Biology Department, Massachusetts Institute
of Technology, Cambridge, Massachusetts 02139
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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.
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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 pGYDC (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).
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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
UmuD2C.
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 × 104 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 105 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 UmuD2C 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 UmuD2C 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
UmuD2C-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
UmuD2 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
UmuD2C 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 181CA (Q61K) (Table 3),
611TA (M204K) (Table 3), 685GC (the
silent mutation in S107L) (Table 3), and 45GT (resulting
in the E15nonsense 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
[16TAGGAGGT9]
[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
UmuD2C-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 UmuD2C, which
presumably involves, at least in part, interaction of
UmuD2C 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 UmuD2C. However, a definitive answer to the
question of whether or not these mutant proteins are deficient in
interaction with UmuD2C 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 UmuD2C 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 UmuD2C and the Pol III
multiprotein complex. Rather, these observations suggest that
UmuD2C exerts its checkpoint role by competing
with for binding to and . Viewed in this way, the
UmuD2C-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 UmuD2C 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
UmuD2 homodimer (in the form of the
UmuD2C 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), UmuD2C, , 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
UmuD2C 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
UmuD2C (or possibly a
UmuD2C- 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 UmuD2C-- 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
UmuD2C 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
UmuD2C in mutually exclusive fashions, such
that two separate complexes are formed. In such a case, one could
imagine either that the UmuD2C- complex
acts to recruit an additional factor(s) (Fig. 5C), as described above
for the UmuD2C-- complex, or that the
UmuD2C- and UmuD2C-
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 UmuD2C- complex,
which may be less efficient than UmuD2C- at
conferring cold sensitivity (see above). This would have the effect
of shifting the equilibrium away from formation of the
UmuD2C- 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 UmuD2C. Likewise,
both of these models (Fig. 5C and D) would predict that
overexpression of would act to shift the equilibrium toward
formation of the UmuD2C- (or
UmuD2C--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
UmuD2C 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 UmuD2C 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 
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Processivity Clamp of
the Replicative DNA Polymerase
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Abstract
Introduction
