(Received for publication, January 19, 1996; and in revised form, February 25, 1996)
From the
The Escherichia coli UmuD` and UmuC proteins play essential roles in SOS-induced mutagenesis. Previous studies investigating the molecular mechanisms of mutagenesis have been hindered by the lack of availability of a soluble UmuC protein. We report the extensive purification of a soluble UmuD`C complex and its interactions with DNA. The molecular mass of the complex is estimated to be 70 kDa, suggesting that the complex consists of one UmuC (46 kDa) and two UmuD` (12 kDa) molecules. In contrast to its inability to bind to double-stranded DNA, UmuD`C binds cooperatively to single-stranded DNA as measured by agarose gel electrophoresis and confirmed by steady-state fluorescence depolarization. A Hill coefficient, n = 3, characterizes the binding of UmuD`C to M13 DNA and to a 600 nucleotide DNA oligomer, suggesting that at least three protein complexes may interact cooperatively when binding to DNA. The apparent equilibrium binding constant of UmuD`C to single-stranded DNA is approximately 300 nM. Binding of the complex to a short, 80 nucleotide, DNA oligonucleotide was detectable by fluorescence depolarization, but it did not appear to be cooperative. Binding of UmuD`C to single-stranded M13 DNA causes an acceleration of the protein-DNA complex, suggesting that the longer DNA may undergo compaction. The UmuD`C complex associates with RecA-coated DNA, and the UmuD`C complex remains bound to DNA in the presence of RecA.
The SOS regulon present in Escherichia coli is
triggered as part of the cell's response to exogenous DNA
damage(1, 2) . As a consequence of attempting to
replicate past DNA lesions, an inducing signal is generated that
results in the activation of RecA protein
(RecA*)()(3) . In its activated state, RecA*
enhances the self-cleavage of the LexA transcriptional
repressor(4) , thus leading to the expression of more than 20
proteins that help the cell to avoid the lethal effects of DNA damage.
Recent reviews of the SOS response are contained in (5) and (6) .
An important, yet poorly understood, feature of the SOS response is error-prone repair or ``SOS-induced'' mutagenesis. The UmuD and UmuC proteins are essential participants in this process(7, 8) , and the absence of either protein reduces mutagenesis by more than 100-fold to spontaneous background levels(2, 9) . The UmuDC operon is induced following LexA cleavage, and the process is further regulated by the need for the UmuD protein to undergo a RecA*-mediated cleavage. This event is mechanistically similar to LexA autodigestion, but in the case of UmuD (and its homologs), cleavage leads to activation of its mutagenesis function(s)(10, 11, 12) . Activated UmuD` forms homodimers and associates with UmuC to form a UmuD`C complex(13) . Both UmuD` and UmuC are believed to interact with RecA* (14, 15) in such a way as to target the relatively small number of mutagenically active UmuD`C molecules to lesions within DNA(15, 16, 17) .
Replicative bypass of the RecA*-UmuD`C-coated lesion is most likely performed by pol III holoenzyme(18, 19) . Although pol II is induced in response to DNA damage as part of the SOS regulon(20, 21) , its role in either lesion bypass or DNA repair has yet to be firmly established. Recent data suggest that pol II may be required for the bypass of abasic lesions provided that heat shock proteins are not induced(22) . A two-step model describing error-prone synthesis was proposed by Bridges and Woodgate (23, 24) in which pol III incorporates a nucleotide opposite a template lesion but cannot continue synthesis. Generation of a UmuD`C-RecA* ``mutasome'' would enable the stalled pol III molecule to continue synthesis past the lesion(5, 13) .
The ability of E. coli to facilitate error-prone translesion synthesis depends upon the cellular levels of the Umu proteins. Under normal conditions the proteins are expressed at low levels(25) . Genetic experiments in which the in vivo levels of the UmuD`C proteins have been artificially manipulated leads to a variety of phenotypes. Overproduction of the Umu proteins in fully SOS-induced cells can cause a cold-sensitive phenotype that is associated with the rapid cessation of DNA replication(26) . Expression of excess UmuC results in a nonmutable phenotype similar to what is seen when there is a deficiency of UmuC(27) . Modest overproduction of UmuD`C appears to inhibit recombinational functions of RecA and promotes a switch from error-free recombinational repair pathways to those that are error prone(5, 28) .
Testing of these models has been severely hampered by the lack of reconstituted ``error-prone'' and ``error-free'' lesion bypass assays. While pol III holoenzyme, RecA, and UmuD` have been extensively purified and characterized, UmuC has been more difficult to study because of its apparent insolubility and its lack of defined enzymatic activity. Previous studies of UmuC and UmuD`C complexes were performed with UmuC that had been purified from a denatured form and renatured in the presence of chaperone proteins(13, 29) . In this paper we describe the first purification of a soluble, intact UmuD`C complex and characterize its interactions with DNA.
Cells were harvested and resuspended in equal weight/volume (gm/ml) amount of storage buffer (50 mM Tris-HCl (pH 7.5), 10% sucrose, 10 mM EDTA). The cells were then quickly frozen by dropwise addition of cell suspension into liquid nitrogen and stored at -70 °C. Cells were thawed at 4 °C in 2.5 volumes of lysis buffer (50 mM Tris-HCl (pH 7.5), 10% sucrose, 0.1 M NaCl). Once thawed, lysozyme was added to a final concentration of 0.4 mg/ml. The cell slurry was incubated for 1 h at 4 °C followed by a 4-min incubation at 37 °C and centrifuged at 11,800 rpm for 1 h in a Sorvall GSA rotor.
A solution of 10% polyethyleneimine HCl (pH 7.6) was added to lysate supernatant in 10-ml portions to a final concentration of 1.1%. The solution was stirred for 15 min after each addition. At the end of the last addition, the suspension was kept on ice for 20 min, and the precipitate was collected by centrifugation for 10 min at 9,000 rpm in a Sorvall SS34 rotor. Proteins were extracted by stirring the pellet for 15 min in 50 ml of R buffer (20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol) containing 1 M NaCl. Following centrifugation (10 min at 6,000 rpm) the supernatant was collected and the extraction repeated until all proteins were eluted from the pellet.
Supernatants were combined
and dialyzed overnight against R buffer containing 50 mM NaCl
(from this point on the concentration of glycerol was increased to 20%
glycerol (v/v)). The dialyzed sample was loaded onto a 200-ml
DEAE-Sephacel (Pharmacia) column (3 15 cm) at a flow rate of 1
ml/min. The column was washed with 2 column volumes of R buffer
containing 50 mM NaCl, and protein was eluted from the column
with three column volumes of R buffer containing 500 mM NaCl.
Fractions containing high concentrations of UmuD`C were combined,
dialyzed overnight against R buffer containing 250 mM NaCl,
loaded onto a 150-170-ml phosphocellulose (Whatman) column (3
24 cm), and extensively washed. UmuC was eluted from the column
with 2 volumes of R buffer containing 1 M NaCl. Fractions
containing protein were combined and concentrated to 6 ml using
Centriplus-30 concentrators (Amicon), and the total protein was loaded
onto a 600-ml Superdex-75 (Pharmacia) column (2
160 cm)
equilibrated with R buffer containing 1 M NaCl. The column was
run at a flow rate of 0.25 ml/min, and 2.0-ml fractions were collected.
Fractions highly enriched for UmuD`C were combined and concentrated
using Centriplus-30 concentrators to final concentration of at least 1
mg/ml. These fractions were divided into aliquots (100 µl) and
quickly frozen at -70 °C. A final yield of 2-4 mg of
UmuD`C complex was obtained from 30 liters of cells.
Microsequencing was performed
as suggested (31) on one sample, while the other was processed
for Western blot analysis with UmuC antiserum. Amino-terminal analysis
for putative UmuC was performed by Lynn Williams (USC Microchemical
Facilities) and found to be:
NH-MFALXDVNAFYASXE (X represents
an unidentified residue), in agreement with the reported DNA sequence
for UmuC(32, 33) .
Previous attempts to purify UmuC involved extracting insoluble UmuC protein from inclusion bodies, solubilizing in the presence of 8 M urea and subsequently refolding of the purified polypeptide using either S9 ribosomal protein (13) or by sequential incubation with Hsp70 and Hsp60 heat shock chaperones(29) . The effects of denaturation and renaturation on enzymatic and biochemical properties of UmuC are not known. Therefore, a major goal of this study was to purify and characterize a complex containing UmuC and UmuD` (UmuD`C complex) while maintaining the complex in a soluble form throughout purification.
Figure 1: Purification and detection of UmuD`C complex. Fractions from each purification step were separated on a 15% SDS-polyacrylamide gel. A, Coomassie Brilliant Blue G-250-stained purification gel. Lane M, prestained molecular weight markers; lane 1, crude lysate (20 µg of total protein); lane 2, polyethyleneimine extract (20 µg of total protein); lane 3, DEAE pool (20 µg of total protein); lane 4, phosphocellulose pool (10 µg of total protein); lane 5, Superdex 75 concentrated pool (10 µg of total protein). B, Western immunodetection of the proteins in the purification steps. Proteins were separated by 15% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, probed with either UmuC antiserum or UmuD`,D antiserum, and visualized with enhanced chemiluminescence. Lane 1, crude cell lysate; lane 2, polyethylenimine extract; lane 3, DEAE pool; lane 4, phosphocellulose pool; lane 5, Superdex 75 concentrated pool. Five micrograms of each protein fraction were loaded.
In the initial purification step, nucleic acids were precipitated by addition of polyethyleneimine. UmuC and UmuD` coprecipitated with the nucleic acid and were extracted in the presence of 1 M salt and dialyzed into 50 mM NaCl (see ``Experimental Procedures''). The remaining purification steps involved separation on DEAE-Sephacel, phosphocellulose, and Superdex-75 columns (see ``Experimental Procedures''). UmuD`C was present in 1 M NaCl following fractionation on Superdex-75 and attempts to reduce the salt concentration subsequent to gel filtration, e.g. by dialysis, were hampered by the formation of an insoluble protein precipitate containing both UmuC and UmuD`.
The UmuD`C fractions from each stage of purification were
fractionated by SDS-polyacrylamide gel electrophoresis and analyzed by
Western immunodetection (Fig. 1). The UmuD`C complex appeared to
be greater than 80% pure (based on integration of densitometric band
intensities in the linear range) following fractionation with Superdex
75 (Fig. 1A, lane 5). Microsequencing was used to
confirm the true identity of the soluble UmuC protein (see
``Experimental Procedures''): 15 residues identified from the
NH terminus coincided with the published UmuC amino acid
sequence(32, 33) .
Antibodies to both UmuD` and
UmuC showed that at least a fraction of the overproduced UmuD` protein
copurified with UmuC (Fig. 1B). Gel filtration of the
phosphocellulose pool on a Superdex-75 column in 1 M NaCl
failed to separate UmuD` from UmuC and analytical gel filtration using
a Sephadex G100 column in the presence of 1 M NaCl, suggested
that the proteins associated in a complex with a molecular weight of
approximately 70 kDa (Fig. 2). This molecular mass is consistent
with a composition of one UmuC (46 kDa) and two UmuD` (12 kDa)
molecules(13) . The elution volumes of the protein standards (Fig. 2) were determined by running a mixture of the standards
alone and in the presence of the Superdex 75 fraction. It was possible
to purify a small amount of soluble UmuC from RW82F`IQ harboring a
plasmid that overproduced UmuC in the absence of UmuD`. Elution of UmuC
on an analytical Sephadex G100 column gave a value of K slightly larger than that of ovalbumin (43 kDa). The apparent
molecular weight for UmuC in the range of 45-47 kDa is in
agreement with a previous determination (13) .
Figure 2: Size analysis of UmuD`C complex. The purified UmuD`C complex was gel-filtered on a Sephadex G-100 column, as described under ``Experimental Procedures.'' The apparent molecular weights of the UmuD`C protein complex and UmuC protein were estimated based on the migration of protein standards of known molecular weight. The position of gel filtration standards was determined separately from purified protein runs. Ribonuclease A (Ribo A) (13.7 kDa), chymotrypsinogen A (Chy A) (25 kDa), ovalbumin (Ova) (43 kDa), bovine serum albumin (BSA) (67 kDa).
The
properties of UmuC and the UmuD`C complex were investigated using a low
ionic strength native agarose gel. Proteins were resolved by gel
electrophoresis, transferred to nitrocellulose, and incubated with UmuC
antiserum. UmuC is a basic protein having a calculated isoelectric
point of about 9.6 based on amino acid composition. Therefore, UmuC
should migrate toward the cathode in an agarose gel. When UmuC and two
highly acidic UmuD` molecules (pI 4.5) combine to form a UmuD`C
complex, migration should be toward the anode. A fraction of soluble
UmuC, concentrated after purification through a phosphocellulose column
(see ``Experimental Procedures''), migrated toward the
cathode, but only after treatment with RNase (DNase-free) (Fig. 3, lane 3). An untreated sample of UmuC or one
treated with DNase (RNase-free) remained in the wells (lanes 1 and 2, respectively). It appears that in the absence of
UmuD`, and especially when UmuC was extracted from a crude cell lysate
at low salt concentration (
50 mM NaCl), it was tightly
bound to RNA. The UmuD`C complex migrated toward the anode as expected (Fig. 3, lane 4), and this pattern was essentially
unaffected by treatment with DNase or RNase (Fig. 3, lanes 5 and 6, respectively).
Figure 3: Charge-dependent gel mobility of the UmuD`C complex. UmuC protein and UmuD`C protein complexes were electrophoresed on a 0.9% agarose gel and immunodetected with UmuC antiserum. Lane 1, UmuC protein untreated; lane 2, UmuC protein incubated with DNase I (RNase-free); lane 3, UmuC protein incubated with RNase A (DNase-free); lane 4, UmuD`C protein complex untreated; lane 5, UmuD`C protein complex incubated with DNase I (RNase-free); lane 6, UmuD`C protein complex incubated with RNase A (DNase-free).
The results indicate that UmuD`C
bound cooperatively to a single-stranded 600 mer (Fig. 4). No
detectable protein was bound at low UmuD`C/DNA ratios (Fig. 4A, lanes 1-3) while in the ``steep
binding'' range, binding increased from about 1 UmuD`C bound per
300 nucleotides to about 1 per 50 nucleotides. Saturation of the
600-mer occurred when approximately one UmuD`C was bound per 20
nucleotides. Binding of UmuD`C to the ss600-mer resulted in measurable
retardation as a function of increasing UmuD`C/DNA ratios. To
quantitate the binding of UmuD`C to the ss600-mer, gel band intensities
corresponding to free UmuD`C and UmuD`C complexed to DNA were
integrated in the linear region of the film (see ``Experimental
Procedures''). A logarithmic Hill plot was fit by a straight line
with a slope corresponding to a Hill coefficient n = 3 (Fig. 4B). We estimated a value of K = 260 nM for the apparent binding constant of the
UmuD`C complex to the 600-mer.
Figure 4:
Cooperative binding of the UmuD`C protein
complex to an ss600-mer. A, the UmuD`C-600-mer complex was
separated on a 0.9% agarose gel and immunovisualized with UmuC
antiserum. Each lane represents addition of UmuD`C in increasing
increments from 25 to 450 nM added protein complex with a
constant amount of 600-mer (5.4 nM). The last lane on the right-hand side is free UmuD`C complex. B, Hill plot
for the data in A. Y is the amount of bound UmuD`C
for a given input concentration (UmuD`C), Y is the maximum amount of UmuD`C complex bound to the DNA. The
slope of the line fit by linear least squares is the Hill coefficient, n = 3.
The qualitative binding profile of UmuD`C to M13 DNA was similar to that for the 600 mer. There was no detectable binding of UmuD`C to the DNA at low UmuD`C/DNA ratios (Fig. 5, lanes 1-5). Saturation of the M13 DNA (7250 nt) was difficult to quantitate in the gel binding study, caused, in part, by an accelerated migration of the UmuD`C-M13 DNA complex; the maximum clear attainable binding was roughly 1 UmuD`C per 100 nucleotides occurring at an input level of about 1 UmuD`C per 40 nucleotides. Since the binding measurements were carried out using salt concentrations near 50 mM NaCl, the high salt concentration of the UmuD`C sample coupled with the requirement to maintain a low reaction volume limited the amount of of UmuD`C that could be used in attempting to saturate the M13 DNA.
Figure 5: Cooperative binding of the UmuD`C protein complex to ssM13 DNA. The UmuD`C-ssM13 complex was separated on a 0.9% agarose gel and immunovisualized with UmuC antiserum. Each lane represents addition of UmuD`C in increasing increments from 25 to 200 nM added protein complex with constant amount of ssM13 DNA (0.8 nM). The last lane on the right-hand side is free UmuD`C complex. M13 DNA C and L forms on the left-hand side refer to circular and linear forms of ssM13 DNA, respectively.
A more sensitive measurement for the binding of UmuD`C to M13 DNA was performed using fluorescence depolarization, see e.g. Refs. 40 and 43. In this assay, the protein was incubated in the presence of chemically modified M13 DNA containing a random distribution of the fluorescent base ethenoadenine and to a lesser extent ethenocytosine (see ``Experimental Procedures''). Protein-DNA binding was observed as an increase in the steady-state anisotropy of the protein-DNA complex with increasing amounts of UmuD`C. Much more protein can be used in the fluorescence assay because of the increased reaction volume (180 µl) compared with the gel assay (30 µl). We were able to observe a saturation in the fraction of UmuD`C bound to M13 DNA (Fig. 6). The data fit by a Hill plot gives an apparent binding constant in the range of 300-350 nM and a slope of 3, similar to the Hill coefficient determined for the 600-mer. A plot for the increase in fluorescence intensity with increasing UmuD`C overlapped with the anisotropy plot offering independent evidence for the cooperative nature of the binding of UmuD`C to DNA (data not shown). In contrast to the retardation observed when UmuD`C was bound to a 600-mer, binding of UmuD`C to M13 DNA appeared to accelerate migration of the protein-DNA complex on an agarose gel at high UmuD`C/DNA nucleotide ratios (Fig. 7) suggesting that the longer DNA may undergo compaction.
Figure 6: Cooperative binding of the UmuD`C complex to etheno-M13 DNA. Titration of the UmuD`C complex (0.1-1.0 µM) on etheno-M13 DNA (1.0 nM) was performed as described under ``Experimental Procedures.'' The calculation of the fraction of UmuD`C bound to DNA obtained from the raw anisotropy data is described under ``Experimental Procedures.''
Figure 7: Accelerated mobility of the UmuD`C-ssM13 complex in agarose gels. Binding of UmuD`C to ssM13 DNA is shown as a function of DNA concentration. UmuD`C-ss M13 DNA complexes were separated on a 0.9% agarose gel and immunodetected with UmuC antiserum. A constant concentration of UmuD`C (300 nM) complex was used in each lane in which ssM13 DNA concentration (nucleotide ssM13 DNA) was increased as follows: lane 1, 560 nM ssM13 DNA; lane 2, 1.1 µM ssM13 DNA; lane 3, 1.6 µM ssM13 DNA; lane 4, 2.2 µM ssM13 DNA; lane 5, 2.8 µM ssM13 DNA; lane 6, 3.2 µM ssM13 DNA; lane 7, 3.9 µM ssM13 DNA; lane 8; 4.5 µM ssM13 DNA; lane 9, 5 µM ssM13 DNA; lane 10, 5.6 µM ssM13 DNA; lane 11, represents migration of the free UmuD`C complex.
We observed no detectable binding of UmuD`C to ssDNA less than 600
nucleotides long in the agarose gel assay. However, using fluorescence
depolarization we observed binding of UmuD`C to an 80-mer, and in
contrast to the binding of UmuD`C to either the 600-mer or to M13 DNA,
binding to the 80-mer did not appear to be cooperative (data not
shown). The K for binding the 80-mer was near 150
nM, and ATP was not required for protein-DNA binding. We were
unable to detect any significant difference in the binding of UmuD`C to
either UV-irradiated or unirradiated M13 single-stranded DNA using the
agarose gel assay, and binding of UmuD`C to irradiated DNA did not
protect against degradation by T4 endonuclease V (data not shown). No
detectable binding of UmuD`C was observed either to UV-irradiated or
unirradiated double-stranded DNA (data not shown).
Figure 8:
Binding of the UmuD`C complex and RecA*
protein to ssM13 DNA. Binding of UmuD`C to ssM13 DNA in presence of
RecA* is examined using a mobility gel shift assay (0.9% agarose gel)
probed with UmuC antiserum. A, lane 1, UmuD`C (300
nM) complex incubated with DNA in presence of 1 mM ATPS; lane 2: same as lane 1 except that 30 nM UmuD` protein was added to the reaction; lane 3, RecA*
(560 nM) was preincubated with ssM13 DNA (560 nM in
the DNA nucleotide) for 10 min at at 37 °C before the UmuD`C
complex was added to the reaction; lane 4, same as lane 3 except that 30 nM UmuD` protein was added to the
reaction. B, UmuD`C (300 nM) was preincubated with
ssM13 DNA (560 nM in the DNA nucleotide) for 10 min at 37
°C prior to the addition of RecA protein. Lane 1, UmuD`C
complex bound to ssM13 DNA; lane 2, UmuD`C-ssM13 DNA with 46
nM RecA protein; lane 3, UmuD`C complex with 140
nM RecA protein; lane 4, UmuD`C-ssM13 DNA complex
with 560 nM RecA protein. The three levels of RecA protein
used correspond to 1 RecA per 12, 4, and 1 nucleotide(s), respectively.
The position of free ssM13 DNA on the gel was determined by staining
with ethidium bromide (indicated by the arrow).
The UmuD`C-M13 DNA
complex formed in the absence of RecA* caused an acceleration of the
DNA (Fig. 8A, lane 1, Complex I). Under these
conditions, the amount of UmuD`C complex in the reaction was limiting
as all of it appeared to be bound to the ssDNA. Addition of excess
UmuD` to the reaction gave an essentially similar result, although
there was a slight increase in the amount of detectable free UmuD`C
complex (Fig. 8A, lane 2). In contrast, however, when
RecA was preincubated with DNA (in the presence of ATPS) to form a
RecA*-nucleoprotein filament and UmuD`C subsequently added to the
reaction, a retarded complex was observed (Complex II) that is
consistent with it being a UmuD`C-RecA*-DNA complex (Fig. 8A, lane 3). Further addition of excess UmuD` to
the reaction had no measurable effect on the formation of complex II (Fig. 8A, lane 4). It is interesting to note that under
these conditions, where a significant fraction of the ssDNA would be
expected to be bound by RecA*, there was a significant increase in the
amount of free UmuD`C complex (Fig. 8A, lanes 3 and 4). This result suggests that UmuD`C is unable to compete RecA
from ssDNA once a RecA*-nucleoprotein filament has formed.
Preincubation of UmuD`C with DNA followed by the addition of three different levels of RecA produced a series of complexes that were consistent with UmuD`C-DNA complexes associated with variable amounts of RecA protein (Fig. 8B, lanes 2-4). Since UmuD`C remained bound to the DNA following the addition of RecA, and UmuD`C was limiting in the reactions, it is likely that RecA does not compete off the preassembled UmuD`C-DNA complex but simply binds regions free from UmuD`C. The complex formed by preincubating DNA with RecA (Fig. 8A, lanes 3 and 4) migrated more slowly than the complex II formed by preincubation with UmuD`C (Fig. 8B, lanes 2-4), suggesting that prior binding of UmuD`C to DNA limited the ability of RecA to form a nucleoprotein filament.
Previously, Echols and co-workers succeeded in purifying denatured UmuC(13) ; upon renaturation it was shown that UmuC bound ssDNA(29) . Although these results represented an important initial step for reconstituting lesion bypass in vitro(44) , it is necessary to isolate and purify UmuC alone or complexed with UmuD` in soluble form to investigate the full range of biological interactions of these proteins.
We coexpressed the UmuD`C proteins from a ptac promoter and purified the UmuC protein from a strain carrying a deletion of the entire chromosomal umuDC operon. Under these conditions a sizable fraction of a 46-kDa protein was soluble, and this protein was shown to be UmuC by antibody binding and microsequencing.
UmuC remained soluble throughout each purification step and copurified as a tightly bound complex with UmuD` (Fig. 1). The UmuD`C complex with an estimated molecular mass of 70 kDa remained intact during chromatography on Superdex 75 in the presence of 1 M NaCl. These results suggest that this complex is composed of a UmuC monomer (46 kDa) and UmuD` dimer (24 kDa), which are tightly associated.
It was also possible to purify a small amount of soluble UmuC by overproducing UmuC alone in the absence of UmuD`. Free UmuC has an apparent molecular mass of 46 kDa based on gel filtration (Fig. 2), a finding suggesting that it exists as a monomer when in solution(13) . UmuC is a basic protein and should therefore migrate in an electric field toward the cathode in a neutral agarose gel. However, the UmuC fraction obtained from the phosphocellulose column remained in the wells during electrophoresis unless it was first treated with RNaseA (Fig. 3). Following a pretreatment of the phosphocellulose fraction with RNaseA, UmuC migrated toward the cathode (Fig. 3, lane 3), suggesting that free UmuC is bound to RNA in crude cell lysates. UmuC can be released from RNA in the presence of 1 M NaCl (Fig. 2), and this property can be exploited in the future to purify substantially larger quantities of soluble UmuC. Our data suggest that UmuC binds preferentially to UmuD` precluding formation of significant amounts of the UmuC-RNA complex. Pretreatment of the UmuD`C complex with RNase had negligible effect on its migration toward the anode in a neutral agarose gel (Fig. 3, compare lanes 5 and 6).
The binding studies with ssDNA offer potentially important insights into the properties of the UmuD`C complex. Binding of the protein complex, either to a 600-mer fragment of M13 DNA (Fig. 4), or to full-length M13 DNA (Fig. 5), occurred cooperatively with a Hill coefficient of 3. Such cooperativity was observed using two independent measurements: (i) agarose gel electrophoresis and immunodetection of free UmuD`C or UmuD`C complexed with DNA ( Fig. 4and Fig. 5) and (ii) steady-state fluorescence depolarization of ethenoadenine-labeled M13 DNA at increasing concentration of UmuD`C (Fig. 6). The depolarization data were also confirmed by an independent measurement of an increase in the fluorescence of ethenoadenine as a function of increasing UmuD`C concentration (data not shown). As reported previously(29) , in the gel assay UmuD`C did not bind irradiated or unirradiated double-stranded DNA.
Fluorescence properties of etheno-modified DNA have been used successfully to calculate DNA binding parameters of RecA protein (37, 38, 45) and bacteriophage T4-coded Gene 32 protein(46) . Since parameters for most activities attributed to these proteins have been confirmed by other methods, it is believed that higher affinity of binding to etheno-modified DNA over natural ssDNA for RecA and gp32 is caused by a disruption of secondary structure of ssDNA. In our experiments, using two independent techniques, we found that UmuD`C bound cooperatively to both ethenoadenine-modified ssDNA (Fig. 6) and to unmodified ssDNA (Fig. 4) with similar values for the Hill coefficient.
In addition to its ability to interact with free ss DNA, UmuD`C interacts with ssDNA that had been pre-coated with RecA (Fig. 8A). These two properties, direct cooperative binding of UmuD`C to DNA, and binding to a RecA nucleoprotein filament, may help to explain some of the intriguing phenotypes exhibited when the Umu proteins are overexpressed in vivo(26, 27, 28) . Under physiological conditions, the UmuD`C proteins are maintained at low basal levels(25) . Since UmuD` is normally expressed at about 12-fold higher levels than UmuC(25) , most, if not all, of the UmuC is likely to exist as a UmuD`C complex. Thus, even in fully SOS-induced cells there may be only about 200 UmuD`C molecules per cell(25) .
In contrast, under similar conditions, there are likely to be 30,000-50,000 molecules of RecA protein/cell(3, 25) . Thus, under normal cellular conditions, UmuD`C may associate with a RecA nucleoprotein filament, perhaps through an interaction between UmuD`-RecA* (15) or UmuC-RecA*(14) . When a RecA* nucleoprotein filament has formed at a lesion in double-stranded DNA(47) , a UmuD`C-RecA* interaction would provide an efficient way to target Umu proteins to locations within the cell where they are required to facilitate error-prone translesion DNA synthesis(15) .
Under conditions where UmuD`C is moderately overproduced, e.g. when expressed from an operator constitutive mutant on a low copy number plasmid, some UmuD`C might begin to bind directly to regions of ssDNA and inhibit the extent of RecA*-nucleoprotein filament formation. This interaction might be sufficient to inhibit RecA's recombinatorial activities directly (28) while allowing for the formation of a lesion-localized nucleoprotein structure (a putative mutasome) containing RecA, UmuD`C and pol III (5, 13) or perhaps pol II (20, 22) .
The nonmutable phenotype of cells that slightly overexpress UmuC alone (27) may result from increased formation of UmuD`C-DNA complexes at regions of ssDNA generated when a cell attempts to replicate damaged DNA, which may hinder continued replication rather than enhance it and ultimately lead to killing of cells with premutagenic lesions, thereby reducing the number of mutants. Significant overproduction of UmuD`C, e.g. from a multicopy plasmid in fully SOS-induced cells, may result in levels of UmuD`C that can bind directly and cooperatively to regions of ssDNA, even in undamaged cells. Encountering UmuD`C coated DNA rather than E. coli single-stranded binding protein-coated DNA may impede replication and thus result in the rapid cessation of DNA synthesis that is associated with cold sensitivity (26) .
The nature of the interactions between pol III or pol II and the SOS-induced proteins described above, that lead to either error-free or error-prone lesion bypass, remain entirely speculative. Our aim is to elucidate the precise interactions between polymerase holoenzymes, RecA*, UmuD`C, and any additional proteins that facilitate bypass of DNA template lesions by reconstituting an SOS lesion bypass system in vitro. The availability of an extensively purified source of soluble UmuD`C complex is therefore a critical step toward achieving this goal.