Section on DNA Replication, Repair and
Mutagenesis, National Institute of Child Health and Human Development,
National Institutes of Health, Bethesda, Maryland
20892-2725,1 and Department of
Biology, University of Louisiana, Lafayette, Louisiana
705042
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INTRODUCTION |
In a wild-type Escherichia
coli cell, most cellular mutagenesis is dependent on the
UmuD'2C complex, which together with RecA allows DNA
polymerase III to traverse otherwise replication-blocking lesions
(33, 43, 44). Both UmuD' and UmuC are induced as part of the
global SOS response to DNA damage (13, 21). The activity of
the Umu proteins is normally kept to a minimum by a variety of
transcriptional and posttranslational mechanisms (reviewed in reference
48). An essential step in this process is the
generation of UmuD': the protein is synthesized as a larger mutagenically inactive precursor, UmuD, and achieves its mutagenically active form only after a RecA-mediated self-processing reaction that is
believed to be mechanistically similar to that by which signal
peptidases act on their substrates (4, 26, 27, 30, 31, 37).
Many homologs of the E. coli umuDC genes have now been
identified, and a recent search of GenBank (release 110.0) identified what appear to be 11 bona fide homologs. In such cases, these homologs
are arranged in an operon and are likely to be negatively regulated at
the transcriptional level by LexA. Perhaps not too surprisingly,
certain proteins with structural similarities are identified as
homologs, but they lack the characteristic operon arrangement or do not
appear to be regulated by LexA. As a consequence, in the absence of
functional data, it is difficult to determine whether they are indeed
functional homologs.
One such case is the bacteriophage P1 humD gene. This
putative homolog was identified by Lewis et al., in 1994 (25), in a search for genes that might be regulated by LexA.
Although a potential LexA-binding site was identified upstream of
humD, the downstream region lacked an associated P1
umuC homolog. Perhaps the most striking observation was the
fact that the translational start signals predict a protein with
identity to the posttranslationally generated or recombinant UmuD'
protein (Fig. 1); therefore, although negatively regulated by LexA, once expressed, HumD might be able to
functionally substitute for UmuD' without the need to undergo any
posttranslational modification.
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FIG. 1.
Alignment of E. coli UmuD and bacteriophage
P1 HumD proteins. The E. coli UmuD and bacteriophage P1 HumD
proteins were aligned by using the program Geneworks 2.51 (Oxford
Molecular, Campbell, Calif.). Residues that are identical are shaded
grey and boxed, while highly conserved residues are shaded in grey.
Interestingly, the start of the HumD corresponds exactly to the
mutagenically active posttranslational cleavage product of UmuD, UmuD'.
Overall, E. coli UmuD and P1 HumD are approximately 33%
identical to each other, but as can be seen, most identity is in the
N-terminal tail, in which 24 of the first 29 residues are identical.
HumD has an extended C-terminal tail, which may be important for
dimerization of the protein.
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Since the mutagenically active Umu complex consists of a heterotrimer
of UmuD'2C (43, 44), one would have to postulate that if HumD is functionally similar to UmuD', it should be a dimer in
solution as well as physically interact with UmuC to form a
HumD2-UmuC complex in such a way as to promote error-prone translesion DNA synthesis. To test this hypothesis, we have subcloned a
~1-kb fragment of the P1 genome containing humD and have
characterized HumD both in vivo and in vitro. All of our experimental
results support the conclusion that HumD is, in fact, a functional
homolog of UmuD' and substitutes for UmuD' in damage-induced mutagenesis.
During the course of this work, the entire genome (46,375 bp) of the
lambdoid bacteriophage N15 was sequenced (GenBank accession no.
AF64539). Sequence analysis revealed that like P1, N15 carries a gene
predicted to encode a HumD protein related to the UmuD' family of
proteins. Presumably, humD (umuD'-like) genes are
maintained on the relatively small genomes of N15 (~46 kb) and P1
(~100 kb) because they provide some function that is evolutionarily
advantageous to both phages. Here, we hypothesize as to what these
functions might be.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
The E. coli
strains used in this study are listed in Table
1. Strains RW260, RW262, RW264, and RW406
were made by P1 transduction (29) of the
(umuDC)595::cat allele from RW82
(47) into EWRP1, JS431, EW181 (42), and WP2uvrA
(17), respectively, by selecting for chloramphenicol
resistance and screening for loss of damage-induced mutagenesis.
Plasmids used in this study (Table 2)
were constructed by using standard methods of recombinant DNA
technology (34). pOS27, a low-copy-number plasmid expressing
humD, was constructed as follows. Plasmid pAW711
(24) was digested with XmnI and
BssHII, and the 1,035-bp humD-containing fragment
was gel purified. The BssHII ends were blunt ended with DNA
polymerase I (Klenow fragment), and EcoRI linkers (New
England Biolabs, Beverly, Mass.) were added. The 1,055-bp
EcoRI fragment was subsequently cloned into the unique EcoRI site of pGB2 (5).
High-copy-number vectors expressing UmuD'-like proteins were
constructed by cloning the various genes into the polylinker of
pBluescript KS+ (Stratagene, La Jolla, Calif.) as follows:
pOS30 and pOS31 were generated by cloning the EcoRI
humD fragment from pOS27 into the unique EcoRI
site of pBluescript KS+. HumD is translated in the same
orientation as LacZ' in pOS30 and in the opposite direction in pOS31.
pOS32 was generated by cloning a 980-bp BglII E. coli
umuD'-containing fragment from pGW2123 (30) into the
BamHI site of pBluescript KS+. pOS33 was
similarly constructed by cloning a 1,491-bp HindIII rumA' fragment from pRW320 (23) into
HindIII digested pBluescript KS+. pOS34 was
constructed by cloning a 489-bp mucA'-containing
BglII-HindIII fragment from pRW294
(23) into BamHI-HindIII-digested
pBluescript KS+.
Low-copy-number vectors expressing UmuC-like proteins were constructed
by introducing frameshift mutations in their cognate UmuD'-like partner
as follows: pRW274 (umuC+) by digesting pRW134
(7) with NcoI, filling the ends, and subsequent
religation (14); pOS35 (rumB+) by
digesting pRW320 (23) with TthIII, filling the
ends, and subsequent religation; and pOS37
(mucB+) by partial digestion of pRW294
(23) with BspMI, followed by end filling and religation.
A HumD-overproducing plasmid was constructed by PCR amplification of
the humD gene from pOS30, using the 22-mer T7 sequencing primer (Stratagene) and a 39-mer,
5'-GAGGTGAAAACGCCATGGGCTTCCCTTCTCCTGCGGCGG-3', which is identical to the start of the humD gene
except that it contains a T
C transition at the 
1 position relative
to the ATG codon. This change introduces an NcoI restriction
enzyme site (underlined) at the start of the humD gene. The
PCR product was subsequently digested with NcoI and
EcoRI and subcloned into a similarly digested T7 expression
vector, pEC46 (12). The humD gene in this new
plasmid, pOS36, was sequenced (Lark Technologies, Houston, Tex.) to
confirm that no mutations were inadvertently generated during PCR.
pOS36 therefore expresses HumD from an
isopropyl-
-D-thiogalactopyranoside (IPTG)-inducible T7
RNA polymerase promoter.
UV survival assays.
To test the ability of HumD to
complement UmuD' functions and restore UV resistance to normally
UV-sensitive recA718 umuDC cells (46), cultures
were grown in Luria-Bertani (LB) medium until they reached a cell
density of 108/ml, at which point they were harvested and
resuspended in SM buffer (50 mM Tris-HCl [pH 7.5], 8 mM
MgSO4, 100 mM NaCl, 0.01% gelatin) (29). Cells
were exposed to UV light at a fluence of 0.25 J/m2/s, and
appropriate dilutions were plated on LB agar plates. Surviving colonies
were scored after overnight incubation at 37°C.
Qualitative spontaneous mutagenesis plate assay.
Assays were
performed essentially as described elsewhere (49). Aliquots
(1 ml) from a fresh overnight culture were centrifuged and resuspended
in an equal volume of SM buffer (29). The ability of
particular plasmid-bearing strains to promote Umu-dependent SOS mutator
activity in the absence of exogenous DNA damage was judged by plating
100-µl aliquots on Davis-Mingioli minimal agar plates supplemented
with a trace amount of tryptophan (0.75 µg/ml) (3).
Umu-dependent chemically induced mutagenesis was determined as
described above except that 1 µl of methyl methanesulfonate (MMS;
Sigma, St. Louis, Mo.) was applied to a small sterile disk in the
center of the plate. Both spontaneously arising and MMS-induced Trp+ mutants were scored after 3 days of incubation at
37°C.
Purification of the bacteriophage P1 HumD protein.
HumD
protein was purified from a 3-liter culture of E. coli
BL21(
DE3)/pOS36 that had been induced with 1.0 mM IPTG for 3 h
prior to harvesting. The purification strategy was based upon that
successfully used to isolate the structurally homologous UmuD' and
MucA' proteins (12) and includes selective precipitation with ammonium sulfate followed by DEAE-Sephacel (Pharmacia, Piscataway, N.J.) and hydroxyapatite (Bio-Rad, Hercules, Calif.) ion-exchange chromatography and AcA54 (BioSepra, Cergy St. Christopher, France) gel filtration.
Polyclonal antisera to the highly purified HumD protein were raised in
rabbits, using standard methods, by Covance (Vienna, Va.). The serum
was subsequently affinity purified by standard methods (34).
Steady-state levels of HumD in vivo.
The steady-state level
of HumD expressed from pOS30 in various genetic backgrounds was
determined as described previously (11). Briefly, cells were
grown in LB broth at 37°C to early exponential phase, at which time
1.5-ml aliquots were harvested and resuspended in sample buffer (50 mM
Tris-HCl [pH 6.8], 10% glycerol, 2.0% sodium dodecyl sulfate
[SDS], 0.1% bromophenol blue, 10 mM dithiothreitol). Aliquots
representing equal cell numbers were electrophoresed on SDS-17%
polyacrylamide gels. Proteins were then transferred to an Immobilon P
membrane (Millipore, Bedford, Mass.) and subsequently probed with a
1:10,000 dilution of the affinity-purified polyclonal antibodies raised
against HumD. The transferred proteins were subsequently visualized
using the CSPD-Star chemiluminescence assay (Tropix, Bedford, Mass.).
Membranes were exposed to Kodak Bio-Max MR film for periods of 1 to 10 min.
Stability of HumD and UmuC in vivo.
The relative stability
of the HumD and UmuC proteins was measured essentially as described
above except that when the cells reached mid-log phase (time zero),
chloramphenicol (100 µg/ml) was added to the medium to inhibit
further protein synthesis. Aliquots of 1.5 ml were removed at various
time points thereafter, and samples were processed as described above.
The apparent half-life of HumD (or UmuC) was determined by simple
analysis of the relative steady-state level of the protein at each time
point (11).
Glutaraldehyde cross-linking studies.
To determine if HumD
is capable of forming a dimer with itself, ~250 ng of HumD was
incubated at 37°C for 30 min in 20 mM Tris (pH 7.5)-1 mM EDTA-10%
glycerol-50 mM NaCl-1 mM dithiothreitol, after which time protein
complexes were chemically cross-linked by adding glutaraldehyde to a
final concentration of 0.05%. After 15 min of further incubation at
room temperature, complexes were separated in an SDS-15%
polyacrylamide gel and electrotransferred to an Immobilon P membrane.
The monomeric/multimeric state of HumD was determined after probing the
membrane with polyclonal antibodies to HumD and subsequently visualized
with the CSPD-Star chemiluminescence kit as described above.
Ability of HumD to bind to a RecA nucleoprotein filament.
The ability of HumD to bind to a RecA nucleoprotein filament was
determined essentially as previously described for UmuD' (12). Reaction mixtures (10 µl) contained 40 ng of 
X174
DNA (New England Biolabs), 20 mM HEPES buffer (pH 7.5, 20 mM NaCl, 1 mM
EDTA, 1 mM ATP
S, 1.5 µg of RecA (added where indicated), 1 mM
dithiothreitol, and 50 µg of bovine serum albumin. To initiate nucleoprotein formation, MgCl2 was added to a final
concentration of 10 mM and the mixture incubated at 37°C for 20 min,
after which time 300 ng of UmuD' or HumD was added to the reaction
mixture and incubated for an additional 20 min at 37°C. Nucleoprotein complexes were chemically cross-linked by adding glutaraldehyde (final
concentration of 0.05%) at room temperature for 10 min. These
complexes were separated by electrophoresis in a nondenaturing 0.95%
agarose gel, and the relative mobility of the nucleoprotein complex was
determined after chemiluminescence immunoanalysis as described above.
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RESULTS |
Restoration of cellular mutagenesis by P1 HumD to normally
nonmutable lexA(Def) recA430 strains.
To
test the hypothesis that HumD might encode a protein with a function
similar to that of the UmuD'-like mutagenesis proteins, we subcloned a
~1-kb fragment of the P1 genome, containing humD, into a
low-copy-number plasmid and introduced the plasmid, pOS27, into the
lexA(Def) recA430 strain, JS431 (42).
Although this strain expresses derepressed levels of the chromosomally
encoded UmuDC proteins, it does not exhibit any cellular mutagenesis
because RecA430 is unable to mediate cleavage of UmuD (4,
37). Both spontaneous and damage-induced mutagenesis can,
however, be restored, if UmuD' is provided in trans from a
high copy-number plasmid (30, 42) and to a lesser extent by
a low-copy-number plasmid (7; Fig. 2). Unlike the
low-copy-number E. coli plasmid, pRW66, which resulted in an
5-fold increase in spontaneous mutagenesis and 13-fold increase in
MMS-induced mutagenesis over background, the low-copy-number HumD
plasmid, pOS27, resulted in no discernible increase in spontaneous
mutagenesis and only a 3-fold increase (from 74 to 245 Trp+
mutants) in MMS mutagenesis (Fig. 2).
Although these effects are clearly smaller than those observed for the
low-copy-number UmuD' plasmid, we were nevertheless encouraged by these
results since the qualitative plate assay is a sensitive and accurate reflection of the ability of cells to promote damage-induced
mutagenesis (15).
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FIG. 2.
Restoration of mutagenesis functions to a normally
nonmutable lexA(Def) recA430 strain by P1 HumD.
The ability of E. coli UmuD' and P1 HumD to restore
mutagenesis functions to JS431, a recA430 lexA(Def) strain,
was monitored in a qualitative plate assay by following reversion of
the trpE65(oc) allele in the absence of exogenous DNA damage
() or after exposing the cells to MMS (+). recA430
lexA(Def) strains are nonmutable because they are unable to
posttranslationally mediate the cleavage of UmuD to UmuD' and therefore
give rise to essentially the same number of Trp+ revertants
as the isogenic umuDC strain, RW262. Mutagenesis can,
however, be restored to JS431 by providing a recombinant UmuD' in
trans. As can be seen, the extent of restoration is
dependent on the copy number of the plasmid; pRW66 is a low-copy-number
UmuD' plasmid, and pOS32 is a high-copy-number UmuD' plasmid.
Similarly, HumD also restores mutagenesis to JS431, with the extent of
mutagenesis related to the copy number/expression of the relevant HumD
plasmid: pOS31, low-copy-number HumD; pOS30, high-copy-number HumD
translated in the same direction as LacZ' (expressed from the vector
polylinker); pOS31, high-copy-number HumD translated in the opposite
direction to LacZ'. The data presented are the means from three
independent isolates and three plates per isolate. The error bars
represent the standard error of the mean for each experiment.
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As noted above, the extent to which UmuD' restores mutagenesis to
recA430 cells depends on the copy-number plasmid from which it is expressed (7) (Fig. 2). We were interested in
determining if the same is true for HumD. As a consequence, we
subcloned the humD gene from pOS27 into the high-copy-number
plasmid pBluescript KS+. Plasmids were obtained with the
humD gene transcribed in the same and opposite directions as
lacZ', and one plasmid from each group was chosen for
further study. Introduction of these plasmids (pOS30 and pOS31)
resulted in a dramatic increase in the extent of both spontaneous and
MMS-induced Trp+ reversion (Fig. 2). The greatest increase
was observed with pOS30, in which HumD is translated in the same
direction as LacZ'. Indeed, the amount of mutagenesis promoted by pOS30
was comparable to that seen with the high-copy-number E. coli UmuD' plasmid, pOS32. By comparison, pOS31, in which HumD is
translated in the opposite direction to LacZ', yielded twofold fewer
Trp+ revertants, but this was still significantly higher
than that seen with the low-copy-number plasmid, pOS27 (Fig. 2). Based
on these observations, we conclude that HumD can substitute for
E. coli UmuD' in cellular mutagenesis. The fact that very
little mutagenesis is seen with a low-copy-number plasmid, and the most is found when translation is in the same orientation and distal to the
lac promoter from a high-copy-number plasmid, suggests, however, that production of high levels of HumD-dependent mutagenesis requires maximal expression of HumD.
Specificity of HumD's ability to restore SOS mutagenesis.
HumD clearly has the ability to restore mutagenesis functions to a
lexA(Def) recA430 strain, and we were interested
in determining if the effect might be greater in other recA
mutant strains. An excellent background to assay for Umu-like activity
is in a lexA(Def) recA718 strain (18,
42). When fully derepressed, the RecA718 protein exhibits a
modest coprotease activity that results in a spontaneous mutator
activity (18, 42) (Fig. 3).
This activity is clearly Umu dependent, as strains carrying a
umuDC mutation do not exhibit the spontaneous mutator
phenotype (18, 42) (Fig. 3). Based on genetic studies, it
has been hypothesized that the mutator activity reflects the ability of
the Umu-like proteins to promote extension from mispaired bases
generated during normal replication (9).
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FIG. 3.
Specificity of HumD's ability to restore mutagenesis
functions in a umuDC recA718 lexA(Def) strain. The
specificity of HumD's ability to restore mutagenesis functions to
RW264 was assayed by introducing compatible plasmids expressing various
combinations of E. coli UmuD' and/or UmuC, pKM101 MucA'
and/or MucB, and R391 RumA' and/or RumB together with high-copy-number
HumD. Mutagenesis was monitored as described in the legend to Fig. 2 by
following spontaneous reversion of the trpE65(oc) allele.
Strain EW181 demonstrates the level of spontaneous mutagenesis promoted
by the chromosomally encoded UmuD(D') proteins, and RW264 is the
isogenic umuDC strain. As can be seen, background levels
of spontaneous mutagenesis were observed with RW264 harboring pRW274
(low-copy-number E. coli UmuC), pOS30 (high-copy-number
HumD), pOS32 (high-copy-number E. coli UmuD'), pOS33
(high-copy-number R391 RumA'), pOS34 (high-copy-number pKM101 MucA'),
pOS35 (low-copy-number R391 RumB), pOS37 (low-copy-number pKM101 MucB)
alone or with pRW274/pOS33 (low-copy-number E. coli UmuC and
high-copy-number R391 RumA'), pRW274/pOS34 (low-copy-number E. coli UmuC and high-copy-number pKM101 MucA'), and pOS37/pOS30
(low-copy-number pKM101 and high-copy-number HumD). A twofold increase
over background was observed with pOS35/pOS30 (low-copy-number R391
RumB and high-copy-number HumD). In dramatic contrast, significant
levels of spontaneous mutagenesis, comparable to that seen in the
umu+ strain, were observed with pRW274/pOS32
(low-copy-number E. coli UmuC and high-copy number E. coli UmuD') and pRW274/pOS30 (low-copy-number E. coli
UmuC and high-copy-number HumD). The data presented are the means from
three independent isolates and three plates per isolate. The error bars
represent the standard error of the mean for each experiment.
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Many UmuD'C-like homologs have now been cloned, and we were interested
in determining if bacteriophage P1 HumD could interact with these
homologs in addition to E. coli UmuC. The Salmonella typhimurium Umu proteins are closely related to their E. coli counterparts (38, 45), and one might intuitively
think that they would be the best candidates to determine if HumD has a
broad specificity in promoting mutagenesis. Unfortunately, S. typhimurium is poorly mutable because it appears to have acquired
mutations in its umuC gene that render the protein less
active (20, 36). As a consequence, we decided to look at the
ability of HumD to promote mutagenesis in combination with the more
diverged but normally very active MucB and RumB proteins
(22). Lack of complementation between Umu homologs has
previously been demonstrated and is understandable given their
evolutionary divergence (36). Indeed, no complementation is
seen with a high-copy-number HumD plasmid and a low-copy-number MucB
plasmid, and only a very slight increase in MMS mutagenesis is seen
with a high-copy-number HumD plasmid and a low-copy-number RumB plasmid
(Fig. 3). Likewise, no mutagenesis is seen with a low-copy-number
E. coli UmuC plasmid and a compatible MucA' or RumA' plasmid
(Fig. 3). In fact, the only combination of plasmids that resulted in
significant levels of spontaneous mutagenesis in the
umuDC background was the high-copy-number HumD plasmid or
high-copy-number UmuD' plasmid in combination with the low-copy-number UmuC plasmid (Fig. 3).
HumD shares only 39% identity with E. coli UmuD' (Fig. 1)
and slightly less with MucA' (35%) and RumA' (30%), suggesting that it is not the overall degree of identity that is important for the
HumD-UmuC interaction, but rather that the residues conserved between
the HumD and UmuD' proteins presumably play a critical role in their
ability to interact with UmuC.
Ability of HumD to restore UV resistance to UV-sensitive
recA718 umuDC bacteria.
Several years ago, Witkin
and colleagues demonstrated that a recA718 umuDC uvrA155
strain was very sensitive to the killing effects of UV light because
the strain was unable to resume replication after sustaining DNA damage
(46). Similar to the mutator phenotype described above, this
UV-sensitive phenotype is dependent on the Umu-like proteins, as
resistance to UV light is restored in the presence of the chromosomally
encoded Umu proteins (46) or by Umu-complementing plasmids
(18). As can be seen in Fig.
4, the recA718 umuDC
lexA(Def) uvrA155 strain, RW264, is very sensitive to
UV light, with survival dropping 6 orders of magnitude after a dose of
approximately 6 J/m2. Transformation with either the
low-copy-number UmuC plasmid, pRW274, or the high-copy-number HumD
plasmid, pOS30, alone has no obvious effect on UV survival. If,
however, the strain is cotransformed with both plasmids, UV resistance
increases dramatically (103-fold at 6 Jm
2),
but is roughly 10-fold less than that seen with pRW274 and the
compatible high-copy-number UmuD' plasmid, pOS32 (Fig. 4). These
findings are consistent with the data from the mutagenesis assays,
described above, by suggesting that HumD is able to interact with UmuC.
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FIG. 4.
Restoration of UV resistance to a normally UV-sensitive
umuDC lexA(Def) recA718 strain by HumD-UmuC.
The ability of various plasmids to restore UV resistance to RW264
[umuDC lexA(Def) recA718 uvrA155] was
assayed by exposing plasmid containing cultures to UV light and plating
serial dilutions on LB agar plates. Surviving colonies were scored
after 24 h of incubation at 37°C. The data presented are the
means from three independent isolates and three plates per UV dose.
Strains analyzed were RW264 alone ( ), RW264/pOS30 (high-copy-number
HumD) ( ), RW264/pRW274 (low-copy-number E. coli UmuC)
( ), RW264/pRW274/pOS30 (low-copy-number E. coli UmuC with
high-copy-number HumD) (X), and RW264/pRW274/pOS32 (low-copy-number
E. coli UmuC with high-copy-number E. coli UmuD')
( ).
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Purification of HumD.
The genetic data strongly suggest that
HumD can functionally substitute for E. coli UmuD'. To
better characterize the HumD protein and its interactions with other
components of the mutasome, we overproduced bacteriophage P1 HumD by
placing the humD gene under the control of an IPTG-inducible
T7 RNA polymerase promoter (41). Addition of IPTG to
exponentially growing cells resulted in the induction of a protein with
the expected molecular mass of HumD (Fig.
5). A large proportion of the
overproduced protein was found in the soluble fraction of cell
extracts, thereby allowing us to follow purification by simple
visualization with Coomassie brilliant blue. HumD was purified to
greater than 95% homogeneity (Fig. 5) by using the same protocol as
used for other UmuD'-like proteins (12). N-terminal sequence
analysis of the highly purified protein confirmed its identity as HumD
and revealed that like UmuD' (12), the formylmethionyl had
been removed by amino-terminal peptidase (1) (data not
shown). Like other homologs, HumD eluted from the gel filtration at a
position consistent with it being a dimer in solution.
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FIG. 5.
Overproduction and purification of HumD. Bacteriophage
P1 HumD was purified from BL21(DE3) cells carrying the
HumD-overproducing plasmid pOS36. Proteins were separated in SDS-15%
polyacrylamide gels, and proteins were visualized after staining with
Coomassie blue. Lanes U and I are whole-cell extracts from uninduced
and IPTG-induced cells, respectively; lanes A, D, H, and G are
fractions obtained after selective ammonium sulfate precipitation and
DEAE-Sephacel, hydroxyapatite, and AcA54 gel filtration chromatography,
respectively. The position of monomeric HumD is marked on the right,
and the molecular masses of marker proteins are indicated (in
kilodaltons) on the left.
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We further analyzed the ability of HumD to form dimers by chemically
cross-linking HumD (Fig. 6). Most of the
uncross-linked HumD migrated at a position consistent with it being a
monomer (under denaturing conditions). However, a significant quantity of HumD migrated at a position of dimeric HumD (even under apparently denaturing conditions). The intensity of this dimeric HumD band increased dramatically upon cross-linking, as did the appearance of
higher-order structures (Fig. 6) that we have previously seen with
UmuD' under similar conditions (32). We conclude, therefore, that like UmuD', HumD is a dimer in solution.
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FIG. 6.
HumD is a dimer in solution. To determine if HumD is
capable of forming a dimer with itself, HumD was chemically
cross-linked with glutaraldehyde as described in Materials and Methods.
Untreated () and cross-linked (+) samples were separated in an
SDS-15% polyacrylamide gel and transferred to an Immobilon P
membrane. The monomeric/multimeric state of HumD was determined by
probing the membrane with polyclonal antibodies to HumD and subsequent
visualization with the CSPD-Star chemiluminescent substrate. The
positions of HumD monomers, dimers, and higher-order structures are
indicated on the right.
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Expression of HumD in vivo.
The location of a LexA-binding
site immediately upstream of HumD (25) (Fig.
7) strongly suggests that under normal
conditions, HumD is negatively regulated by LexA and is damage
inducible. To confirm these suggestions, we analyzed the in vivo level
of HumD expressed from pOS30 in a wild-type
lexA+ cell, with and without exposing the cells
to the DNA-damaging agent MMC (Fig. 7). Like the umu operon,
the humD gene has a potential LexA-binding site that has a
very good match with the consensus binding site (25). In the
case of HumD, it deviates from this consensus by only three nucleotides
and as a consequence would be expected to be tightly regulated by LexA.
Surprisingly, the basal level of expression was higher than expected,
and probably arose from the inability of the chromosomally expressed
LexA to bind to all of the humD operator sites on the
multicopy plasmid pOS30 (>100 copies per cell). Exposure of the
wild-type cells to MMC resulted in only an ~1.7-fold induction of
HumD and presumably reflects incomplete derepression and/or tight
binding by LexA to the operator sequence, as the level of HumD
expressed in a lexA(Def) strain was 10- to 20-fold greater
than in the induced lexA+ background. These
observations therefore support the hypothesis that HumD is part of the
LexA regulon and is induced as a consequence of cellular DNA damage.
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FIG. 7.
Damage-inducible expression of HumD. (A) Nucleotide
sequence of the humD promoter/operator region. The initiator
codon (formylmethionyl [fmet]) of HumD is at the far right and boxed;
positions of the 35 and 10 promoter elements and of the ribosome
binding site (RBS) are overlined. The proposed LexA-binding site is
located between the 10 promoter element and the RBS and is also
boxed. It should be noted that this LexA-binding site deviates from the
consensus at only three positions. (B) Steady-state levels of HumD
expressed from pOS30 in an undamaged or MMC-treated
lexA+ strain (RW406) or in an undamaged
lexA51(Def) strain (RW260). The position of HumD is
indicated on the right. As can be seen, the HumD antiserum recognizes
another cellular protein in addition to HumD. The identity of this
protein is unknown, but it serves as a useful internal control,
ensuring that equal amounts of protein extract have been applied to the
gel.
|
|
Perhaps the most surprising aspect of these studies was the actual
amount of HumD protein expressed in the cell. As noted above, pOS30 is
a high-copy-number plasmid, and so we expected that HumD would be
significantly overproduced. In fact, even when fully derepressed, the
amount of HumD expressed from pOS30 was roughly similar to that of
UmuD' from a low-copy-number plasmid (11) (data not shown).
The poor expression of HumD can potentially be explained by weak
promoter activity, especially as the sequence of the 35 box deviates
from the consensus (19) and similar substitutions in the
35 box of the umu operon resulted in reduced expression of
UmuD' (28). The limited expression of HumD from its native
promoter might also explain why complementation of mutagenesis
functions is greatly enhanced in the presence of the high-copy-number
plasmid: there is simply not enough HumD expressed from the
low-copy-number plasmid for functional complementation.
Stability of HumD in vivo.
The intracellular levels of
E. coli UmuD and UmuD' proteins are exquisitely regulated by
targeted proteolysis (10). UmuD is relatively labile in vivo
and is targeted for degradation by the Lon protease (10,
14). Homodimeric UmuD' is, in contrast, relatively insensitive to
Lon, and it is stable in vivo until it forms a heterodimer with UmuD
and is rapidly degraded by ClpXP (10). Analysis of the
stability of HumD (Fig. 8) revealed that like homodimeric UmuD', it is very stable in vivo, with a half-life estimated to be greater than 50 min. Similar results were obtained when
HumD was coexpressed with UmuD, suggesting that HumD and UmuD
heterodimers are insensitive to proteolysis or that HumD and UmuD are
unable to form heterodimers (data not shown).
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FIG. 8.
In vivo stability of HumD. Plasmid pOS30 was introduced
into the (umuDC)596::ermGT
recA+ lexA51(Def) strain EC10, and the relative
stability of HumD was measured after protein synthesis was inhibited by
the addition of chloramphenicol (100 µg/ml) at time zero. Additional
aliquots were removed at 10-min intervals. Whole-cell extracts were
separated in an SDS-15% polyacrylamide gel, and proteins were
visualized using HumD antibodies and the CSPD-Star chemiluminescence
assay. Track S is ~20 ng of highly purified HumD protein; track is an extract of the strain lacking pOS30. As shown in Fig. 7, the HumD
antiserum recognizes another cellular protein in addition to HumD that
serves as a useful internal control, ensuring that equal amounts of
protein extract have been applied to the gel. The position of HumD is
indicated on the right.
|
|
In vivo interaction of HumD and UmuC.
Our observation that
HumD is able to restore mutagenesis functions specifically in
combination with E. coli UmuC strongly suggests that the
proteins physically interact with each other. Like UmuD, UmuC is labile
in vivo because it is rapidly degraded by Lon (10) (Fig.
9). However, in the presence of UmuD',
the stability of UmuC increases dramatically (11),
presumably as a result of UmuD' protecting UmuC from Lon degradation
(10). Similarly, when HumD and UmuC are coexpressed, UmuC
exhibits a dramatic increase in its observed half-life (Fig. 9). Thus,
although not direct evidence of a protein-protein interaction, taken
together with the genetic complementation studies, these observations
suggest that HumD and UmuC are able to physically interact.
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FIG. 9.
Stabilization of E. coli UmuC by HumD. A
plasmid expressing UmuC alone (pRW274) or coexpressing HumD (pOS30) was
introduced into the (umuDC)596::ermGT
recA+ lexA51(Def) strain, EC10. The relative stability
of UmuC was assayed after protein synthesis was inhibited by the
addition of chloramphenicol (100 µg/ml) at time zero. Whole-cell
extracts were separated in an SDS-15% polyacrylamide gel, and
proteins were visualized by using UmuC antibodies and the CSPD-Star
chemiluminescence assay. The position of UmuC is indicated by an arrow
at the left.
|
|
Ability of HumD to interact with a RecA nucleoprotein
filament.
As noted in the introduction, SOS mutagenesis requires
the formation of a multiprotein "mutasome" (6). A key
step in the formation of the mutasome is an interaction between
UmuD'2C and RecA that most likely positions the Umu complex
at the site of a DNA lesion (2, 39, 40). HumD, Like UmuD',
is an acidic protein with a predicted pI of ~5.1, and it does not
bind directly to DNA (Fig. 10). If the DNA is, however, coated with
RecA, HumD migrates at a position similar to that of RecA-DNA-UmuD'
complexes, suggesting that it too binds to a RecA nucleoprotein
filament (Fig. 10). We hypothesize,
therefore, that like UmuD', HumD interacts with a RecA nucleoprotein
filament in such a way as to target the limited number of HumD
molecules to sites where they may promote translesion DNA synthesis.
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FIG. 10.
Interaction of HumD with a RecA-nucleoprotein filament.
Nucleoprotein complexes were formed as described in Materials and
Methods. After electrophoretic separation in 0.9% agarose,
nucleoprotein complexes were transferred to a support membrane. The
membrane was cut, and the blots were probed with UmuD' and HumD
antibodies, as indicated. Both blots were subsequently visualized using
the CSPD-star chemiluminescence immunoassay. Tracks labeled lack RecA protein, whereas those labeled + contain RecA and are
therefore able to form RecA nucleoprotein filaments. Because of their
large size, these nucleoprotein complexes have limited mobility in the
agarose gel and are therefore retained at the top of the gel. In
contrast, the free protein migrates much more quickly. These gels are
run under native conditions, and the smear observed in the presence of
the nucleoprotein filaments presumably arises from the dissociation of
some of the un-cross-linked UmuD' and HumD from the filament during
electrophoresis. The positions of free UmuD' and HumD as well as the
binding complex are indicated at the right.
|
|
| |
DISCUSSION |
Bacteriophage P1 HumD is a functional homolog of UmuD'.
Based
on sequence comparisons, Lewis et al. (25) suggested that
the bacteriophage P1 humD gene might encode a protein
similar in function to UmuD'-like proteins. We have tested this
hypothesis directly and find that HumD can in fact functionally
substitute for E. coli UmuD' in vivo. Complementation
between Umu-like orthologs is rare (16, 36), and it could be
argued that since HumD does not have a natural cognate partner, it may
have a more relaxed specificity for the necessary protein-protein
interactions required for mutasome formation. However, we observed no
restoration of mutagenesis functions with HumD and MucB and only very
slight complementation with HumD and RumB, suggesting that the
HumD-UmuC-RecA interaction is quite specific. Clues as to the sites of
these protein-protein interactions come from comparison of HumD's
predicted structure to that of UmuD' (32). Although overall
homology is quite low (~39%) between UmuD' and HumD, when one aligns
the primary amino acid sequences of both proteins (Fig. 1), it is clear
that most homology is in the N-terminal tail, where 24 of 29 residues (~83%) are identical. This region has been implicated as a RecA binding site as deletion of the first 20 amino acids of UmuD' results
in a protein that has a reduced capacity to interact with a
RecA-nucleoprotein filament (32). In addition, the longer N-terminal tail, along with the shorter C-terminal tail, is important for UmuD' dimerization (8, 32). In particular, the short C-terminal tails of the two UmuD' protomers form a -sheet that helps
dimerization. While there is limited identity between HumD and UmuD' in
their C termini, the C terminus of HumD is 10 amino acids longer than
that of UmuD', suggesting that it may be able to form a more extensive
sheet with a cognate protomer, thereby explaining the appearance of
a HumD dimer, even under apparently denaturing conditions (Fig. 5).
The sites on UmuD' required for an interaction with UmuC are unknown,
but it is clear from our functional studies that they are conserved in
HumD. All of the available evidence is therefore consistent with the
hypothesis that HumD is a bona fide homolog of the UmuD' mutagenesis protein.
At first glance, one might consider whether the existence of
humD on P1 is a peculiarity of nature. Very recently,
however, a gene (GenBank accession no. AF064539) that is approximately 70% identical to P1 HumD has been identified on the lambdoid phage N15
(not shown), and it seems likely that other bacteriophage orthologs
will be identified. One obvious question is, why do P1 and N15 carry
such orthologs on their small chromosomes? Like all organisms, both
phage are subject to evolutionary pressures, and so humD
presumably confers some advantage over phage that have lost the gene.
Our in vivo complementation studies revealed that HumD's mutagenesis
promoting activity is manifested, albeit at low levels, when expressed
from a low-copy-number plasmid. Both P1 and N15 are normally maintained
at one to two copies in a lysogenic state, and so by analogy to the
phenotype observed with the low-copy-number plasmid, it is possible
that HumD's function is to allow the host to survive the potentially
fatal consequences of replication-inhibiting DNA lesions, thereby
obviating the need for the phage to enter a lytic cycle. Such an
activity seems hardly warranted in E. coli, which already
possesses active UmuD'2C proteins, but what about additional hosts of P1, N15, and other humD-bearing phage?
Many enteric bacteria possess Umu homologs which, based upon their cross-reactivity to E. coli Umu antibodies (35),
are clearly closely related to their E. coli counterparts.
Moreover, unlike the common E. coli laboratory strains with
which most of us work, many naturally occurring E. coli
strains are poorly mutable (35), suggesting ineffective Umu
systems. Furthermore, some bacteria, such as Klebsiella
aerogenes and Citrobacter intermedius, are apparently
rendered nonmutable because they are unable to posttranslationally process UmuD to mutagenically active UmuD' (35) and are
therefore analogous to the E. coli recA430 lexA(Def) strain
used for Fig. 2. Under such conditions, any activity provided by HumD
may, in fact, provide a significant evolutionary advantage to both the host and the phage.
Of course, the other possibility is that the function of HumD is to
protect the phage itself, rather than its host. After all, once the
phage has entered the lytic cycle, the cell is doomed anyway. Under
lytic conditions, the copy number of these temperate phage increases
dramatically (over 100-fold) before eventual lysis, and this could be
considered analogous to the conditions wherein we observed the
considerable mutagenesis-promoting activity of HumD when expressed from
a high-copy-number plasmid (Fig. 2 and 3). Presumably, if the phage has
entered the lytic cycle because of host DNA damage, it may have
acquired damage itself, and the role of HumD is to protect the phage
genome by allowing translesion DNA synthesis across otherwise
unrepairable lesions.
Another formal possibility is that the retention of humD on
phage genomes is completely unrelated to HumD's translesion-promoting activity. For example, we have recently demonstrated that E. coli UmuD' is an enzyme that possesses the ability to cleave
intact UmuD in vitro and in vivo (26, 27). Given the
structural similarities between UmuD' and HumD (32),
including the active-site residues necessary for cleavage, it is likely
that HumD possesses similar enzymatic activities and that it is this
property which provides the selective pressure for its retention on
certain phage genomes.
We thank Arun Alagappan for technical help with the bacterial
mutagenesis assays shown in Fig. 2 and 3, Michael Yarmolinsky for
plasmid pAW711 and helpful insights into P1 biology and Martín Gonzalez for stimulating discussions during the course of this work.
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Abstract
Introduction
Present address: Molecular Biology Research Program, Henry Ford
Hospital, Detroit, MI 48202.
Present address: School of Medicine, University of Pittsburgh,
Pittsburgh, PA 15261.
