated time points after beginning the
chase. The details are described under ``Experimental
Procedures.'' The graph shows the results of tracing the
fluorogram with a Molecular Dynamics 300A computing
densitometer.
Mutating ATG (Met2) had essentially no
effect on the rate of synthesis of
* (Fig. 3). In contrast,
mutating
ATG (Met1) caused a 3-fold reduction in the
rate of synthesis of
* (Fig. 3), suggesting that
ATG is the initiation codon of dnaN*.
To
further support this conclusion, we have constructed another set of
plasmids, in which the dnaN* gene was cloned under the lac promoter in plasmid pUC18. In this case we detected * by
Western blot analysis of cell extracts, using affinity-purified
anti-
antibodies. As can be seen in Fig. 5(lanes
7-12), when the lac promoter was repressed by
glucose,
* was not produced. Upon induction by IPTG, the dnaN* plasmid yielded two products, a major product that
comigrated with a sample of
* purified from an overproducing cell
and a minor product that migrated slightly faster (Fig. 5, lane 2). The major product comigrated with
* synthesized in vivo from the chromosome(48) . As can be seen in Fig. 5(lane 3), mutating the
ATG codon
resulted in disappearance of the major
* band, whereas the minor
*-related band remained unchanged. On the other hand, mutating
ATG (Fig. 5, lane 4) eliminated the
minor band, and caused also a reduction in
*. These results
indicate that
ATG is the initiation codon of dnaN*. The minor band seems to be the result of an alternative
initiation from
ATG when
* was present on a
plasmid.
Figure 5:
The effects of site-directed mutations in
the translation control elements of dnaN* on the synthesis of
*. E. coli AB1157XL cells harboring mutated dnaN* genes cloned under the lac promoter in plasmid
pBluescript SK
were grown to mid-logarithmic phase and
then treated with IPTG to induce the synthesis of
*. Total cell
lysates were fractionated by SDS-PAGE, blotted to a nitrocellulose
membrane, and probed with affinity-purified anti-
subunit
antibodies using the enhanced chemiluminescence method for detection.
The details are given under ``Experimental Procedures.'' Lanes 1-6 show IPTG-treated cells, whereas lanes
7-12 show controls with glucose repression. Lanes 1 and 7, cells harboring the vector pBluescript
SK
; lanes 2 and 8, cells harboring
the wild-type dnaN* plasmid pBSOW1; lanes 3 and 9, cells harboring plasmid pBSOM11, with the
ATG
CTG dnaN* mutation
(Met1); lanes 4 and 10, cells harboring plasmid
pBSOM22 carrying the
ATG
CTG dnaN* mutation (Met2); lanes 5 and 11, cells
with plasmid pBSORS1 carrying the
GCAGG
ACAAG mutation in the dnaN* Shine-Dalgarno
sequence (SD-S); lanes 6 and 12, cells with plasmid
pBSORM1 carrying the dnaN* mutation SD-M
GCAGG
GCAAA mutation in the dnaN*
Shine-Dalgarno sequence (SD-M). Lane M contains purified
subunit and
* as markers.
The assignment of ATG as the initiation
codon of dnaN* pointed to the GCAGG sequence as a likely
Shine-Dalgarno sequence involved in ribosome binding. In order to
examine this possibility we prepared two Shine-Dalgarno double mutants:
GCA GG
ACA AG (SD-S) and GCA GG
GCA AA (SD-M). As can be
seen in Fig. 5(lanes 5 and 6), both mutants
exhibited reduced expression of dnaN*, consistent with the
suggested role of the GCAGG sequence in ribosome binding.
The
dnaN* Transcript Is Induced by Nalidixic Acid-Total RNA was
isolated from E. coli cells, and the 5` termini of mRNAs
initiating at the promoter region of dnaN* were analyzed using
the RNase protection techniques. Several transcription initiation sites
could be detected in the region analyzed, including the major recF transcript initiating at promoter P1, and a fully protected RNA
probe, which represents the overlapping dnaN mRNA (Fig. 6, P). In the dnaN* promoter
region, a band of approximately 130 bases was detected, suggesting that
transcription of dnaN* starts near position 2013 (Fig. 2).
Figure 6:
Mapping of transcription initiation sites
in the dnaN* control region by the RNase protection technique. Upper panel, cellular RNA was extracted from MC4100 wild-type
cells at the indicated time points after induction of the SOS response
by nalidixic acid (40 µg/ml). The RNA was purified and then
hybridized to a uniformly labeled RNA probe transcribed from plasmid
pRPHF11. The hybrids were digested with RNase A and RNase
T, then treated with proteinase K, extracted with phenol,
and separated on a denaturing 6% urea-polyacrylamide gel, after which
the gel was dried and autoradiographed. P,
P
and P
represent transcription initiation at the promoters of dnaN, dnaN*, and the major promoter of the recF gene, respectively. The weak second promoter of recF, P
, is hardly seen under our conditions.
The details are presented under ``Experimental Procedures.'' Lower panel, the riboprobe used in the assay shown in the upper panel and the predicted sizes of its protected regions
that hybridize to mRNAs initiating inside the dnaN*
gene.
The promoter region of the dnaN* gene
contains the sequence 5`- CGCTGTCTACCCTGCCAGCG-3` (positions
1960-1979; Fig. 2), resembling the consensus sequence of
the binding site of the LexA repressor,
5`-NNCTGTNTatNcaNNCAGNN-3`(3) . The most conserved 8
nucleotides are present in the dnaN* SOS box-like sequence,
including the inverted repeat CTGNCAG, which in our case
is part of a pentanucleotide inverted repeat, CGCTGN
CAGCG.
If indeed this sequence binds LexA, it is expected that the gene will
be inducible by agents that induce the SOS regulon. In agreement with
such a prediction, UV irradiation of E. coli cells was found
to cause induction of
*, the dnaN* gene
product(48) . As can be seen in Fig. 6, treatment of
cells with nalidixic acid, a potent inducer of the SOS and the heat
shock responses, caused a 4-5-fold induction in the dnaN* transcript. Thus, induction of dnaN* expression
is regulated, at least in part, at the transcriptional level.
In order to examine whether dnaN* is controlled directly by LexA, the global SOS repressor, we studied the binding of purified LexA repressor to the promoter region of dnaN*, using the gel mobility shift assay(22) . Binding of LexA to the promoter region of recA has been demonstrated by this technique(24) . Indeed, the LexA protein caused specific retardation of a 148-bp MspI restriction DNA fragment carrying the recA promoter which served as a positive control (data not shown). However, we could not to detect any specific binding of LexA to the dnaN* promoter region under a variety of condition (data not shown). This suggests that the inducibility of the dnaN* gene is not regulated directly by LexA.
The region of the recF promoters located inside the dnaN* gene contains an
antisense promoter, termed P(25) (Fig. 2).
The transcript directed by this promoter is complementary the first 86
nucleotides of the dnaN* transcript. We have confirmed the
activity of this promoter in our cells and found that its transcript
was unaffected by treatment with nalidixic acid (data not shown). The
role of this transcript is not clear, although it may function to
regulate dnaN* and/or dnaN expression.
We have used the recA promoter, a
classical SOS promoter, to serve as a positive control for a
UV-inducible gene (Fig. 7). The CAT activity of cells harboring
plasmid pRC5, containing the recA-cat fusion, was 4 units/mg
of protein. UV irradiation at various doses led to the induction of CAT
activity peaking at 30 min after irradiation. The extent of induction
increased with increasing UV dose, up to an effect of 8-fold at 30 J
m (Fig. 7). The increase in recA transcription as assayed by this recA-cat gene fusion is
similar to the results obtained by assaying directly the level of recA mRNA, where a maximal 8-9-fold induction was found
after 20 min(26) . Based on these results we used an inducing
dose of 30 J m
for examining the UV inducibility of dnaN*.
Figure 7:
UV-dose dependence of the induction of CAT
activity from a recA-cat fusion. AB1157 cells harboring the recA-cat fusion plasmid pRC5 were UV-irradiated at various
doses and assayed for CAT activity at the indicated time points after
irradiation as described under ``Experimental Procedures.''
The inducing UV doses were 0 (open circles); 2 J
m (black squares); 5 J m
(white triangles); 15 J m
(black
circles); 30 J m
(white
squares).
We have constructed three dnaN*-cat fusions
plasmids, containing various portions of the dnaN* gene (Fig. 8). Plasmid pNCB17 contains the 332-bp BstUI
(1896)-BstUI(2228) fragment of dnaN containing the
two recF promoters, and 147 nucleotides upstream to the
initiation codon of dnaN* including the SOS box-like sequence,
the promoter, and the Shine-Dalgarno sequence. Plasmid pNCS14 contains
the 172-bp SfaNI-(1918-2090) DNA fragment of dnaN, including the control region of dnaN*, but
lacking the recF promoters. Plasmid pNCH6 contains the 141-bp BstUI-(1896)-HgaI-(2037) fragment, containing
the SOS box-like sequence and the promoters of dnaN*, but no
coding sequences. All three dnaN* gene fragments exhibited
weak promoter activities as judged by the level of CAT activity (Fig. 9). The activity varied from 0.04 to 0.1 unit, which is
2-4-fold higher than the background activity of the control
plasmid without a promoter (pNCH20). UV irradiation of cells harboring
the dnaN*-cat gene fusion plasmids caused a 3-fold induction
of CAT activity (Fig. 9), consistent with the dnaN*
transcript analysis (Fig. 6). This included plasmid pNCS14 which
did not contain the recF promoters. In order to analyze the
P promoter we have constructed a P
-cat fusion using the 138-bp BstUI-(2228)-SfaNI-(2090) DNA fragment (Fig. 8), containing the P
promoter. The resultant
plasmid, termed pPC1, had a basal activity of 0.35 unit, which was
3-4-fold higher than the dnaN* gene fusion (Fig. 9). UV irradiation of cells harboring plasmid pPC1 did not
affect CAT activity (Fig. 9). Thus, in contrast to the dnaN*-cat fusions, the antisense P
-cat fusion was not inducible by UV light, in agreement with the
P
transcript analysis.
Figure 8:
Structure of dnaN* gene fusions.
Fragments containing various portions of dnaN* were fused to
the cat gene in plasmid pCAT to form transcriptional gene
fusions (above the dotted line) or to the 8th codon of the lacZ gene in plasmid pMC1403 to form translational gene
fusions (below the dotted line). The constructions are
described in detail under ``Experimental Procedures.'' The
fused parts of the reporter genes are indicated by striped (cat) or gray (lacZ) bars. The arrows inside the bars indicate the direction of transcription
of dnaN*. Thus, in plasmid pPC1 cat is transcribed
from the antisense promoter P, and in the control plasmid
pNCH20 there is no known promoter to transcribe cat.
Figure 9:
UV-induction of CAT activity from plasmids
carrying dnaN*-cat transcriptional gene fusions. E. coli AB1157 cells harboring the indicated dnaN*-cat gene fusion plasmids were UV-irradiated at a UV dose of 30 J
m and assayed for CAT activity 75 min after
irradiation as described under ``Experimental Procedures.'' Light bars, unirradiated cells; dark bars,
UV-irradiated cells.
Plasmids pTEN5 and pNB3 gave rise
to similar constitutive levels of -galactosidase activity,
indicating that the recF promoters did not contribute to the
expression of the gene fusion (Fig. 10, A and C). Upon UV irradiation of cells harboring these gene fusions,
an increase of
-galactosidase activity was observed. The level of
induction was 5-10-fold, and was the same also for plasmid pSB6,
in which the 5`-half of the SOS box-like sequence was deleted (Fig. 10D). The UV induction was completely abolished
in isogenic mutant cells with either a lexA3 or recA56 mutation (Fig. 10). The lexA3 mutation renders the
LexA repressor non-cleavable by activated RecA protein, whereas the recA56 mutation inactivates the RecA protein, thus the SOS
response cannot be induced in cells carrying either of these mutations.
The noninducibility of dnaN* in these strains suggested that
its expression is under the control of the SOS stress regulon. The uvrA6 mutation, which inactivates nucleotide excision repair,
and the umuC36 mutation, that inactivates UV mutagenesis, did
not affect the UV inducibility of the dnaN*-lacZ fusions,
indicating that the induction was not dependent on excision repair or
UV mutagenesis (data not shown).
Figure 10:
Kinetics of UV-induction of
-galactosidase activity from dnaN*-lacZ translational gene fusions. Cells harboring plasmids with lacZ translational fusions were UV-irradiated and assayed for the
amount of
-galactosidase activity at the indicated time points
after irradiation as described under ``Experimental
Procedures.'' A, cells harboring plasmid pTEN5; B, cells harboring the control plasmids pHSA2 (dnaA-lacZ; circles and triangles), and
pHSC6 (cI-lacZ; diamonds); C, cells with
plasmid pNB3; D, cells with plasmid pSB6. Open
symbols, unirradiated cells; closed symbols,
UV-irradiated cells. Circles, E. coli KY700
(wild-type); squares, E. coli KY703 (lexA3); triangles, E. coli KY705 (recA56). The
inducing UV doses were 60, 5 and 1 J m
for strains
KY700, KY703, and KY705, respectively.
The control fusion of the dnaA gene did show UV induction consistent with the report on the inducibility of dnaA by mitomycin C(27) . However, as can be seen in Fig. 10B, the UV induction was not dependent on the recA gene product, implying that the SOS response was not involved. The negative control for induction was the cI-lacZ fusion that was noninducible by UV irradiation (Fig. 10B).
The kinetics of induction of the dnaN*-lacZ fusions showed peak levels of -galactosidase
activities at 3-5 h after irradiation (Fig. 10), whereas
many SOS functions(26) , as well as the UV induction of
* (48) peak an hour or less after irradiation. A similarly slow
induction of SOS-inducible genes fused to lacZ was observed
before(28) . This may be the result of the fact that the active
structure of
-galactosidase is a tetramer(29) , and that
oligomerization of the fused
-galactosidase molecules might be
slow, particularly when their concentration is low. Indeed, when the
induction of the
*-
-galactosidase protein was examined at the
protein level, by Western blot analysis using polyclonal antibodies
against
-galactosidase, maximal induction of
-galactosidase
occurred approximately 60-90 min after UV irradiation (Fig. 11). This time period is close to the time of induction of
the
* protein(48) . This result shows that the UV-induced
increase in the activity of
-galactosidase was indeed due to an
increase in the synthesis of the enzyme, and it is consistent with the
suggestion that the slower kinetics of induction of the activity of the
fused enzyme was due to the slow rate of assembly of the active
tetrameric structure.
Figure 11:
Kinetics of UV-induction of a
*-
-galactosidase fused protein assayed by immunoblot
analysis. E. coli MC4100 cells harboring plasmid pTEN5 or the
control plasmid pMC1403 were UV-irradiated at 50 J m
and assayed for the induction of the
*-
-galactosidase
protein by immunoblot analysis using anti
-galactosidase
antibodies and the enhanced chemiluminescence method for detection. Lane M contains a marker of
-galactosidase.
Figure 12:
Effect of the activator of the heat shock response on the induction of a dnaN*-lacZ translational fusion. Cells harboring
plasmid pTEN5 were UV-irradiated at 50 J m
and
assayed for
-galactosidase activity at the indicated time points
after irradiation as described under ``Experimental
Procedures.'' White symbols, unirradiated cells; black symbols, UV-irradiated cells. A, E. coli MC4100 (wild-type) cells harboring plasmid pTEN5 (triangles) or both plasmids pCM
1 (carrying the rpoH gene) and pTEN5 (circles). B, E. coli R40NL8 (
rpoH) cells carrying plasmid pTEN5 (triangles) or both plasmids pCM
1 and pTEN5 (circles).
We attempted transcribe dnaN* in vitro using
purified RNA polymerase. We were unable to detect any in vitro initiation of transcription from the dnaN* promoter using
either the regular RNA polymerase, or the heat
shock-specific
RNA polymerase, although the recF transcripts were observed (data not shown). Thus, it
seems that transcription of dnaN* requires additional factors,
or possibly another
subunit. Possible candidates are
, which is specific for some heat-induced
genes(30, 31) , and
, which
transcribes stationary phase genes (32, 33) .
Consistent with such a possibility we found a higher amount of
*
in stationary phase cells(48) .
We have previously shown that a smaller form of the
subunit of DNA polymerase III holoenzyme is induced in E. coli by UV irradiation(48) . Such a protein can be generated by
proteolytic processing, like the mutagenesis protein UmuD`, that is
formed from UmuD by specific proteolysis promoted by the RecA
protein(10) . Alternatively, the protein can be translated from
the dnaN mRNA by a de novo translational start, or it
can be expressed from an internal in-frame gene.
The data presented
here suggests that * is expressed from an internal in-frame gene
termed dnaN*. This is based on the following observations. 1)
The ATG initiation codon of dnaN* and its Shine-Dalgarno
sequence were identified by site-directed mutagenesis. 2) A
transcription initiation site was mapped inside dnaN, upstream
to a Shine-Dalgarno sequence. 3) Plasmids carrying the dnaN*
gene expressed
*. 4) When cloned into a plasmid, the promoter
region of dnaN* directed the expression of a promoter-less cat gene. 5) When the control region of dnaN*,
including the beginning of its coding region, was fused in-frame to a
portion of the lacZ gene lacking all transcriptional and
translational control elements as well as its first 8 codons, it
directed the synthesis of a fused
*-
-galactosidase protein.
The expression of dnaN* is complex and is likely to be
regulated via several mechanisms. Transcription of dnaN* was
not observed in vitro using either or
RNA polymerase, suggesting that another
transcription factor is required. Internal initiation of translation at
the dnaN* ATG initiation codon on the intact dnaN mRNA seems to be very inefficient. This is indicated by the fact
that overexpressing dnaN mRNA from the lac promoter
on a plasmid did not yield any detectable
*. Only after
introducing a frameshift mutation into dnaN, upstream to dnaN*, that eliminated overproduction of the
subunit,
expression of
* was observed from dnaN mRNA(48) .
Thus, it seems that, under normal conditions, synthesis of
* from dnaN mRNA is strongly inhibited, e.g. due to its
engagement in translation of the
subunit or due to direct
inhibition by the
subunit. The antisense transcript originating
from P
, may also be involved in the down-regulation of the
expression of dnaN*.
UV induction of dnaN* is
regulated at the transcriptional level, and subjected to control by
both the SOS and heat shock responses, as indicated by the dependence
of UV induction of dnaN*-lacZ gene fusions on recA, lexA, and rpoH. However, this dual regulation is
indirect, since dnaN* did not bind LexA, and it was not
transcribed by RNA polymerase. Thus, another
factor(s) that is controlled by these major stress responses, is
responsible for the UV induction of dnaN*. The role of the SOS
box-like sequence in the promoter region of dnaN* is puzzling.
It may represent a degenerated LexA binding site, or it may be a
coincidental homology of no functional role, especially since its
5`-half was found to be dispensable for UV induction of dnaN*-lacZ gene fusions. It should be noted that if the sequence
5`-TACTGTATATATATACAGTA-3` is taken as the consensus LexA binding site,
then based of the differences between it and the dnaN* SOS
box-like sequence(34) , the latter is predicted to have no
specific binding to LexA. Similar SOS box-like sequences, that did not
bind LexA, were found in the phr gene, encoding DNA
photolyase(35) , and in the uvrC gene, encoding a
subunit of the UvrABC repair excinuclease(36) ; however, their
significance remains unclear. In addition to dnaN* at least
three other genes are inducible by DNA-damaging agents in a recA- and lexA-dependent pathway, but are not directly
regulated by LexA: The phr gene mentioned above(35) ,
the dnaQ gene encoding the proofreading
subunit of DNA
polymerase III(37) , and the dnaN gene(37, 38) . The mechanism of this regulation
is unknown yet, representing another layer of complexity of the SOS
regulatory network. It may be performed by a factor which is by itself
repressed directly by LexA.
Genes whose coding sequences overlap are
not rare; however, extensively overlapping genes, or genes nested
within other genes, are not common in the chromosome(39) . A
well documented case is the phage T7 gene gp4, encoding a
helicase-primase. The gene encodes two proteins of 63 and 56 kDa, the
latter generated by an internal in-frame start site(40) . The dnaX gene encodes two subunits of DNA polymerase III
holoenzyme: and
. They both start at the same site, but
is terminated before
by a mechanism of ribosomal
frameshifting, leading to the production of proteins of 47.5 and 71 kDa (7) .
The expression of the internal dnaN* gene
produces a protein that lacks precisely one of the three repeating
domains of the subunit. In this respect it belongs to a family of
mechanisms such as alternative splicing, that produce from a single
gene more than one protein, differing by a one or more defined
functional domains. Such mechanisms generate a protein (or more) with a
subset of the properties of the parental intact protein. They might be
required to fulfill biochemically similar reactions under different
conditions, or with conjunction with different counterpart proteins.
Such are the cases of the dnaX gene and the T7 gp4 genes. The intact
subunit forms a
ring-shaped sliding DNA clamp, that confers high processivity on
DNA polymerase III holoenzyme by tethering it to the
DNA(41, 42) . As shown in a companion
study(49) ,
* forms an alternative DNA clamp for DNA
polymerase III that may have a specialized function connected to DNA
synthesis in the UV-irradiated cell. The increase in UV resistance
caused by overproducing
* is consistent with such a
model(48) .