(Received for publication, August 11, 1995; and in revised form, November 15, 1995 )
From the
The subunit of DNA polymerase III holoenzyme of Escherichia coli is a 40.6-kDa protein that functions as a
sliding DNA clamp (Stukenberg, P. T., Studwell-Vaughan, P. S., and
O'Donnell, M.(1991) J. Biol. Chem. 266,
11328-11334). It is responsible for tethering the polymerase to
DNA and endowing it with the high processivity required for DNA
replication. Here and in a companion study (Paz-Elizur, T., Skaliter,
R., Blumenstein, S., and Livneh, Z.(1996) J. Biol. Chem. 271,
2482-2490) we report that the dnaN gene, encoding the
subunit, contains an internal in-frame gene, termed dnaN*, that encodes a smaller form of the
subunit. The
novel 26-kDa protein, termed
*, is UV-inducible, and when
overexpressed from a plasmid under an inducible promoter, it increases
up to 6-fold the UV resistance of E. coli cells. These
findings suggest that the
* protein functions in a reaction
associated with DNA repair or recovery of DNA replication in
UV-irradiated cells.
UV irradiation of Escherichia coli cells produces in
DNA primarily cyclobutyl pyrimidine dimers and pyrimidine-pyrimidone ) ()adducts that are responsible for most of the mutagenic and
inactivating effects of UV irradiation(1) . The immediate
cellular response to UV irradiation is an arrest of chromosome
replication in order to allow a period of elimination of DNA damage by
DNA repair mechanisms(2) . Many of the genes known to be
involved in these processes, such as uvrA, uvrB, recA, umuD, and umuC are regulated by the
SOS regulatory network, whose primary function is to help the cell in
coping with DNA damage(3, 4) . However, UV irradiation
induces also heat shock genes (5) and other genes (6) which affect the post-UV physiology of the cell.
We have
previously examined in detail the replication of UV-irradiated DNA with
purified proteins(7, 8, 9, 10) .
These studies revealed that the subunit of DNA polymerase III
holoenzyme, the major replicase of the E. coli chromosome(11) , limits the ability of the purified
polymerase to bypass UV lesions during in vitro replication of
UV-irradiated single-stranded DNA(10) . 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(12) . 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. Here and in two companion
studies(27, 28) , we report that UV irradiation
induces a shorter form of the
subunit that functions in vitro as an alternative DNA polymerase clamp. We suggest that this
protein functions in DNA synthesis associated with post-UV recovery in E. coli.
Figure 1: The control region of the putative dnaN* gene residing inside the coding sequence of dnaN. The sequence that shows homology to a LexA binding site (SOS boxlike sequence), the -35 and -10 regions of the putative promoter, the Shine-Dalgarno (SD) sequence and the putative ATG initiation codon are underlined. Also shown are the two promoters of the adjacent downstream recF gene. Those were previously mapped inside the coding sequence of dnaN(26) . Sequence coordinates are according to Ohmori et al.(18) . The lower panel shows schematically the relationship between dnaN and dnaN*, and their protein products.
As can be seen in Fig. 2(lane 4), the subunit
was overproduced; however, no protein of 26 kDa was detected. Puzzled
by the inability of this plasmid to express
*, we examined its
expression from plasmid pUN234FS2, which contains a +4
insertion/frameshift mutation inside the dnaN gene upstream to
the beginning of the dnaN* gene. As can be seen (Fig. 2, lane 6), the mutation essentially eliminated
the synthesis of the
subunit from the plasmid, and at the same
time a band of
* could be detected. This represents most likely
* synthesized from the mutated plasmid. A possible explanation is
that the
subunit itself is a negative regulator of
*
expression, and when present in large amounts, it inhibits the
expression of
*. This experiment also suggests that the 26-kDa
protein was not formed by proteolysis of the
subunit either in vivo or during extract preparation and handling, otherwise
it would have been expected to appear in lane 4. Notice that a
26-kDa protein was not observed also in lanes 2 or 3 in Fig. 2suggesting that cellular
* is present in
much smaller amount than the
subunit.
Figure 2:
Expression of proteins from dnaN plasmids. E. coli MC4100XL cells harboring plasmids with
the dnaN gene cloned under the lac promoter were
treated with IPTG to induce expression from the lac promoter.
Equal amounts of total cellular proteins were fractionated by SDS-PAGE,
transferred to a nitrocellulose membrane and detected with affinity
purified anti- antibodies using enhanced chemiluminescence as
described under ``Materials and Methods.'' The plasmids
tested were plasmid pUN234, carrying the dnaN gene cloned
under the lac promoter in plasmid pUC18 (lanes 3 and 4), plasmid pUN234FS2, a derivative of pUN234, containing a
+4 insertion/frameshift mutation in the dnaN upstream to dnaN* (lanes 5 and 6), and the control
vector pUC18 (lane 2). Lane 1 contains purified
subunit and
* markers.
Figure 3:
Expression of proteins from dnaN* plasmids. E. coli MC4100XL cells harboring plasmids with
the dnaN* gene cloned under the lac promoter were
treated with IPTG to induce expression from the lac promoter.
Total cellular proteins were fractionated by SDS-PAGE, transferred to a
nitrocellulose membrane, and detected with affinity-purified anti-
antibodies using enhanced chemiluminescence as described under
``Materials and Methods.'' In addition to the
subunit
and
* the antibodies cross-reacted with two other proteins whose
signals on the immunoblots exhibited large variations. Plasmid pBSOW1
contains dnaN* expressed under the lac promoter in
plasmid pBluescript SK
, and plasmid pBSW7 is similar
to pBSOW1 except that dnaN* was cloned in the opposite
orientation.
Figure 4:
Identification of cellular * by
two-dimensional gel electrophoresis. Total protein was extracted from a
late logarithmic E. coli MC4100 culture and fractionated by
two-dimensional gel electrophoresis. The gels were assayed by Western
blot analysis for the presence of the
subunit and
*. The
detailed procedures are described under ``Materials and
Methods.'' Lane M contains purified
subunit and
* as markers. Lane E contains an extract fractionated by
one-dimensional SDS-PAGE. Arrows 1 and 2 mark
*
and the
subunit, respectively.
Figure 5:
Induction of * by UV irradiation. E. coli MC4100 were grown to OD
= 0.3,
UV-irradiated at a dose of 50 J m
, and returned for
further growth. At the indicated time points, cell samples were
withdrawn, and total cell extracts containing equal amounts of protein
were fractionated by 10% SDS-PAGE. The presence of
* was assayed
by immunoblotting. The detailed procedure is presented under
``Materials and Methods.''
As can be seen in Table 1, induction of the synthesis of * from plasmid pBSOW1
by IPTG caused an increase of up to 6-fold in the UV resistance of two
different strains, compared to the same cells grown with glucose and in
the absence of IPTG, conditions under which the lac promoter
was repressed. Such increase in UV survival was not observed with the
control plasmid pBSW7, in which dnaN* was cloned opposite to
the lac promoter, or with the cells without any plasmid (Table 1). It is noteworthy that when the intact
subunit
was overproduced from the same plasmid, it caused a decrease in UV
resistance of the cells, opposite to the effect of
*(12) .
Overproduction of the
subunit of DNA polymerase III had no effect
on UV resistance(12) . These results are consistent with a
model in which
* participates in a recovery process in the cell,
which is limited by the amount of
*. Overproduction of
*
would be expected to facilitate this recovery reaction, and thus
increase UV resistance. Possible pathways to be affected are DNA repair
or the reactivation of DNA replication(2) .
It was recently
shown by x-ray crystallography that the subunit is composed of
three structurally similar domains, and it dimerizes to form a
hexagon-like ring(20) .
* contains precisely two of the
three domains of the
subunit, raising the possibility that it
forms a trimeric alternative clamp that functions in the UV-irradiated
cell with one of the DNA polymerases. Such a clamp activity is
demonstrated in our companion study(28) . The function of the
subunit is carried out in eukaryotes by the proliferating cell
nuclear antigen (PCNA), which serves as the processivity clamp of DNA
polymerase
(21) . PCNA and the
subunit are
structurally very similar, forming nearly identical hexagonal
rings(22, 23) . However, in contrast to the dimeric
structure of the 40.6 kDa
subunit, PCNA is 29 kDa and forms a
trimer(23) . Thus
* bears resemblance to PCNA with a
possible evolutionary link. In this context it is interesting to note
that PCNA is the target for several regulatory mechanisms that
coordinate the response of mammalian cells to UV
irradiation(24, 25) .