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Journal of Bacteriology, May 2001, p. 2897-2909, Vol. 183, No. 9
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.9.2897-2909.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Genetic Interactions between the Escherichia coli
umuDC Gene Products and the
Processivity Clamp of
the Replicative DNA Polymerase
Mark D.
Sutton,
Mary F.
Farrow,
Briana M.
Burton, and
Graham C.
Walker*
Biology Department, Massachusetts Institute
of Technology, Cambridge, Massachusetts 02139
Received 5 December 2000/Accepted 22 January 2001
 |
ABSTRACT |
The Escherichia coli umuDC gene products encode DNA
polymerase V, which participates in both translesion DNA synthesis
(TLS) and a DNA damage checkpoint control. These two temporally
distinct roles of the umuDC gene products are regulated
by RecA-single-stranded DNA-facilitated self-cleavage of UmuD (which
participates in the checkpoint control) to yield UmuD' (which enables
TLS). In addition, even modest overexpression of the
umuDC gene products leads to a cold-sensitive growth
phenotype, apparently due to the inappropriate expression of the DNA
damage checkpoint control activity of UmuD2C. We have
previously reported that overexpression of the
proofreading subunit
of DNA polymerase III suppresses umuDC-mediated cold
sensitivity, suggesting that interaction of
with UmuD2C
is important for the DNA damage checkpoint control function of the
umuDC gene products. Here, we report that overexpression
of the
processivity clamp of the E. coli replicative
DNA polymerase (encoded by the dnaN gene) not only
exacerbates the cold sensitivity conferred by elevated levels of the
umuDC gene products but, in addition, confers a severe
cold-sensitive phenotype upon a strain expressing moderately elevated
levels of the umuD'C gene products. Such
a strain is not otherwise normally cold sensitive. To identify mutant
proteins possibly deficient for physical interactions with the
umuDC gene products, we selected for novel
dnaN alleles unable to confer a cold-sensitive growth
phenotype upon a umuD'C-overexpressing strain. In all, we identified 75 dnaN alleles, 62 of
which either reduced the expression of
or prematurely truncated its
synthesis, while the remaining alleles defined eight unique missense
mutations of dnaN. Each of the dnaN
missense mutations retained at least a partial ability to function in
chromosomal DNA replication in vivo. In addition, these eight
dnaN alleles were also unable to exacerbate the cold
sensitivity conferred by modestly elevated levels of the
umuDC gene products, suggesting that the interactions between UmuD' and
are a subset of those between UmuD and
. Taken
together, these findings suggest that interaction of
with UmuD2C is important for the DNA damage checkpoint function
of the umuDC gene products. Four possible models for how
interactions of UmuD2C with the
and the
subunits of
DNA polymerase III might help to regulate DNA replication in response
to DNA damage are discussed.
 |
INTRODUCTION |
The umuDC genes encode a
DNA polymerase, DNA polymerase V (Pol V), that has a remarkable ability
to copy over abasic sites (43, 63), cyclobutane dimers
(62), and pyrimidine-pyrimidone [6-4] photoproducts
(62), a process referred to as translesion DNA synthesis
(TLS). This ability, however, comes at the cost of reduced fidelity.
Thus, replication by Pol V is inherently less accurate, leading to the
formation of mutations, even when replicating undamaged templates
(29, 62). Therefore, to limit the ability of Pol V to
introduce mutations into the host genome, expression of the
umuDC genes is tightly regulated as part of the
Escherichia coli stress-induced SOS response
(12). This highly regulated response helps the cell
maintain the integrity of its genome following treatments that lead
either directly or indirectly to DNA damage (12).
The E. coli SOS response consists of at least 30 unlinked
genes (9, 12), collectively referred to as the SOS
regulon. Expression of the various SOS-regulated genes is coordinately regulated at the level of their transcription by the LexA and RecA
proteins (28). In the absence of DNA damage, LexA acts to
repress the expression of the members of the SOS regulon
(27). RecA protein, the main bacterial recombinase
required for essentially all homologous recombination (reviewed in
reference 22), binds to single-stranded DNA (ssDNA)
generated by the cell's failed attempts to replicate past lesions in
its genome, thus forming RecA-ssDNA nucleoprotein
filaments (47). These RecA-ssDNA filaments, in
addition to acting in homologous recombination, also act to facilitate
the latent capacity of LexA to autodigest (26).
Autodigestion of LexA serves to inactivate it as a transcriptional
repressor, leading to the concomitant increase in expression of
LexA-regulated genes (12).
The UmuD protein similarly undergoes a RecA-ssDNA-facilitated
autodigestion that serves to remove its first 24 residues to yield
UmuD' (3, 34, 48). The UmuD'2
homodimer then interacts with UmuC (18, 59, 68), which has
an ability to catalyze the formation of phosphodiester bonds, in such a
way that the UmuD'2C complex is able to
participate in TLS (43). In addition to participating in
TLS, UmuC together with the full-length UmuD2 homodimer participates in a DNA damage checkpoint control that acts to
regulate DNA replication in response to DNA damage, thereby allowing
additional time for nucleotide excision repair to accurately remove
lesions in the DNA prior to continued replication (37). Thus, self-cleavage of UmuD to UmuD' can be regarded as a molecular switch that acts to temporally regulate these two distinct activities of the UmuD2C and UmuD'2C
complexes (37, 57).
In addition to participating in a DNA damage checkpoint control and
enabling TLS, overexpression of the umuDC gene products confers a cold sensitivity for growth (30, 38, 59). Our recent characterizations of umuDC-mediated cold sensitivity
indicated that (i) moderately elevated levels of the umuDC
gene products confer a cold-sensitive growth phenotype, while similarly
elevated levels of the umuD'C gene products do
not (59), and (ii) the catalytic DNA polymerase activity
of UmuC is not required for this cold sensitivity (59).
These findings, together with others, suggest that the cold sensitivity
conferred by elevated levels of the umuDC gene products is a
manifestation of the inappropriate expression of
UmuD2C functions involved in the DNA damage
checkpoint control (37, 38, 59). Therefore, in an effort
to better characterize the components of the
UmuD2C-dependent checkpoint control, we have
embarked on an analysis of the genetic requirements of
umuDC-mediated cold sensitivity.
We have previously suggested that interactions of the umuDC
gene products with components of the E. coli replicative DNA
polymerase, DNA polymerase III holoenzyme (Pol III), could serve as a
convenient mechanism for regulating the checkpoint and TLS roles of the
umuDC gene products (37, 57). On the basis of
this hypothesis, we reasoned that if interactions involving the
umuDC gene products and components of Pol III were important
for the checkpoint role of UmuD2C, we might then
be able to observe an effect on the extent of the cold sensitivity
conferred by umuDC by the simultaneous overexpression of
certain (i.e., relevant) components of Pol III. Using such an approach,
we have recently reported that overproduction of the
proofreading
subunit of Pol III or deletion of its structural gene (dnaQ)
suppresses umuDC-mediated cold sensitivity
(56). A systematic analysis of the remaining nine Pol III
subunits indicated that the homodimeric
processivity clamp was the
only other Pol III subunit that when overexpressed affected the extent
of umuDC-mediated cold sensitivity (56).
In this report, we describe how overexpression of the
processivity
clamp (encoded by dnaN) strongly exacerbates
umuDC-mediated cold sensitivity. We have exploited this
ability of the
clamp to confer a cold-sensitive growth phenotype
upon a umuD'C-expressing E. coli
strain (a strain that is not normally cold sensitive
[59]) to identify novel dnaN alleles unable
to confer this phenotype. Our genetic characterizations of these novel
dnaN alleles indicate that they are also unable to
exacerbate the cold sensitivity conferred by elevated levels of the
umuDC gene products. Our results described in this report,
taken together with others (37, 38, 56, 57, 59), suggest
that the UmuD2C-dependent DNA damage checkpoint control is a manifestation of protein-protein interactions
involving UmuD2C and the
and the
subunits
of Pol III.
 |
MATERIALS AND METHODS |
Bacteriological techniques.
E. coli strains and
plasmid DNAs used in this study are described in Table
1. Bacterial strains were routinely grown
in Luria-Bertani (LB) medium as described previously
(46), unless otherwise stated. When necessary, LB medium
was supplemented with the following antibiotics at the indicated
concentrations: ampicillin, 150 µg/ml; spectinomycin, 60 µg/ml; and
kanamycin, 80 µg/ml. Because strains expressing elevated levels of
the umuDC gene products do not grow well at 30°C
(30, 38, 59), the strains bearing a
umuDC-expressing plasmid used in this study were routinely
grown at 42°C, unless otherwise stated. pGYD'
C was constructed by
the same method used for construction of pGYD
C (59).
Briefly, pGY9738 was digested with MluI, followed by end
filling of the linear DNA with all four deoxynucleoside triphosphates
and the Klenow fragment of DNA Pol I (New England BioLabs) prior to its
ligation with T4 DNA ligase (New England BioLabs). Bacterial
transformation was by either calcium chloride treatment
(46) or electroporation using a GenePulser (Bio-Rad) as
per the manufacturer's recommendation. Plasmid DNAs were isolated
using the QIA-Spin Prep kit (Qiagen) as per the manufacturer's
recommendation.
Genetic assay for the selection of novel dnaN
alleles.
A derivative of AB1157 bearing the plasmid pGY9738, which
constitutively expresses elevated levels of the
umuD'C gene products (51), grows
well at 30°C. However, if AB1157(pGY9738) also carries the
compatible plasmid pJRC210, which overexpresses the
processivity clamp of Pol III from the Ptac promoter,
its growth is cold sensitive (Table 2).
To select for dnaN alleles unable to confer the
cold-sensitive phenotype, we selected for derivatives of AB1157 bearing
both the umuD'C-expressing plasmid and the
-expressing plasmid that were able to grow at 30°C. In this
analysis, we used a derivative of the
-expressing plasmid (pJRC210)
that had been chemically mutagenized in vitro with hydroxylamine as
described previously (31) before transformation into
AB1157(pGY9738). To minimize the isolation of siblings, transformation
reaction mixtures were aliquoted prior to their outgrowth; automated
DNA sequence analysis revealed that all of the dnaN missense
alleles identified by this approach arose independently. Following a
60-min outgrowth period, a portion of each aliquot was plated at 42°C
(the permissive temperature) to permit a calculation of the total
number of transformants obtained. The remainder of each transformation
reaction mixture was plated directly at 30°C to select for
plasmid-encoded dnaN alleles that allowed growth at this
otherwise nonpermissive temperature. To confirm that growth at 30°C
was attributable to the plasmid-encoded dnaN allele, plasmid
DNAs were purified and subsequently retransformed back into AB1157
bearing the umuD'C-expressing plasmid (pGY9738). One half of each transformation reaction mixture was plated at 30°C,
and the other half was plated at 42°C. Plates were scored after
incubation overnight (~20 h).
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TABLE 2.
Overexpression of exacerbates the cold sensitivity
conferred by elevated levels of the umuDC
gene productsa
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It should be stressed that high-level overproduction of

by addition
of IPTG (isopropyl-

-
D-thiogalactopyranoside) was not
necessary for expression of the synthetic lethal phenotype. The
noninduced level of

expressed from the
Ptac promoter of pJRC210
was sufficient.
Consequently, all of the experiments described
in this report designed
to measure the phenotypes of the wild-type
and mutant
dnaN
alleles were performed in the absence of added
IPTG, unless otherwise
stated.
Nucleotide sequence analysis of dnaN alleles.
The promoter region and entire coding sequence for each plasmid-encoded
dnaN allele were determined by automated DNA sequence analysis using at least four of the following six different
oligonucleotide primers (GIBCO-BRL) corresponding to the indicated
nucleotide positions (with position 1 corresponding to the first base
of the coding sequence of dnaN): BETA-R1, positions 292 to
331 of the noncoding strand; BETA-F2, positions 236 to 258 of the
coding strand; BETA-F3, positions 815 to 836 of the coding strand;
BETA-R4, positions 879 to 901 of the noncoding strand; BETA-F5,
positions 97 to 120 of the coding strand; and BETA-R6, positions 394 to 415 of the noncoding strand. Analyses of the nucleotide sequences were
performed using the Lasergene software package (version 3.6.0), and
mutations were identified by comparison of the sequence data files to
the wild-type dnaN sequence deposited in GenBank (accession number J01602).
 |
RESULTS |
Overexpression of the
processivity clamp of Pol III exacerbates
umuDC-mediated cold sensitivity.
We have previously
suggested that interaction of UmuD with the
processivity clamp of
Pol III is important for the checkpoint role of
UmuD2C (57). We have also suggested
that the cold sensitivity conferred by elevated levels of the
umuDC gene products is due to the inappropriate expression
of UmuD2C functions involved in this checkpoint
(59). To explore this hypothesis further, we used the
previously described quantitative transformation assay (38) to investigate whether or not overexpression of the
clamp would affect the extent of the cold sensitivity conferred by
elevated levels of the umuDC gene products. In these
experiments, elevated levels of the umuDC gene products were
supplied from a pSC101 derivative named pGY9739 (Table 1)
(52) that contains a single base change in the operator
site near the umuDC promoter (the oc1 mutation). This
mutation eliminates much of the repression normally conferred by the
LexA repressor (52). As controls, we used either pGB2, the
parent to pGY9739 which lacks a umuDC operon, as well as a
comparable pSC101-based plasmid named pSE115, which expresses the
umuDC gene products from their wild-type, LexA-regulated
promoter (8). Thus, in AB1157, expression of the
umuDC gene products from pSE115 is efficiently blocked by
LexA protein, while expression of the umuDC gene products from the
oc1umuDC
allele contained in pGY9739 is not.
In this analysis, competent cells of AB1157 bearing either pGY9739,
pGB2, or pSE115 were prepared following their growth at
42°C.
Consistent with our previous observations that overexpression
of
umuDC causes cold sensitivity (
59), AB1157
bearing pGY9739
does not grow well at 30°C (Table
2). We then
transformed these
two AB1157 derivatives with either pBR322 (as a
control) or a
pBR322 derivative that expresses the

processivity
clamp of Pol
III from the
Ptac promoter
(pJRC210); no IPTG was added to induce

expression in these
experiments. Following transformation and
a 60-min period of outgrowth
at 42°C, the reaction mixtures were
split and aliquots were plated at
both 30 and 42°C. As expected,
the extent of the growth defect at
30°C of the AB1157 derivative
bearing pGY9739, which expresses
elevated levels of the
umuDC gene products, was unaffected
by transformation with pBR322; it
still plated nearly 300-fold less
efficiently at 30°C than at
42°C (Table
2). The lack of a
cold-sensitive growth phenotype
after transformation with pBR322 for
AB1157 bearing either pGB2,
which does not encode a
umuDC
operon, or pSE115, which does not
express detectable levels of the
umuDC gene products in AB1157
due to the presence of the
LexA repressor (data not shown), confirms
that the cold sensitivity
conferred by pGY9739 was due to the
elevated levels of
UmuD
2C.
However, when the
umuDC-expressing strain was transformed
with pJRC210, the pBR322 derivative that overexpresses

from the
Ptac promoter, it became more cold
sensitive for growth (i.e.,
from a plating efficiency ratio at
30°C/42°C of 3.5 × 10
3 with pBR322 to
5.1 × 10
4 with pJRC210 [Table
2]),
despite the fact that the
Ptac promoter
was not induced. This enhancement in
umuDC-mediated cold
sensitivity
was independent of the order in which the two plasmids were
introduced
into AB1157; comparable plating efficiencies were observed
whether
the

-expressing plasmid was transformed into the
umuDC-expressing
strain or vice versa (data not shown).
Despite the enhanced cold-sensitive
growth phenotype of the strain
overexpressing the
umuDC gene products
following its
transformation with the

-expressing plasmid, its
transformation
efficiency with the same plasmid at 42°C was roughly
10
5 CFU per µg of plasmid DNA (see Table
2,
footnote
c). This efficiency
is similar to that
observed with the same strain with pBR322 in
place of pJRC210,
indicating that growth of the strain expressing
elevated levels of both

and the
umuDC gene products was essentially
unaffected
at 42°C. The control strain bearing pSE115, which did
not express
elevated levels of UmuD
2C due to the presence of
the
LexA repressor, did not become cold sensitive when transformed
with
pJRC210. This indicated that the exacerbation of the cold
sensitivity
of the
umuDC-expressing strain was due to the combination
of
elevated levels of UmuD
2C together with elevated
levels of

.
These results, indicating that the

processivity clamp of Pol III
might be involved in
umuDC-mediated cold sensitivity,
prompted
us to evaluate the effect of overexpression of

on a strain
that
expressed elevated levels of the
umuD'
C gene
products. In this
analysis, we used a plasmid named pGY9738 that is
similar to the
one described above containing the
oc1 mutation except
that it results in
constitutive expression of the
umuD'
C gene products instead of
the
umuDC gene products. Such a strain is not normally cold
sensitive
(
59), presumably due to the inability of
UmuD'
2C to function
in the
UmuD
2C-dependent checkpoint control
(
37). Consistent
with our previous results
(
59), AB1157 expressing elevated levels
of the
umuD'
C gene products was not cold sensitive when
transformed
with pBR322 (Table
2). However, transformation of this
umuD'
C-expressing
strain with the

-expressing
plasmid (pJRC210) resulted in a severe
cold-sensitive growth phenotype,
as indicated by the nearly 4,000-fold
reduction in its plating
efficiency at 30°C (Table
2). This plating
efficiency is similar to
that which we observed for the
umuDC-expressing
strain
following its transformation with the

-expressing plasmid
(pJRC210).
However, one important difference between the
umuDC-
and
umuD'
C-expressing strains is that the latter is
cold sensitive
only following its transformation with the

-expressing plasmid
(Table
2), while the former is cold sensitive
regardless of whether
or not it bears the

-expressing plasmid and
becomes more cold
sensitive after transformation with the

-expressing
plasmid.
We have previously reported that
umuDC-mediated cold
sensitivity requires the
umuC gene product but not its
catalytic proficiency
as a DNA polymerase (
59). Likewise,
we found that overexpression
of

together with elevated levels of
UmuD
2 or UmuD'
2 alone did
not confer cold sensitivity; the
umuC gene product was
absolutely
required for cold sensitivity (Table
2). Finally, the extent
of cold sensitivity conferred upon AB1157 expressing elevated
levels of
the
umuDC or
umuD'
C gene products by
the

-expressing
plasmid (pJRC210) was unaffected by addition of
IPTG, which induces
high-level overproduction of

(data not shown);
the noninduced
level of

expressed from pJRC210 was sufficient
(Table
2). Thus,
it is important to stress that all the experiments
described in
this report designed to measure the phenotype of the
pJRC210-encoded
dnaN allele were performed in the absence of
IPTG. Finally, although
we have not yet carefully quantitated the level
of expression
of

from pJRC210 in the absence of IPTG, it is clear
that strains
bearing this plasmid and grown in rich medium, such as LB
medium,
express at least ~10- to 100-fold more

from the plasmid
than
they do from their chromosomal allele (data not
shown).
The cold sensitivity conferred by the overexpression of
together with elevated levels of the umuDC gene products
is more severe than that conferred by
together with
umuD'C.
The quantitative plating
assay that we typically use to gauge the extent of cold sensitivity
measures the ability of a strain to form a colony on selective medium
after overnight incubation (38). Consequently, it is a
relatively insensitive method for characterizing small differences
between strains, such as growth rates. In addition, we have previously
found that depending upon the selectable antibiotic marker being used,
the upper limit with respect to the extent of cold sensitivity that can
be observed may vary. For example, using the same E. coli
strain (GW8018) and the same umuDC-expressing plasmid
(pSE117), which confers resistance to both ampicillin and kanamycin, we
observed a 659-fold range in the extent of the cold sensitivity
depending on whether we selected for growth using solid medium
supplemented with ampicillin (32) or kanamycin
(38). Therefore, to establish whether the umuDC- and umuD'C-expressing strains
were similarly affected by the simultaneous overproduction of
, we
further characterized the growth defects at 30°C relative to those at
42°C of some of the same strains described in Table 2 by monitoring
their growth rates in liquid culture. Our focus in these experiments
was on comparing the effects of elevated levels of umuDC to
elevated levels of umuD'C with or without
simultaneous expression of the
clamp. The AB1157 derivative bearing
pSE115, which did not express detectable levels of
UmuD2C (due to the presence of the LexA repressor protein), with or without the
-expressing plasmid, served as the
control. Briefly, overnight cultures of each strain grown at 42°C in
M9 minimal medium supplemented with 0.5% Casamino Acids, 0.2%
glucose, and the appropriate antibiotics were subcultured 1:125 into
the same prewarmed medium and grown at 42°C for 60 min. Cultures were
then split in half, and one of each pair was shifted to 30°C while
its partner was maintained at 42°C. One-milliliter aliquots of each
culture were then removed at various times for determination of their
optical density at 600 nm (Fig. 1).

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FIG. 1.
The cold sensitivity conferred upon a
umuDC-expressing strain by overexpression of is more
severe than that conferred upon a
umuD'C-expressing strain. Growth curve
analyses were performed as described in the text. The arrow indicates
the time at which cultures were spilt into two equal parts and one of
each pair was shifted to 30°C (B) while the other was maintained at
42°C (A). Symbols: , AB1157(pGY9738)(pBR322); ,
AB1157(pGY9738)(pJRC210); , AB1157(pGY9739)(pBR322); ,
AB1157(pGY9739)(pJRC210); , AB1157(pSE115)(pBR322); ,
AB1157(pSE115)(pJRC210). E. coli AB1157 and plasmid DNAs
are described in Table 1. OD600, optical density at 600 nm.
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|
The growth rates of AB1157 bearing pBR322 and expressing elevated
levels of either the
umuDC (pGY9739) or the
umuD'
C (pGY9738)
gene products were slightly
retarded at 42°C relative to that
of the control strain bearing the
umuDC genes under control of
their native, LexA-regulated
promoter (pSE115), which grew well
at both temperatures (Fig.
1).
Consistent with previous reports
(
38), the growth defect
of the
umuDC-expressing strain was more
pronounced at
30°C, while the growth rate of the strain expressing
elevated levels
of the
umuD'
C gene products was somewhat less
affected (Fig.
1, compare panels A and
B).
In contrast to these findings, the growth rates of the strains
expressing elevated levels of the
umuDC or
umuD'
C gene products
together with elevated
levels of

were significantly more retarded
at both 30 and 42°C
compared to the same strains bearing pBR322
in place of the

-expressing plasmid. Interestingly, the growth
rate of the strain
expressing
umuDC together with

was considerably
more
retarded at both 30 and 42°C than was that of the strain
expressing
umuD'
C together with

(Fig.
1). The control
strain
bearing pSE115, which does not express detectable levels of the
umuDC gene products because of the LexA repressor, together
with
the

-expressing plasmid was only slightly cold sensitive
compared
to the same strain bearing pBR322 in place of the

-expressing
plasmid.
Taken together, these results (Fig.
1) confirm that exacerbation of the
cold sensitivity requires elevated levels of both

and the
umuDC gene products and indicate that the cold sensitivity
conferred by the combination of elevated levels of
UmuD
2C together
with elevated levels of

is
more severe than that conferred by
elevated levels of
UmuD'
2C together with

. Our inability to
detect
this difference in our plating assay is presumably due to its
insensitivity with respect to small differences in growth rate
(see
above).
Direct selection for novel dnaN alleles deficient in
conferring a cold-sensitive phenotype upon a
umuD'C-expressing E. coli
strain.
The ability of the
-expressing plasmid (pJRC210) to
confer cold sensitivity upon AB1157 expressing elevated levels of the umuD'C gene products provided us with an
extremely powerful means of identifying dnaN (which encodes
) alleles deficient in conferring this cold sensitivity. Based on
our findings that (i) the umuD gene products interact with
in vitro and (ii) UmuD has a greater affinity for
than does
UmuD' (57) (an observation that correlates well with their
respective degrees of cold sensitivity [Fig. 1]), we reasoned that
the exacerbation of umuDC-mediated cold sensitivity conferred by elevated levels of
was in fact a manifestation of
these physical interactions. Thus, we hoped that by selecting for
dnaN alleles unable to exacerbate
umuD'C-mediated cold sensitivity, we would be
able to identify missense mutant
proteins deficient in physical
interactions with the umuDC gene products. We therefore used
hydroxylamine to chemically mutagenize the
-expressing plasmid in
vitro and then transformed this chemically mutagenized plasmid into
AB1157 bearing pGY9738, which constitutively expresses the umuD'C gene products. Transformation reaction
mixtures were plated at the normally nonpermissive temperature of
30°C to select for plasmid-encoded dnaN alleles deficient
in conferring cold sensitivity. To minimize selecting for siblings,
transformation reaction mixtures were aliquoted prior to their
outgrowth; nucleotide sequence analysis (see below) confirmed that none
of the missense dnaN alleles we identified were siblings.
By this approach, we identified 76 strains whose plating efficiencies
at 30 and 42°C were similar. Although we had selected
these clones by
directly plating the transformation reaction mixtures
at 30°C, we had
also plated an aliquot of each reaction mixture
at the permissive
temperature of 42°C to allow for a determination
of the total number
of viable transformants. This analysis indicated
that these 76 putative
mutants were selected from a total pool
of ~60,000 independent
transformants. To confirm that the observed
phenotypes of these 76 presumed mutant
dnaN alleles were conferred
by the
respective pJRC210 derivatives and not by a mutation mapping
to the
host chromosome, we isolated the plasmid DNA from each
of the 76 clones
and transformed it back into AB1157 bearing the
umuD'
C-expressing plasmid. Of the 76 pJRC210
plasmids, 75 were
unable to confer cold sensitivity upon AB1157
expressing elevated
levels of the
umuD'
C gene
products, indicating that they carried
the mutation responsible for the
observed
phenotype.
To characterize these 75 plasmid-encoded
dnaN alleles
deficient in conferring the cold-sensitive phenotype further, we
assessed
their respective abilities to overproduce

in response to
addition
of IPTG; the
dnaN gene in pJRC210 is under the
control of the
IPTG-inducible
Ptac
promoter. The purpose of this analysis was
to differentiate between
those pJRC210 derivatives unable to confer
cold sensitivity because of
an expression defect (i.e., a mutation
affecting the expression of the
dnaN gene product) or a nonsense
mutation (which was not
suppressed by the
supE44 allele of AB1157
and would result
in expression of a truncated form of

) from
those expressing a
full-length

protein that is expressed at
a level comparable to that
observed for the
dnaN+ allele. The latter
class represented the type of allele we were
interested in and
presumably corresponds to those containing one
or more missense
mutations that affect the ability of the

derivatives
they encode to
interact physically with the
umuD'
C gene
products.
pJRC210, the parent plasmid, overexpresses

to very high levels in
response to IPTG (>20% total soluble protein [data not
shown]).
Thus, we were able to easily distinguish those pJRC210
derivatives able
to overexpress the

clamp from those unable
to do so by Coomassie
blue staining of sodium dodecyl sulfate-polyacrylamide
gel
electrophoresis-fractionated whole-cell lysates prepared from
cultures
of AB1157 derivatives bearing the respective

-expressing
plasmids
after their treatment with IPTG (data not shown). This
analysis
indicated that of the 75 plasmids identified by our genetic
assay, 13 were capable of overproducing a full-length

protein
(data not
shown). Thus, only 13 of the ~60,000 transformants carrying
a
hydroxylamine-treated pJRC210 derivative possessed the desired
phenotypes of (i) expressing a full-length

protein and (ii)
being
unable to confer a cold-sensitive phenotype upon a
umuD'
C-expressing
strain.
Nucleotide sequence determination of novel dnaN
alleles.
To determine the specific nucleotide alterations in each
of the 13 dnaN alleles presumed to contain a missense
mutation, we sequenced the promoter region and entire coding sequence
of each using at least four different oligonucleotide primers (see
Materials and Methods). In addition, we also sequenced 14 alleles that
we suspected, based on analysis of whole-cell lysates (see above), to
be either expression mutations (i.e., mutations mapping to the promoter
region that affect expression) or alleles that expressed a truncated
form of the
clamp (i.e., nonsense mutations). This analysis,
summarized in both Table 3 and Fig.
2, confirmed our hypothesis regarding the
alleles we suspected to contain either one or more mutations in their
promoter region, thus affecting expression of dnaN, or a
nonsense mutation resulting in expression of a truncated form of the
protein. In addition, it indicated that the 13 alleles presumed to
contain a missense mutation(s) actually represent eight unique and
novel missense dnaN alleles. These eight alleles identify a
total of eight different amino acid residues that, when changed, affect
the ability of
to confer cold sensitivity upon the
umuD'C-expressing strain. Of the 35 mutations
identified by nucleotide sequence analysis (see the legend to Fig. 2),
only four could not be accounted for by the known propensity of in
vitro hydroxylamine treatment to cause C-to-T transitions
(31). Finally, this analysis indicated that, with the
exceptions of pJRCHA-5.1 (S107L) and pJRCHA-6.2 (P363S), all eight
alleles contained only a single missense mutation; the two exceptions
contained, in addition, one silent mutation each (Table 3).

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FIG. 2.
Stick representation of the dnaN gene,
summarizing the relative positions of all mutations identified in this
study. The positions of all nucleotide alterations identified in the 27 dnaN alleles subjected to automated DNA sequence
analysis are shown. Approximate positions of amino acid residues in and the approximate delineation of each of the three structurally
similar domains (I, II, and III) comprising each monomer
(21) are indicated. The light-shaded rectangles represent
the coding sequences of the dnaN and
dnaN* (see text) genes, while the dark-shaded rectangles
represent their respective promoters. Approximate positions of those
mutations resulting in deduced amino acid substitutions and described
in Table 3 are indicated by filled triangles; approximate positions of
silent mutations, including those which map to the promoter region, are
indicated by open triangles; and approximate positions of nonsense
mutations are indicated by open diamonds. Except for the F75 and D229
silent mutations that were identified together with the P363S and S107L
mutations, respectively (indicated by the lines connecting these
respective positions), the alleles containing silent or nonsense
mutations are not described in Table 3. The silent mutation L236
results in formation of a rare Leu codon leading to a significantly
reduced steady-state level of the expressed protein (data not
shown). The silent mutations S104, N221, and L248 were identified in
combination with at least one other mutation mapping in the vicinity of
the ribosome binding site (which maps to nucleotide positions 16 to
9) and appear to also express significantly lower levels of (data
not shown). The four mutations that cannot be accounted for by
hydroxylamine treatment (which promotes C T transitions when used in
vitro [31]) are 181C A (Q61K) (Table 3),
611T A (M204K) (Table 3), 685G C (the
silent mutation in S107L) (Table 3), and 45G T (resulting
in the E15 nonsense mutation).
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All eight amino acid substitutions identified by our genetic assay
correspond to residues located at or very close to the
surface of the
molecule (Fig.
3) based on the crystal
structure
of the

clamp reported by Kong et al. (
21),
with the majority
of them affecting the face of

that is believed to
interact with
DNA Pol III (Fig.
3B) (
23,
24,
41,
55).
Incidentally,
some of the native residues that were mutated are
not easily visible
in the surface representations of the

clamp
(Fig.
3C to G).
This is because their position either is located just
below the
surface or is obstructed by other surface features of the

clamp
in the views shown.

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FIG. 3.
Relative position of each deduced amino acid
substitution in the crystal. (A and B) Worm representation of the
clamp showing the positions of missense mutations as viewed from
the face bearing the extruding C-terminal tails of each protomer (A),
and the side (B), corresponding to a 90° rotation of that shown in
panel A. The crystal structure of the homodimeric clamp, solved by
Kong et al. (21), is shown with one protomer in green
and the other in blue. Positions of deduced amino acid substitutions
are in red. The complex clamp loader and the subunit of Pol III
are both believed to interact with the face of bearing the
extruding C-terminal tails (B). See the text for further details. (C
through G) Surface representations of the crystal in successive
rotations of 45° each (i.e., 45°, 90°, 135°, and 180°,
respectively) relative to the angle shown in panel A. This view bears
the extruding C-terminal tails and is arbitrarily defined as 0°.
Again, one protomer is in green and the other is in blue, and
positions of deduced amino acid substitutions are in red. Note that not
all of the missense mutations (for example, E204) are easily visible in
the surface representation. This is because their position either is
located just below the surface or is obstructed by other surface
features of the clamp in the views shown. This figure was generated
with GRASP (Molecular Simulations, Inc.) using the atom coordinates of
the crystal (2POL) from the Protein Data Bank and a Silicon
Graphics R10000 workstation.
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The
dnaN alleles described in this report likely represent a
majority of the total number of
dnaN alleles possible with
this
phenotype following treatment of the plasmid-encoded
dnaN+ gene with hydroxylamine in vitro.
This conclusion is based on
the fact that the D150N and G157S alleles
were identified two
and five independent times, respectively,
indicating that our
selection-screen was partially saturating (Table
3). The large
number of mutations mapping to the same small number of
residues
within the promoter region, including the ribosome binding
site
(a region of 8 bp
[
16TAGGAGGT
9]
[Fig.
2]), combined with the fact that 48 more alleles unable
to overproduce
dnaN to a detectable level were identified
but
not sequenced (see above), indicates that the target size for
amino
acid substitutions in

that can affect its ability to confer
cold
sensitivity must be very
small.
Finally, it is interesting that each of the eight
dnaN
alleles encodes a mutant

protein with a comparatively larger
residue
in place of the smaller, native residue (e.g., G157S).
In addition,
some also radically affect the charge characteristics of
the native
residue (e.g., E202K), and many of the deduced amino acid
substitutions
identified affect residues well conserved among other
prokaryotic

homologs (Table
3). It is also important to point out
that
mutations were identified in all three structural domains of

;
each

protomer contains threefold structural similarity
(
21)
(Fig.
2). In addition, two (Q61K and S107L) of the
eight missense
mutations are located in the first structural domain of

(domain
I), which is lacking in an alternative form of the

clamp referred
to as

* (see Fig.
2 and Discussion for further
details) (
39,
50).
Steady-state levels of mutant
proteins.
Prior to their
nucleotide sequencing, we had screened each dnaN allele for
its ability to overexpress a full-length
protein. Their ability to
do so suggested that their promoter regions did not contain mutations
that affected their respective abilities to express their gene
products, an assumption corroborated by their nucleotide sequence
analysis (see above). To confirm that the mutant
proteins were
present at comparable levels, relative both to each other and to the
wild-type control (pJRC210), in the absence of their induced expression
by IPTG, we measured their respective steady-levels by qualitative
Western blot analysis using a polyclonal antibody specific to
. The
steady-state level of each of the eight mutant
proteins was found
to be similar to that observed for the wild-type control in the absence
of added IPTG (Fig. 4), indicating that
their deduced amino acid substitutions do not significantly affect
their overall stability in vivo. This result confirms that the
respective deficiencies of these dnaN alleles to confer cold
sensitivity in our genetic assay are due to their deduced amino acid
substitutions and not to a large change in their abundance. Finally, it
is worthwhile emphasizing that the level of
expressed from pJRC210
(as well as the levels of the mutants expressed from the pJRC210
derivatives) in the absence of IPTG is far greater than the level
expressed from the chromosomally carried
dnaN+ or dnaN59(Ts) allele (Fig.
4). Initial attempts to quantitate the level of expression from pJRC210
suggest that it is at least 10- to 100-fold higher than that observed
from the chromosomal dnaN+ allele (data not
shown).

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FIG. 4.
Steady-state levels of mutant proteins in the
absence of IPTG. The steady-state level of each mutant protein was
measured in the absence of added IPTG by Western blot analysis using
polyclonal antibodies specific to and chemiluminescent detection
(Tropix) as described previously (36, 57). Approximately
108 cells of each strain grown in LB medium at 30°C to
mid-exponential phase (optical density at 600 nm, ~0.5) were
electrophoresed in a sodium dodecyl sulfate-15% polyacrylamide
gel and then transferred to a polyvinylidene difluoride membrane
as described previously (36, 57). Lane 1, AB1157(pJRC210); lane 2, AB1157(pBR322); lane 3, HC123(pJRC210); lane
4, HC123(pBR322); lane 5, HC123(pJRCHA-4.1); lane 6, HC123(pJRCHA-5.1);
lane 7, HC123(pJRCHA-8.1); lane 8, HC123(pJRCHA-5G11); lane 9, HC123(pJRCHA-8I11); lane 10, HC123(pJRCHA-7.1); lane 11, HC123(pJRCHA-6F11); and lane 12, HC123(pJRCHA-6.2). E.
coli AB1157 and HC123 are described in Table 1; plasmids are
described in Tables 1 and 3. Note that under the conditions used in
this analysis, endogenous levels of expressed from the chromosomal
dnaN+ allele (AB1157) (lane 2) were only
barely detectable, while those expressed from the
dnaN59(Ts) allele (HC123) (lane 4) were not
detectable.
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Novel dnaN alleles are deficient in exacerbating
umuDC-mediated cold sensitivity.
As discussed in
the report of our previous in vitro analysis (57), UmuD
interacts more strongly with
than does UmuD'. Two possible
explanations for this difference are (i) UmuD and UmuD' require similar
structural features of
for their interactions, but the extra
N-terminal 24 amino acids in UmuD enable it to make additional contacts
with
, thus accounting for its stronger interaction (57), or (ii) UmuD and UmuD' each interact with different
structural features of
, and hence their different affinities are
due to the different natures of their interactions. To begin to
differentiate between these two possibilities, we investigated whether
or not the eight dnaN alleles identified by virtue of their
inability to interact genetically with the umuD'C
gene products were similarly affected with respect to their
interactions with the umuDC gene products.
If UmuD and UmuD' interact with different structural features of

,
we might have expected that most, if not all, of the
dnaN alleles that we isolated by virtue of their inability to confer
cold
sensitivity for growth upon a
umuD'
C-expressing
strain would
be proficient in causing the

-dependent exacerbation of
the cold
sensitivity of a
umuDC-expressing strain. However,
as shown in
Table
4, all eight
dnaN alleles that were deficient in conferring
cold
sensitivity upon AB1157 expressing elevated levels of
umuD'
C were also deficient in their ability to
exacerbate the cold sensitivity
conferred by elevated levels of the
umuDC gene products. These
observations strongly suggest
that the interactions between UmuD'
and

are a subset of those
between UmuD and

. Thus, we suggest
that UmuD has a higher affinity
for

than does UmuD' because
of additional, important interactions
of

with the N-terminal
24 residues of UmuD, which are missing from
UmuD'. Strikingly,
some of the
dnaN alleles (i.e., G157S,
M204K, and, to a lesser
extent, E202K) not only were deficient in
exacerbation of the
cold sensitivity but actually suppressed the
inherent cold sensitivity
conferred by elevated levels of the
umuDC gene products in the
absence of elevated levels of

(Table
4). This phenotype is
interpreted more thoroughly in Discussion.
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TABLE 4.
Novel dnaN alleles deficient in conferring
cold sensitivity upon a umuD'C-expressing strain are unable
to exacerbate the cold sensitivity of a
umuDC-expressing strain
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Novel dnaN alleles deficient in exacerbating
umuDC-mediated cold sensitivity are proficient for DNA
replication in vivo when overexpressed.
To determine whether or
not the novel dnaN alleles that we identified by our genetic
assay were able to function in chromosomal DNA replication, we
attempted to cross them onto the E. coli chromosome using
the pKO3-based system devised by Link et al. (25). This plasmid was specifically designed for moving in-frame deletions and
mutations onto the bacterial chromosome. Our inability to homogenetize
the five dnaN alleles we tested (Q61K, D150N, G157S, E202K,
and M204K) (data not shown) suggests that they are unable to function
in chromosomal replication at a level adequate for viability when
expressed at the normal, physiological level.
In light of this, we chose to measure the respective abilities of these
dnaN alleles to function in chromosomal DNA replication
when
they were present at an elevated gene dosage by virtue of
residing on a
multicopy plasmid. For this, we used the same plasmids
that we
initially identified by our genetic screen and asked whether
they could
complement the temperature-sensitive phenotype of a
dnaN59(Ts) strain.
E. coli strains bearing the
dnaN59(Ts) allele
are temperature sensitive due to the poor
stability of the mutant

protein (DnaN59) at the elevated
temperature (
4). Using the
previously described
quantitative transformation assay (
38),
we found that all
eight
dnaN alleles were similar to the plasmid-encoded
dnaN+ allele with respect to their
abilities to complement the temperature-sensitive
phenotype of the
dnaN59(Ts) allele (Table
5).
In contrast, pBR322
was unable to substitute for pJRC210 in suppression
of the temperature-sensitive
phenotype of the
dnaN59(Ts)
allele. These results indicate that
each of the novel
dnaN
alleles described in this report retains
at least some biological
activity with respect to DNA replication
in vivo. However, their
apparent inability to function in chromosomal
replication at a level
adequate for viability when expressed at
the normal, physiological
level suggests that it is difficult
to genetically separate the
replication function of

from its
ability to interact with the
umuDC gene products.
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TABLE 5.
Abilities of novel dnaN alleles to complement
the temperature sensitivity of the dnaN59(Ts) E. coli strain HC123
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 |
DISCUSSION |
Interactions of UmuD2C with components of Pol III help
to enable a DNA damage checkpoint control.
The cold sensitivity
conferred by elevated levels of UmuD2C, a
phenomenon that apparently is due to the inappropriate expression of
their DNA damage checkpoint function (59), is exacerbated by overexpression of the
processivity clamp (Table 2). This observation, taken together with our previous findings that
overexpression of the
proofreading subunit of Pol III or deletion
of its structural gene (dnaQ) suppresses
umuDC-mediated cold sensitivity whereas overexpression of
each of the other eight Pol III subunits (
,
,
,
,
,
',
, and
) does not (56), suggests that
UmuD2C associates with the replisome via direct
interactions with
and
and that these interactions serve to
antagonize the DNA polymerase activity of Pol III, thereby arresting
DNA synthesis (30, 37).
Overexpression of

also conferred a severe cold-sensitive growth
phenotype upon a strain expressing moderately elevated levels
of the
umuD'
C gene products. Such a strain is not
otherwise normally
cold sensitive (
59), although
higher-level overexpression of
UmuD'
2C does
confer a cold sensitivity (
38). We have previously
suggested that the cold sensitivity conferred by higher-level
overexpression of
umuD'
C is a result of the
ability of higher
levels of UmuD'
2C to mimic the
UmuD
2C-dependent DNA damage checkpoint
control
(
59). Thus, we now suggest that the

-expressing plasmid
confers cold sensitivity upon an
E. coli strain expressing
moderate
levels of
umuD'
C (a strain that is not
normally cold sensitive
[
59]) by facilitating the
ability of moderate levels of
umuD'
C to mimic the
DNA damage checkpoint function of UmuD
2C, which
presumably
involves, at least in part, interaction of
UmuD
2C with

(
57,
59).
We exploited the ability of the

-expressing plasmid (pJRC210) to
confer cold sensitivity upon a
umuD'
C-expressing
strain
to identify eight unique and novel missense
dnaN
mutations. The
fact that these plasmid-borne mutant
dnaN
alleles could complement
the temperature sensitivity of a
dnaN59(Ts) mutant (Table
5)
suggests that when
overexpressed, these mutant

proteins retain
a partial ability to
participate in normal DNA replication. Some
of the
dnaN
alleles described in this report (i.e., G157S, M204K,
and, to a lesser
extent, E202K) were able to suppress the inherent
cold sensitivity
conferred by elevated levels of the
umuDC gene
products in
the absence of elevated levels of

(Table
4), suggesting
that their
presence in the replisome confers upon it an immunity
to the
replication block normally imposed by elevated levels of
UmuD
2C. However, a definitive answer to the
question of whether
or not these mutant

proteins are deficient in
interaction with
UmuD
2C must await their
biochemical
characterization.
The eight mutant

proteins described in this report bear
substitutions of amino acid residues located at or very close to
the
surface of the molecule (Fig.
3C to G), based on the crystal
structure
of the

clamp reported by Kong et al. (
21). Some
of the
native residues that were mutated are not easily visible
in the surface
representations of the

clamp. This is because
their position either
is located just below the surface or is
obstructed by other surface
features of the

clamp in the views
shown. With respect to the fact
that all of the mutations were
located at or very close to the surface
of the molecule, it is
worthwhile emphasizing the fact that all of the
mutations correspond
to substitutions of a comparatively larger residue
in place of
the smaller, native residue (e.g., G157S). Thus, all
of the mutant

proteins described in this report are expected to
have a slightly
altered surface relative to that of the wild-type

protein due
to the presence of their respective amino acid
substitutions,
consistent with the mutant

proteins being affected
for interaction
with the
umuDC gene
products.
With the exception of Q61K, all of the substitutions reside on the same
face of the

clamp (Fig.
3B). Interestingly, this
face is the same
as that which has been previously suggested to
interact with both the

catalytic subunit (
23,
24,
55)
and the five-subunit
clamp loader, or

complex, of Pol III (
23,
24) (Fig.
3B). This finding suggests that interaction of the
umuDC
gene products with

may eliminate the ability of

to
subsequently
interact with the

catalytic subunit and/or

complex
of Pol
III. Furthermore, our recent observation that the
umuD gene products
interact with the same structural domain
of

that has been previously
shown to interact with

suggests
that a similar situation is
true for the UmuD-

interaction; i.e.,
UmuD and

associate with

in a mutually exclusive fashion
(
56).
If the UmuD
2C complex were to interact with both
the

and the

subunits of Pol III in such a way as to preclude
their subsequent
association with

, then the
umuDC-dependent checkpoint could
not be a manifestation of
interactions involving UmuD
2C and the
Pol III
multiprotein complex. Rather, these observations suggest
that
UmuD
2C exerts its checkpoint role by competing
with

for
binding to

and

. Viewed in this way, the
UmuD
2C-dependent checkpoint
control has certain
aspects in common with the eukaryotic S-phase
checkpoint control. One
component of this surveillance mechanism
is the p21 protein, which acts
not only to regulate cyclin-cyclin-dependent
kinase complexes
(
15,
16) but also to regulate the availability
of the
eukaryotic PCNA processivity clamp (
66). By associating
directly with the same face of PCNA as does Pol

, p21 may preclude
binding of Pol

to PCNA, thereby helping to regulate DNA replication
in response to DNA damage (
66).
The important findings discussed above, taken together with previous
observations (
37,
38,
56,
57,
59), suggest
at least four
different models for how interactions of UmuD
2C
with the

and the

subunits of Pol III could enable a DNA damage
checkpoint control (Fig.
5). With respect
to these four models,
the absolute requirement of
umuC for
exacerbation of the cold
sensitivity (
30,
38,
59) suggests
that UmuC may also interact
directly with

and

, perhaps serving
to impose a directionality
upon the interactions of the symmetrical
UmuD
2 homodimer (in the
form of the
UmuD
2C complex) with

and

.

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FIG. 5.
Four possible models to describe the involvement of the
and the subunits of Pol III in UmuD2C-dependent
cold sensitivity and in the UmuD2C-dependent checkpoint
control. Our results discussed here, taken together with our previous
findings (37, 38, 56, 57, 59), suggest at least four
different models to describe the involvement of the and the subunits of Pol III in UmuD2C-dependent cold sensitivity,
which presumably is a manifestation of functions of UmuD2C
involved in the DNA damage checkpoint control (37, 59).
Previously described genetic interactions between umuC
and (encoded by dnaQ) (10, 11, 17, 19, 35,
56), together with our findings discussed in this report
regarding genetic interactions of with both umuDC
and umuD'C, collectively suggest that
UmuC might interact directly with both and . Therefore, for the
purposes of these models, we have assumed that interaction of UmuC with
or imparts an asymmetry on the UmuD2 homodimer that
is important for defining its subsequent interaction (when in the form
of the UmuD2C complex) with or . The two proposed
structural domains of (40, 61) are represented here as
circles; the N-terminal domain is represented by the large circle,
while the C-terminal domain is represented by the small circle. It has
previously been demonstrated that the small C-terminal domain of interacts with both the subunit of Pol III (40, 61)
and the umuD gene products (56).
Interaction of the unknown factor(s) with UmuC and (B) is arbitrary
and is intended only to imply the possible involvement of additional,
as-yet-unidentified components of the UmuD2C-dependent
checkpoint control pathway. See Discussion for further details
regarding these four different models.
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In the first model (Fig.
5A), UmuD
2C,

, and

interact, forming a single, multiprotein complex. Since
associations of both

(
53) and

(
4,
54)
with

significantly enhance its DNA
polymerase activity, titration
of either or both away from the
replisome by
UmuD
2C would be expected to antagonize the
catalytic
DNA polymerase activity of Pol III. Overexpression of

may
exacerbate
umuDC-mediated cold sensitivity by stabilizing
the multiprotein
complex, thereby effectively removing

from the
replisome.
A variation of this model is suggested by our findings that deletion of
the structural gene encoding

(
dnaQ) results in only
a
partial suppression of
umuDC-mediated cold sensitivity
(
56),
while overexpression of

strongly exacerbates
umuDC-mediated
cold sensitivity. These findings
suggest that

may play a comparatively
less important role in
umuDC-mediated cold sensitivity than does

. In light of
this possibility, it could have been argued that
if
umuDC-mediated cold sensitivity was primarily due to
UmuD
2C
(or possibly a
UmuD
2C-

complex) sequestering

away
from the
replisome, then overexpression of

would be expected to
suppress
much of the cold sensitivity, the opposite to what we
observed.
However, if the unusually high levels of

were
acting to stabilize
a UmuD
2C-

-

complex that
recruits an additional factor(s) which
serves an essential role(s) (for
example, the

subunit of Pol
III), its sequestration into the
multiprotein complex could cause
the exacerbation of cold sensitivity
that we have observed (Fig.
5B).
Two further models to describe how interactions of
UmuD
2C with the

and

subunits of Pol III
might serve to regulate DNA
replication in response to DNA damage
take into account the possibility
that

and

may interact with
UmuD
2C in mutually exclusive fashions,
such
that two separate complexes are formed. In such a case, one
could
imagine either that the UmuD
2C-

complex
acts to recruit
an additional factor(s) (Fig.
5C), as described above
for the
UmuD
2C-

-

complex, or that the
UmuD
2C-

and UmuD
2C-

complexes
simply exist in equilibrium, with neither recruiting
additional
factors (Fig.
5D). In both cases, one might predict that
overexpression
of

would suppress
umuDC-mediated cold
sensitivity (as we have
previously reported [
56]) by
favoring formation of the UmuD
2C-
complex,
which may be less efficient than UmuD
2C-

at
conferring
cold sensitivity (see above). This would have the effect
of shifting
the equilibrium away from formation of the
UmuD
2C-

complex, which
may be
recruiting an additional factor(s). In addition, overexpression
of

would replenish the levels of

initially sequestered away
from Pol
III by elevated levels of UmuD
2C. Likewise,
both of these
models (Fig.
5C and D) would predict that
overexpression of
would act to shift the equilibrium toward
formation of the UmuD
2C-
(or
UmuD
2C-

-unknown factor[s]) complex, thus
exacerbating the
extent of cold
sensitivity.
Although all four of the models discussed above share some common
aspects, they have important differences as well. Experiments
are under
way to distinguish which of these four models most closely
resembles
the events that occur in response to DNA damage in
vivo.
Does the UmuD'2C-
interaction serve an important
role in the living cell?
Despite the finding that
UmuD'2C can carry out efficient lesion bypass in vitro in
the presence of RecA and ssDNA binding protein (SSB) but in the
absence of Pol III (43, 63), various genetic and
biochemical observations suggest that some form of DNA Pol III plays a
role in determining when and where UmuD'2C-dependent TLS
takes place (57; recently reviewed in references
2 and 58). These findings include the
following: (i) some E. coli dnaE alleles
(dnaE encodes the
subunit of Pol III) appeared to
increase UV-induced umuDC-dependent SOS mutagenesis in
vivo (45), (ii) the DNA polymerase activity of the
temperature-sensitive DnaE1026 mutant
protein was stabilized by the
UmuD'2C complex in vitro (63), and (iii) in
vitro UmuD'2C-dependent TLS was significantly enhanced by
small amounts of either the mutant DnaE1026 protein or Pol III core
(63).
In contrast to the role of the

subunit of Pol III in TLS, the role,
if any, of the

processivity clamp is less clear. Although
Reuven et
al., who have successfully reconstituted
UmuD'
2C-dependent
lesion bypass in vitro, found
it to be completely independent
of the

clamp of Pol III
(
43), Tang et al., using a slightly
different approach to
reconstitute UmuD'
2C-dependent TLS in vitro
(
63), reported a strict requirement of both the

clamp
and
the five-subunit clamp loader (

complex) of Pol III for lesion
bypass. This difference with respect to the requirement of the

clamp for TLS in vitro has been attributed to differences in
the
template DNAs and the levels of RecA protein used by these
two groups
(
62,
67), although it could also be due to the
maltose
binding protein-UmuC fusion protein utilized by Reuven
et al.
(
67).
Our results regarding the cold sensitivity conferred upon the
umuD'
C-expressing strain by the

-expressing
plasmid could be
the result of the previous proposal that the
requirement for the

clamp and

complex of Pol III for
UmuD'
2C-dependent TLS in
vitro may not be
physiologically relevant but rather may be due
to the fact that
UmuD
2C interacts with

to enable a DNA damage
checkpoint control (
37,
57), and because of this,
UmuD'
2C
retains a comparatively limited yet
extraneous ability to also
interact with

(
13,
44).
Alternatively, it is possible that
interactions of

with the
umuDC gene products are important for
both the DNA damage
checkpoint control (
57) and TLS (
62,
63)
and
that self-cleavage of UmuD to yield UmuD' serves to modulate
the
interactions of UmuD
2C and
UmuD'
2C with

to effect the release
of the
checkpoint control and enable TLS (
57). In any event,
a
definitive answer regarding the role of the

clamp in TLS in
the
living cell will likely require a combined biochemical and
genetic
approach. Further characterization of the mutant

proteins
described
in this report will help to establish whether or not

serves an
important role in
TLS.
Relationship of the eight novel dnaN alleles to
dnaN*.
Following exposure of E. coli to
various DNA-damaging agents, the level of transcription of the
dnaN gene increases (42, 60). In addition,
expression of a truncated form of
corresponding almost exactly to
the C-terminal two-thirds of the protein is also induced (39,
50). This protein, termed
*, is expressed, in a
recA- and lexA-dependent fashion, from a promoter
located within the coding region of the dnaN gene (Fig. 2)
(39, 50). The gene for
*, which is contained entirely
within the dnaN+ gene, has been termed
dnaN* (39, 50). Biochemical characterization of
* indicates that it can form a trimer in solution (49). Furthermore, in vitro studies have demonstrated that
* can modestly enhance the processivity of Pol III*, a form of Pol III containing all
subunits except the
clamp (49). However, despite these detailed genetic and biochemical analyses, the physiological role of
* is presently unknown. Interestingly, it has recently been suggested that several eukaryotic proteins originally identified because of their roles in cell cycle checkpoints act by serving as an
alternative to PCNA, the normal processivity clamp (33, 64,
65), or as an alternate subunit of the five-subunit
replication factor C complex (65), the normal PCNA clamp loader.
Each

protomer has threefold structural similarity (
21)
(Fig.
2). Because each

* protomer corresponds almost exactly to
the
C-terminal two-thirds of the
dnaN gene product (
39,
50),
it retains the C-terminal two structural domains but is
missing
the N-terminal domain I. It is interesting that two of the
mutations
we identified that reduce interactions with the
umuDC gene products,
Q61K and S107L, both reside within the
N-terminal domain (domain
I), which is lacking in

*. Thus, relative
to intact

,

* may
also be deficient for interactions with the
umuDC gene products.
These results raise the interesting
possibility that a clamp composed
of a trimer of

* might constitute
an efficient mechanism for
conferring processivity upon Pol III, as
well as possibly other
DNA polymerases, including Pol II
(
1), that is insensitive
to the checkpoint function(s) of
UmuD
2C. Such a mechanism might
be particularly
important during replication restart (
7) (or
induced
replisome reactivation [
20]) or following TLS, a time
in
which the cell is presumably involved in reestablishing a replisome
wherein processive and accurate DNA synthesis is carried out primarily
by Pol III (
6,
14,
58). However, further genetic and
biochemical
characterization of

* will be required in order to
establish
its physiological
role(s).
 |
ACKNOWLEDGMENTS |
We thank Mary Berlyn and the E. coli Genetic Stock
Center for E. coli HC123; Suzanne Sommer and Adriana
Bailone for plasmids pGY9738 and pGY9739; Roger Woodgate for plasmid
pGB2; George Church for plasmid pKO3; Charles McHenry for plasmid
pJRC210, the polyclonal anti-
antibodies, and many helpful
discussions; Ann Ferentz for help in making Fig. 3; Veronica Godoy for
help with cloning the dnaN alleles into pKO3; and the
members of our lab for helpful discussions and advice, in particular
Brad Smith for his comments on the manuscript.
This work was supported by Public Health Service grant CA21615 to
G.C.W. from the National Cancer Institute. M.D.S. was supported by a
fellowship (5 F32 CA79161-03) from the National Cancer Institute. B.M.B. was supported by a predoctoral training grant (5 T32 GM07287-26) from the National Institutes of Health. M.F.F. carried out her research
as part of the Undergraduate Research Opportunities Program (UROP) at
the Massachusetts Institute of Technology.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Biology
Department, 68-633, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139. Phone: (617) 253-6716. Fax:
(617) 253-2643. E-mail: gwalker{at}MIT.EDU.
 |
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Journal of Bacteriology, May 2001, p. 2897-2909, Vol. 183, No. 9
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.9.2897-2909.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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