(Received for publication, August 22, 1995; and in revised form, November 15, 1995 )
From the
Control elements located inside the coding sequence of dnaN, the gene encoding the subunit of DNA polymerase
III holoenzyme, direct the synthesis of a shorter and UV-inducible form
of the
subunit (Skaliter, R., Paz-Elizur, T., and Livneh,
Z.(1996) J. Biol. Chem. 271, 2278-2281, and Paz-Elizur,
T., Skaliter, R., Blumenstein, S., and Livneh, Z.(1996) J. Biol.
Chem. 271, 2282-2290). The protein, termed
*, was
overproduced using the phage T7 expression system, leading to its
accumulation as inclusion bodies at 5-10% of the total cellular
proteins.
* was purified in denatured form, followed by refolding
to yield a preparation >95% pure. Denatured
* had a molecular
mass of 26 kDa and contained two isoforms when analyzed by
two-dimensional gel electrophoresis. The major isoform had a pI of
5.45, and comigrated with cellular
*. Size exclusion high
performance liquid chromatography under nondenaturing conditions and
chemical cross-linking experiments indicate that
* is a
homotrimer. DNA synthesis by DNA polymerase III* was stimulated up to
10-fold by
*, primarily due to an increase in the processivity of
polymerization. It is suggested that
* functions as an alternative
sliding DNA clamp in a process associated with DNA synthesis in
UV-irradiated cells.
DNA-damaging agents in general, and UV radiation in particular, affect dramatically the physiology of Escherichia coli(1) . These changes are regulated at the molecular level by several stress regulons, most notably the SOS and heat shock responses(2, 3, 4) . The immediate response is a transient arrest of DNA replication(5, 6, 7) which provides time for the repair of the damaged DNA. DNA replication than recovers in a process that requires SOS-inducible proteins(5, 6, 7) , but its mechanism is largely unknown. During the post-UV recovery period mutations are formed, primarily at sites of DNA damage. This mutagenesis pathway is believed to occur by polymerization through DNA lesions, a process termed bypass synthesis(7, 8, 9) .
A
detailed biochemical analysis of in vitro replication of
UV-irradiated or depurinated DNA with purified DNA polymerase III (Pol
III) ()holoenzyme (10, 11, 12, 13) led us to
concentrate on the
subunit of the polymerase and to conclude that
it modulates UV mutagenesis in vivo(14) and bypass of
UV lesions in vitro(15) . The
subunit is the
major processivity factor of Pol III holoenzyme(16) . It is a
homodimer that forms a ring structure(17) , and once loaded on
DNA by the
complex it functions as a sliding DNA clamp that
tethers the polymerase to the DNA, thus endowing it with high
processivity(18, 19, 20) . In companion
studies (39, 40) we reported that in UV-irradiated E. coli cells a shorter form of the
subunit, termed
*, is produced, which corresponds to the C-terminal two-thirds of
the
subunit. This study describes the overproduction,
purification, and characterization of
* and its activity as an
alternative processivity clamp for DNA polymerase III.
Figure 1:
Overproduction of *. E. coli BL21(DE3) cells harboring plasmid pKT11 were induced by IPTG
treatment, and the synthesis of
* was assayed by Western blot
analysis (left panel) or by pulse labeling with
[
S]methionine (right panel). Cells were
collected at the indicated time points after addition of IPTG (0.5
mM), fractionated on 15% SDS-PAGE, and electroblotted onto a
nitrocellulose membrane. The membranes were probed with anti-
subunit antibodies, and reactive proteins were detected by the enhanced
chemiluminescence method (ECL, Amersham Corp.; left panel). In
parallel, 1-ml samples were withdrawn and labeled with 50 µCi of
[
S]methionine for 10 min at 37 °C. The cells
were then collected, and total cellular proteins were separated by 15%
SDS-PAGE. The gel was dried and fluorographed (right panel). C, control cells harboring the vector
pET3a.
Figure 2:
Purification of *. A Coomassie
Blue-stained gel summarizing the purification process of
*. 1, total proteins from
* overproducing cells; 2,
purified inclusion bodies; 3, the peak fraction eluted from a
Q-Sepharose column; 4, refolded
* after phosphocellulose
chromatography. 5, protein size
standards.
Figure 3:
Analysis of the * preparation for the
presence of the
subunit.
* in increasing amounts was
fractionated by 10% SDS-PAGE. The protein was then transferred to a
nitrocellulose membrane and probed with anti-
antibodies. Lanes 1, 2, 3, and 4 contained 1,
2, 5, and 10 µg of
*, respectively. Lane 4 contained
0.4 µg of the
subunit.
Figure 4:
Immunoblot of a two-dimensional
fractionation of *. Purified
* was separated by
two-dimensional gel electrophoresis, isoelectric focusing in the first
dimension, and 12.5% SDS-PAGE in the second dimension. The
subunit was added as an internal marker. Purified
subunit and
* were fractionated in the second dimension as
markers.
Figure 5:
Size exclusion chromatography of *
under native conditions.
* was analyzed on a 60-cm long TSK3000SW
size exclusion column, as described under ``Materials and
Methods.'' The protein was detected by a Western blot analysis (Panel A) or by absorbance at 214 nm (Panel B). The left lane in Panel A contains a marker of
*.
In order to
further support its suggested trimeric structure, the subunit and
the HPLC peak of
* were each subjected to chemical cross-linking.
These proteins proved to be remarkably resistant to cross-linking
attempts by a variety of chemical agents. Successful cross-linking of
* was achieved with dimethyl suberimidate(25) . The
cross-linked products were separated by SDS-PAGE, transferred onto a
nitrocellulose membrane, and probed with anti-
antibodies. Despite
considerable efforts to reduce smearing, the treated proteins migrated
as smeared bands; still the major result is clearly seen. The
cross-linking of
* produced two forms in addition to the monomeric
*: a
* dimer formed most likely due to partial cross-linking,
and a putative
* trimer representing fully cross-linked
* (Fig. 6, lane 1). The apparent molecular mass of the
putative
* trimer was 107 kDa in this gel, higher than the
calculated molecular mass of 78 kDa. To examine whether this
discrepancy is due to an aberrant migration of the cross-linked protein
in the gel we used cross-linked dimeric
subunit as a marker. The
subunit was cross-linked with ethyl dimethyl carboimidate and N-hydroxysuccinimide since dimethyl suberimidate was
ineffective. As can be seen in Fig. 6(lane 2) the only
band in addition to the monomeric
subunit, presumably the
subunit dimer, had an apparent molecular mass of 107 kDa, identical to
that of the putative
* trimer. Taken together with the HPLC data
these result suggests that the native
* is a trimer.
Figure 6:
Chemical cross-linking of *.
*
was cross-linked with dimethylsuberimidate, and the
subunit was
cross-linked with ethyl dimethyl carboimidate and N-hydroxysuccinimide as described under ``Materials and
Methods.'' The cross-linked proteins were fractionated by 8%
SDS-PAGE, transferred to a nitrocellulose membrane, and probed with
anti-
antibodies. Lane 1, cross-linked
*; lane
2, cross-linked
subunit.
Figure 7:
* stimulates DNA synthesis by DNA
polymerase III*. The effect of
* on DNA synthesis by Pol III* was
tested using oligonucleotide-primed M13mp8 ssDNA as described under
``Materials and Methods.'' DNA synthesis was measured in the
absence (open circles) or presence (closed circles)
of
* by scintillation counting of radiolabeled dTTP that was
incorporated into acid-insoluble replication
products.
Figure 8:
Analysis of DNA products synthesized by
DNA polymerase III* in the presence of *. A time course of the
replication of SSB-coated and primed M13mp8 ssDNA with Pol III* (12
nM) and
* (200 nM). The reaction mixture was
preincubated at 30 °C for 10 min in the absence of dATP and
[
P]dTTP, after which DNA synthesis was initiated
by the addition of the dNTPs. Replication products were separated on a
1% alkaline agarose gel and visualized by autoradiography. The details
are presented under ``Materials and
Methods.''
Can the activity
observed be due to a contamination of the subunit in our
*
preparation? As shown in Fig. 3no
subunit could be
identified in immunoblots of
* preparations. However, in order to
rule out completely this possibility, we fractionated the purified
* on an SDS-polyacrylamide gel, eluted the
* band and
renatured it. This preparation of
*, which was resolved on the gel
from any contamination of the
subunit, stimulated Pol III* (Fig. 9, lane 2 compared to lane 1 at each
time point), although to a lesser extent than the original
*
preparation (Fig. 9, lane 3 compared to lane 2 at each time point). Presumably the harsh treatment led to a
considerable decrease in the activity of
*. Still, the results
clearly show that the stimulation of Pol III* was caused by
*.
Figure 9:
Gel-purified * stimulates Pol III*.
* that was purified by SDS-PAGE followed by renaturation was
examined for its effect on Pol III* as described under ``Materials
and Methods.'' The template used was primed M13mp8 ssDNA in the
absence of SSB. Lanes marked 1 contained Pol III* alone; lanes
marked 2 contained Pol III* and 100 nM
*
(purified by SDS-PAGE); lanes 3 and 4 contained 100
nM and 300 nM
*, respectively, purified by the
standard procedure.
Figure 10:
ATP is required for stimulation of DNA
polymerase III* by *. SSB-coated and oligonucleotide-primed M13mp8
ssDNA was replicated with Pol III* (12 nM), and with
*
(200 nM) or the
subunit (200 nM) as described
under ``Materials and Methods,'' with dATP
S instead of
dATP. The traces of long radiolabeled products synthesized by Pol III*
in the absence of added
* or
subunit were most likely due to
trace contamination of
subunit in the Pol III*
preparation.
Figure 11:
* increases the processivity of DNA
polymerase III*. DNA replication was performed with a 55-fold excess of
primed M13mp8 ssDNA over the polymerase, in the absence of SSB.
Aliquots were withdrawn at the indicated time points and the
replication products were fractionated on a denaturing alkaline agarose
gel. An autoradiogram of radiolabeled products is shown. The details
are described under ``Materials and
Methods.''
Our results suggest that * can act as an alternative
polymerase clamp, and confer high processivity on Pol III* (Fig. 11). This activity requires ATP and is thus likely to
involve loading of a Pol III*
complex on the primer template, similar to the case of Pol III
holoenzyme(18, 19) . The overall slow synthesis
activity of this polymerase (Fig. 7) is likely to be due to a
slower initiation stage, e.g. a slow loading process of
on the DNA by the
complex which is
part of Pol III*(18, 19) . Consistent with this
possibility, when a period of preincubation was allowed, a rapid phase
of polymerization was observed, similar to that of Pol
III*
( Fig. 8and Fig. 10). A
possible explanation is that the preincubation period allows the
assembly of Pol III*
complexes, and
thus upon addition of the dNTPs polymerization resumes immediately.
According to this model the assembly of a Pol
III*
complex on the primer-template
is less efficient than the assembly of a Pol III*
initiation complex. However, once a Pol
III*
complex is assembled on the
DNA, it has a high processivity similar to that of Pol III holoenzyme.
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(17) .
* contains
precisely two of the three domains of the
subunit(40) .
The fact that
* appears as a trimer suggests that it may from a
-like ring structure, composed of three two-domain
proteins, forming an alternative DNA clamp. The three repeating domains
of the
subunit are structurally very similar, although there is
no significant homology at the amino acid sequence level(17) .
Although the overall structure of
may be
similar to that of
, it is expected to show
significant differences in activity as compared to
,
since it is lacking the N-terminal domain of the
subunit.
Polymerase clamps composed of homotrimers are in fact more common
than dimeric clamps. The gp45 gene of bacteriophage T4, which serves as
an auxiliary subunit of T4 DNA polymerase(31) , is an homologue
of the subunit(17) . The gp45 protein has a molecular
mass of 25 kDa, and based on its amino acid sequence it is a homologue
of two of the three repeating domains of the
subunit(17) . Indeed, gp45 forms a trimer in solution (32, 33) and is likely to form a
-like sliding clamp. In eukaryotes, proliferating cell
nuclear antigen (PCNA) serves as a processivity factor of DNA
polymerase
, which is involved in DNA replication and
repair(34, 35, 36) . PCNA has usually a size
of 29 kDa, and it forms a trimeric hexagonal ring-shaped structure very
similar to that of the
subunit(37) . Interestingly, it
was recently reported that two PCNA genes were found in carrot,
encoding putative PCNA molecules of 29 and 40 kDa(38) . Based
on its size, its trimeric structure and its activity as a processivity
factor,
* could thus be viewed as an analog of the T4 gp45 and the
eukaryotic PCNAs, with a possible evolutionary link.
The two major
DNA synthesis processes in UV-irradiated cells are repair synthesis of
short ssDNA gaps(1) , and the resumption of DNA replication,
that involves both reactivation of replication forks stalled at UV
lesions and the activity of new origins of replication (7) . In
addition, trans-lesion synthesis occurs and gives rise to mutations.
The involvement of * in these processes, and its effects on each
of the three E. coli DNA polymerases are currently under
investigation. However, its activity as a processivity clamp, and the
increase in cell UV survival caused by its overproduction (39) suggest that it is involved in a process related to DNA
synthesis in the UV-irradiated cell.