From the Department of Biological Chemistry, Weizmann
Institute of Science, Rehovot 76100, Israel and the
§ Laboratoire d'Analyse Ultrastructurale, Batiment de
Biologie, Universite de Lausanne, CH-1015 Lausanne-Dorigny,
Switzerland
Received for publication, July 31, 2000, and in revised form, November 13, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Translesion replication is carried out in
Escherichia coli by the SOS-inducible DNA polymerase V
(UmuC), an error-prone polymerase, which is specialized for
replicating through lesions in DNA, leading to the formation of
mutations. Lesion bypass by pol V requires the SOS-regulated proteins
UmuD' and RecA and the single-strand DNA-binding protein (SSB). Using
an in vitro assay system for translesion replication based
on a gapped plasmid carrying a site-specific synthetic abasic site, we
show that the assembly of a RecA nucleoprotein filament is required for
lesion bypass by pol V. This is based on the reaction requirements for
stoichiometric amounts of RecA and for single-stranded gaps longer than
100 nucleotides and on direct visualization of RecA-DNA filaments by
electron microscopy. SSB is likely to facilitate the assembly of the
RecA nucleoprotein filament; however, it has at least one additional
role in lesion bypass. ATP Genomic DNA is afflicted by numerous lesions that might interfere
with its propagation and with gene expression (1). Most of these
lesions, which are usually base modifications, are repaired by cellular
DNA repair mechanisms (1). When the replication fork encounters a
blocking DNA lesion that has escaped repair, replication stops forming
a ssDNA1 region in DNA (2).
In Escherichia coli at least two mechanisms, which are
regulated by the SOS response (3), act to repair the gap by converting
the ssDNA region into a dsDNA region without actually removing the
damaged nucleotide. Recombinational repair patches the gap with a
complementary DNA segment from the fully replicated sister chromatid
(4, 5), whereas translesion replication fills in the gap by DNA
synthesis. This pathway, also termed lesion bypass or error-prone
repair, is mutagenic, because DNA lesions often cause misincorporation
by DNA polymerases, leading to the formation of mutations (2, 6).
The in vitro reconstitution of SOS translesion replication
with purified components (7-9) established that SOS-targeted
mutagenesis occurs by replication through DNA lesions by DNA polymerase
V (UmuC)2 in the presence of
UmuD', RecA, and SSB (10, 11). Pol V effectively bypasses a synthetic
abasic site (10, 11), a cyclobutyl TT dimer and a 6-4 TT adduct (12),
leading to targeted mutations. When replicating an undamaged DNA
template pol V is highly mutagenic and forms preferentially
purine-purine and pyrimidine-pyrimidine mismatches, resulting in
transversion mutations (13). These activities of pol V are responsible
for SOS mutagenesis targeted to DNA lesions and for untargeted
mutagenesis, which occurs in undamaged DNA regions.
Proteins similar to UmuC are widespread from E. coli to
humans. Several of them were shown to encode DNA polymerases in
bacteria (14), in Saccharomyces cerevisiae (15, 16), and in
humans (reviewed in Refs. 17-19). Unlike pol V, these polymerases do
not require any additional proteins for their polymerase and/or lesion bypass activities, at least in vitro. In this sense pol V is
more similar to the recently discovered DNA polymerase RI, product of
the plasmidic mucB gene, whose activity also requires RecA and SSB (20). Most intriguing is the requirement for RecA, a protein
known to be required also for gap filling by recombinational repair.
Here we show that the assembly of a RecA nucleoprotein filament is
required for translesion synthesis by pol V.
Proteins--
UmuD' and MBP·UmuC (a fusion of UmuC to
maltose-binding protein) were overexpressed and purified as previously
described (8, 11). The degree of purity was estimated to be >95% for
UmuD' and 90-95% for MBP·UmuC. SSB and RecA were purified as
described (21, 22), except that a phosphocellulose purification step was added for RecA. Their degree of purity was estimated to be >95%.
Restriction nucleases, T4 DNA ligase, and T4 polynucleotide kinase were
from New England BioLabs; T7 gp6 exonuclease was from Amersham
Pharmacia Biotech, and ATP DNA Substrates--
The preparation of the gapped plasmid GP21
carrying a site-specific lesion was recently described (23, 24). GP21
contains a site-specific synthetic (tetrahydrofuran) abasic site and a ssDNA region of ~350 nucleotides (see Fig. 1).
Translesion Replication Assay--
The translesion replication
reaction was performed as previously described (8, 11) with minor
changes. The standard reaction mixture (25 µl) contained 20 mM Tris-HCl, pH 7.5, 8 µg/ml bovine serum albumin, 5 mM dithiothreitol, 0.1 mM EDTA, 4% glycerol, 1 mM ATP, 10 mM MgCl2, 0.1 mM each of dATP, dGTP, dTTP, and dCTP, 0.1 µg (2 nM) of gapped plasmid, 50 nM SSB, 2 µM RecA, 500 nM UmuD', and 5-220
nM pol V (MBP·UmuC). Reactions were carried out at
37 °C as follows. RecA and the DNA substrate were incubated in the
assay buffer for 2 min at 37 °C, and were then transferred to ice.
Next SSB and UmuD' were added, and reactions were incubated for 4 additional min at 37 °C. MBP·UmuC was then added, and incubation continued for the indicated periods of time. Reactions were stopped by
adding 20 mM EDTA, 0.2% SDS, and 100 mM NaCl
(final concentrations) followed by heat inactivation (65 °C for 10 min), and the DNA was purified from the proteins by proteinase K
digestion followed by phenol/chloroform extraction and ethanol
precipitation. The purified DNA was digested with Asp700 (5 units) at 37 °C for 2 h. Then, 5 units of BstXI were
added, and incubation was continued at 55 °C for another 2 h.
This restriction yielded radiolabeled DNA fragments 15, 25, and 43 nucleotides long, representing the unextended primer, the replication
stop at the lesion, and the bypass product, respectively.
Alternatively, a modified procedure was used as follows. The purified
DNA mixture was treated with calf intestine alkaline phosphatase (0.2 units, 1 h, 37 °C) to hydrolyze remaining dNTPs (11). The DNA
was then digested with Asp700 (5 units) and
MspA1I (5 units) at 37 °C instead of BstXI. This produced radiolabeled DNA bands which were 4 nucleotides longer
than those obtained with the Asp700/BstXI
cleavage, namely, fragments 19, 29, and 47 nucleotides long,
representing the unextended primer, the replication stop at the lesion,
and the bypass product, respectively (see Fig. 1). The DNA samples were
fractionated by 15% PAGE-urea, followed by phosphorimager analysis
(Fuji BAS 2500). The extent of bypass was calculated by dividing the
amount of bypass products by the amount of the extended primers (lesion bypass). When indicated, the bypass was also calculated differently, by
dividing the amount of bypass products by the total amount of DNA
(molecules bypassed of total).
Electron Microscopy--
Protein-DNA complexes were fixed by
addition of glutaraldehyde to 0.2% followed by a 15-min incubation at
37 °C (complexes stabilized with ATP RecA Is Needed in Stochiometric Amounts for Lesion
Bypass--
The experimental system for assaying lesion bypass was
previously described (8, 23). It is based on a gapped plasmid carrying
a site-specific synthetic abasic site in the ssDNA region and an
internal radiolabeled phosphate in the primer strand (Fig. 1). Upon addition of pol V the 3'-primer
terminus is extended up to the abasic site, where synthesis may
terminate or proceed through the abasic site (lesion bypass). At the
end of the reaction the DNA is isolated and restricted with
MspA1I, which cleaves 4 nucleotides upstream of the
radiolabel, and with Asp700, which cleaves downstream of the
lesion. This yields radiolabeled DNA fragments of 19, 29, and 47 nucleotides long, for the unextended primer, the block at the lesion,
and the bypass product, respectively (Fig. 1). Bands in the length
range of 20-46 nucleotides represent pol V pause or dissociation sites
(see for example Figs. 2 and 6). In some
cases the DNA products were restricted with Asp700 and
BstXI, yielding products which were 4 nucleotides shorter than with the Asp700/MspA1I cleavage. The DNA
were fractionated by urea-PAGE and visualized and quantified by
phosphorimaging.
The activity of the RecA protein in recombination or SOS induction
requires its binding to DNA. This binding is stoichiometric and
cooperative, leading to the formation of a helical RecA filament along
ssDNA, composed of multiple RecA monomers (reviewed in Refs. 26, 27).
We examined whether the formation of a RecA nucleoprotein filament is
required also for lesion bypass. First, a titration experiment was
performed, to determine the amount of RecA needed to saturate the
bypass reaction. As can be seen in Fig. 2, although lesion bypass could
be observed at 0.5 µM RecA, 2 µM RecA was required to reach a saturation level of bypass. Further increases up to
16 µM RecA had little effect on lesion bypass (Fig. 2). For comparison, a titration of pol V was performed. As can be seen in
Fig. 3, bypass activity reached
saturation at 50-100 nM pol V, 20-40-fold lower than the
saturating concentration of RecA. The concentration of the DNA in the
reaction was 2 nM of gapped molecules, which translates
into 700 nM nucleotides in the ssDNA region. Because a
single RecA molecule binds 3-4 nucleotides (26), this means that
~200 nM RecA will be sufficient to cover the ssDNA region. The result that the saturating concentration of RecA is 10-fold
higher than required to cover the ssDNA region is consistent with the
formation of RecA nucleoprotein filament, reflecting the equilibrium
between bound and free RecA. In addition, the RecA filament may extend
to the double-stranded portion of the DNA molecule, as previously shown
(28-30).
Lesion Bypass Requires Single-stranded Gaps That Are Longer than
100 Nucleotides--
The formation of a stable RecA nucleoprotein
filament requires a ssDNA region longer than 50 nucleotides (26). Thus,
if a stable RecA nucleoprotein filament is required for lesion bypass, this should be reflected in a requirement for a long ssDNA region in
the substrate DNA. This prediction was examined by using DNA substrates
with single-stranded regions 12, 100, 350, or 850 nucleotides long.
Fig. 4 shows the results of lesion bypass
with DNA gaps 12 and 350 nucleotides long. In these experiments, the
primer terminus was located opposite the nucleotide preceding the
lesion in the template strand. This configuration of the DNA substrate gave lesion bypass results similar to those obtained with the standard
substrate where the primer terminus was located 11 nucleotides upstream
of the lesion (data not shown). As can be seen in Fig. 4, when the DNA
substrate contained a small gap of 12 nucleotides, no bypass was
observed by pol V, UmuD', RecA, and SSB (Fig. 4, lane 7).
Furthermore, in a control reaction, bypass by pol I was higher than pol
V on this substrate (Fig. 4, lane 2 versus
lane 7). Attempts to examine whether omission of one of the
protein components will allow lesion bypass on this DNA substrate
showed no bypass under all the conditions examined (Fig. 4, lanes
3-6). No bypass was observed even with a 100-nucleotide-long gap
(data not shown). In contrast, effective bypass was observed with gaps of 350 (Fig. 4, lane 14; see also Figs. 2 and 3) or 850 nucleotides long (see Fig. 8 below).
RecA Nucleoprotein Filaments Are Formed under Bypass Reaction
Conditions--
The formation of RecA nucleoprotein filaments under
the conditions of the bypass reaction was examined using electron
microscopy. The components of the reaction were mixed and incubated at
37 °C. Then the mixture was treated with glutaraldehyde to form
protein-protein and protein-DNA cross-links and visualized by electron
microscopy. As can be seen in Fig.
5C, RecA formed a filament
along the ssDNA region in the DNA substrate. The length of the filament
corresponds to the size of the gap in the plasmid. A similar picture
was obtained when both RecA and SSB were present (Fig. 5D)
or when the complete bypass reaction mixture was analyzed (Fig.
5F), although the size of the protein filament is somewhat
larger. This may reflect coverage of the dsDNA adjacent to the gap;
however, binding of RecA was largely restricted to the ssDNA region.
This is not because of a shortage in RecA, because upon addition of
ATP ATP Requirement for SSB in Lesion Bypass--
SSB was previously shown
to stimulate RecA-catalyzed reactions by facilitating the formation of
a RecA nucleoprotein filament (31-33). This occurs at low SSB
concentrations, which favor a limited cooperativity mode of binding to
DNA, leading to the appearance of beads (octamers of SSB) on a string
(ssDNA Ref. 33). To examine whether the SSB requirement in lesion
bypass is related to the loading of RecA on DNA, a titration of SSB was
performed. As can be seen in Fig. 7, the
highest bypass activity was observed at 50 nM SSB.
Increasing SSB concentration above 50 nM caused a slight decrease in bypass activity, followed by a plateau. Electron microscopy analysis of SSB binding to the gapped plasmid under our reaction conditions (50 nM SSB) revealed the appearance of beads on
a string (see Fig. 5B, above). This is consistent
with the notion that under lesion bypass reaction conditions, SSB binds
ssDNA in the limited cooperativity mode and may facilitate the
formation of a RecA nucleoprotein filament.
If the only function of SSB in bypass is to help assemble the RecA
nucleoprotein filament, then bypass should be observed also in the
absence of SSB. Similar situations exist for other RecA-catalyzed
reactions such as strand exchange. This reaction is stimulated by SSB,
but it occurs also in its absence (26). When SSB is absent, however,
the concentration of Mg2+ should be kept low to prevent
secondary structures from interfering with RecA polymerization on DNA
(34). We have therefore performed a preincubation step of RecA and the
DNA, in the absence of SSB, under 1 mM Mg2+.
Then pol V and UmuD' were added, and the concentration of
Mg2+ was raised to 10 mM to enable optimal
activity of the polymerase. No bypass was observed under these
conditions (data not shown). Other experimental protocols that were
carried out included prolonged preincubation times with RecA prior to
the addition of pol V, usage of higher concentrations of RecA, and
addition of ATP Lesion Bypass Does Not Require Stochiometric Coverage of the RecA
Nucleoprotein Filament by UmuD' or UmuD'C--
Plasmon surface
resonance analysis has shown that complexes of UmuD'2C bind
along a RecA nucleoprotein filament (35). This binding is mediated most
likely through UmuD', which was previously shown to interact with RecA
(36). So far, there was no demonstration of a direct binding between
UmuC and RecA. A report that UmuC was retained on a column of
immobilized activated RecA was based on a cell extract as a source of
UmuC, and therefore binding could have been mediated via a third
protein (37). The binding of UmuD'2C along the RecA
nucleoprotein filament may be related to the tendency of multiple
UmuD'2 molecules to form long filaments in crystalline
state (38). This property of UmuD' might be also reflected in its
structure in solution (39). To examine whether such filaments might be
required for lesion bypass, the following experiments were performed.
In addition to the standard substrate with a gap of 350 nucleotides, a
DNA substrate with an extended gap of 850 nucleotides was also
constructed. A stoichiometric binding of UmuD'2 or
UmuD'2C along the RecA·ssDNA complex would be reflected
in a requirement for higher concentrations of these proteins in the
case of the extra large gap. We performed a titration of UmuD' under a
constant concentration of 100 nM UmuC. As can be seen in
Fig. 8, maximal bypass on the 350 nucleotides gap was obtained with 100 nM
UmuD'2. This concentration is equimolar with the
concentration of UmuC on the one hand, but it is also sufficient to
cover the RecA-bound ssDNA region, assuming that one UmuD'2 molecule binds two RecA monomers (Ref. 35; As discussed above, there is
200 nM RecA bound to the ssDNA region). The UmuD' titration was repeated with a gap of 850 nucleotides, in the presence of a 3-fold
higher RecA concentration (because of the larger size of the gap). It
was found that the saturating amount of UmuD'2 was the
same, 100 nM (Fig. 8), significantly less than the amount required to cover the RecA nucleoprotein filament (at least 240 nM). Moreover, the extent of maximal bypass was similar
with both DNA substrates (Fig. 8). This suggests that under our assay
conditions, binding of either UmuD'2 or UmuD'2C
along the RecA nucleoprotein filament is not required for lesion
bypass.
Two of the most striking features of the activity of pol V are the
ease with which it bypasses lesions that severely block other DNA
polymerases and the absolute requirement for RecA (8-12). As discussed
before, RecA is known to be required for another DNA damage tolerance
mechanism, namely recombinational repair. Numerous studies have
documented the binding of RecA to DNA in the form of a long helical
nucleoprotein filament, with RecA wrapped around the DNA (reviewed in
Refs. 27, 40). The following results argue that the assembly of a RecA
nucleoprotein filament is required for lesion bypass by pol V. 1) RecA
is required at stoichiometric concentrations in the lesion bypass
reaction. 2) Electron microscopy analysis showed the existence of RecA
nucleoprotein filaments under our assay conditions. 3) No bypass was
found with DNA substrates in which the lesion was located on a ssDNA
100 nucleotides or shorter, on which a stable RecA filament is
difficult to assemble.
On gapped DNA, the assembly of the RecA nucleoprotein filament starts
in the ssDNA region, and proceeds in the 5' In the presence of ATP SSB is known to facilitate the formation of RecA nucleoprotein
filaments by removing secondary structures from DNA (27, 40). This
occurs in the beaded limited cooperativity mode of binding of SSB to
DNA. In this mode, octamers of SSB bind ssDNA forming a beaded
structure, representing a binding mode of 65 nucleotides per SSB
octamer (43, 44). Such binding allows entry sites for RecA, which then
forms filaments via cooperative binding, with the displacement of SSB.
Electron micrograph analysis under our reaction conditions revealed the
appearance of complexes (Fig. 5B), which were consistent
with the beaded structure of SSB·DNA complexes reported by Griffith
(43). This supports a role for SSB in facilitating the formation of the
RecA nucleoprotein filament required for lesion bypass. We were unable
to observe lesion bypass in the absence of SSB. This means one of two
possibilities, which are not mutually exclusive. 1) The RecA
nucleoprotein filament in the presence of SSB is different from the
filament of RecA alone, or 2) SSB has an essential role other than the
loading of RecA on DNA. It is possible that mixed RecA·SSB complexes
with DNA are required for lesion bypass. Such mixed complexes were previously described (45).
The importance of the proper assembly of a RecA nucleoprotein filament
is likely to be the reason for the difference between our results and
those of Goodman and co-workers (10). Whereas in our system only UmuC,
UmuD', RecA and SSB were required for lesion bypass (11), in their
system the processivity subunits of DNA polymerase III holoenzyme (the
The inhibitory effect of ATP The RecA nucleoprotein filament is a tool used by the cell to protect
the integrity of its DNA when replication is blocked at DNA lesions and
single-stranded regions are exposed. Coating of the ssDNA with RecA
protects it from degradation on the one hand (46) and provides the
platform for repairing the gap by recombinational repair and
translesion synthesis, on the other hand. Having all these activities
require a common intermediate, i.e. the RecA nucleoprotein
filament, offers an obvious effective mode of regulation and
coordination of these processes. The relationship between
recombinational repair and translesion synthesis is not clear. The two
mechanisms might compete with each other or there might be a switching
mechanism that governs the activities of the two pathways. In this
context it should be mentioned that pol V itself was proposed to switch
the cellular repair systems from recombination to bypass, by inhibiting
recombinational repair (47, 48).
Based on current knowledge, the following model can be drawn for
translesion replication (Fig. 9): When a
replication fork encounters a blocking lesion, the DNA polymerase
stops. At that stage the RecA protein displaces SSB from DNA, forming a
RecA nucleoprotein filament. This nucleoprotein filament serves as a
substrate for DNA polymerase V. The interaction between pol V and RecA
is mediated most likely by UmuD'2, which interacts both
with RecA and pol V. Pol V then replicates across the damaged site,
incorporating a limited number of nucleotides. Because replication of
undamaged regions of the DNA is both inefficient (because of the low
processivity of pol V) and mutagenic, there is a switch back to
error-free and efficient replication by DNA polymerase III holoenzyme.
Obviously, this model provides only a preliminary scenario for
translesion replication by pol V. Further studies are needed to
elucidate the detailed mechanism of action of this fascinating DNA
polymerase.
S, which is known to strongly increase
binding of RecA to DNA, caused a drastic inhibition of pol V activity.
Lesion bypass does not require stoichiometric binding of UmuD' along RecA filaments. In summary, the RecA nucleoprotein filament,
previously known to be required for SOS induction and homologous
recombination, is also a critical intermediate in translesion replication.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
S was from Sigma.
S did not require fixation).
Samples were diluted and washed in 5 mM magnesium acetate
before uranyl acetate staining, as previously described (25). Complexes
were visualized at magnifications of × 20,500 using a Philips
CM100 electron microscope.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (15K):
[in a new window]
Fig. 1.
Outline of the translesion replication assay
using a gap-lesion plasmid. The gap-lesion plasmid substrate is
represented above with restriction sites as indicated.
Products of the lesion bypass assay following cleavage by restriction
nucleases are represented below. Filled
rectangle, synthetic abasic site; filled circle,
radiolabeled phosphate. See text for details.
View larger version (31K):
[in a new window]
Fig. 2.
Effect of RecA concentration on lesion bypass
by pol V. Reactions were performed with the gap-lesion plasmid
GP21, 100 nM pol V (as MBP·UmuC), 1.25 µM
UmuD'2, 600 nM SSB tetramers, and the indicated
concentrations of RecA, for 4 min at 37 °C. Restriction of the DNA
products prior to PAGE analysis was done with Asp700 and
MspA1I. The details are described under "Materials and
Methods." Upper panel, phosphorimage of the gel.
Lower panel, quantification of the results shown in the
phosphorimage.
View larger version (36K):
[in a new window]
Fig. 3.
Effect of pol V concentration on lesion
bypass. Reactions were performed as described in the legend to
Fig. 2, except that RecA was at 4 µM, MBP·UmuC was at
the indicated concentrations, and reaction time was 8 min. Restriction
of the DNA products prior to PAGE analysis was done with
Asp700 and BstXI. The details are described under
"Materials and Methods." Upper panel, phosphorimage of
the gel. Lower panel, quantification of the results shown in
the phosphorimage.
View larger version (71K):
[in a new window]
Fig. 4.
Pol V is unable to perform translesion
synthesis on a substrate with a 12-nucleotide gap. The gapped
plasmid substrates contain a primer ending one nucleotide before the
lesion and gaps of 12 or 350 nucleotides long. Reactions were performed
with the indicated combinations of proteins for 8 min. The
concentrations of the proteins were: MBP·UmuC, 220 nM;
UmuD'2, 1.25 µM; RecA, 4 µM;
SSB, 600 nM (as tetramers); and pol I, 90 nM.
Restriction of the DNA products prior to PAGE analysis was done with
Asp700 and BstXI. The details are described under
"Materials and Methods." The phosphorimage of the gel is
presented.
S, all DNA molecules were entirely covered with RecA (Fig.
5E).
View larger version (210K):
[in a new window]
Fig. 5.
Electron microscopy analysis of complexes
formed between the gap-lesion plasmid and protein components of the
translesion replication reaction. A, the gap-lesion plasmid
GP21. B, GP21 incubated with 50 nM (as
tetramers) SSB. C, GP21 incubated with 2 µM
RecA. D, GP21 incubated with 2 µM RecA and 50 nM SSB. E, same as D but with the
addition of ATP S. F, GP21 incubated under standard
bypass conditions with 100 nM pol V, 500 nM
UmuD'2, 2 µM RecA, and 50 nM SSB.
Samples shown in A-D and F were treated after
incubation with glutaraldehyde, whereas the sample shown in
E was fixed with ATP
S, without glutaraldehyde
cross-linking. The bar represents 100 nm.
S Inhibits Lesion Bypass--
ATP
S is a nonhydrolyzable
ATP analog, which is known to strongly stabilize RecA nucleoprotein
filaments (26). When lesion bypass was examined with ATP
S instead of
ATP, there was a strong reduction in lesion bypass (Fig.
6, lanes 5-7). This effect of ATP
S was also observed in the presence of ATP (Fig. 6, lanes 11-13). Lesion bypass was calculated in this case by two methods. 1) The usual measure of lesion bypass was calculated as the percentage of bypass products of the primers that were extended. This measures lesion bypass directly, correcting for the efficiency of initiation of
synthesis at the primer terminus. Molecules bypassed of
total is the percentage of molecules on which bypass has occurred.
For a substrate in which the primer terminus is located several
nucleotides upstream to the lesion, this will be lower than lesion
bypass, because usually not all primers are extended. As can be see in Fig. 6, the effect of ATP
S is primarily on the initiation of DNA
synthesis, as indicated by the decrease in the fraction of primers that
were extended, and by the molecules bypassed of total (Fig.
6, lanes 2-4 versus lanes 5-7). Additionally,
it seems that the bypass step itself is also inhibited, but the effect
is considerably smaller (Fig. 6; lesion bypassed, %). In
the presence of ATP
S, the RecA filaments extend to cover the entire
DNA molecule under our reaction conditions (see Fig. 5E,
below). Therefore, the inhibition of pol V by ATP
S can be
because of reduced accessibility to the primer terminus caused by the
tight RecA·DNA complex. Alternatively, the altered conformation of
the RecA nucleoprotein in the presence of ATP
S may be inhibitory.
Lesion bypass was also observed in the absence of either ATP or ATP
S
(Fig. 6, lanes 8-10), presumably because of the presence of
dATP. This is consistent with previous results showing that dATP can
replace ATP as a cofactor for RecA activities (26).
View larger version (69K):
[in a new window]
Fig. 6.
Translesion replication by pol V in the
presence of ATP S. Reactions were
performed with the gap-lesion GP21, 90 nM pol V, 500 nM UmuD', 50 nM SSB (as tetramers), 2 µM RecA, and 1 mM ATP or ATP
S for 2, 4, and 6 min. In lanes 11-13, ATP
S was added from the
beginning of the RecA·DNA preincubation, and ATP was added with the
addition of pol V. Restriction of the DNA products prior to PAGE
analysis was done with Asp700 and MspA1I. The
details are described under "Materials and Methods." Bypass
activity was calculated in two ways: 1)as a percentage of the extended
primers (lesion bypass) or 2) as a percentage of total
substrate molecules (molecules bypassed out of total). The
value for percentage of primers initiated (of total substrate
molecules) is also presented.
View larger version (10K):
[in a new window]
Fig. 7.
Effect of SSB concentration on lesion bypass
by pol V. Reactions were performed with the gap-lesion plasmid
GP21, 100 nM pol V, 1.25 µM UmuD', 4 µM RecA, and the indicated concentrations of SSB for 4 min. The graph shows the results of the translesion replication
assay.
S instead of ATP. Under any of these conditions no
bypass was observed in the absence of SSB (data not shown). In an
attempt to examine whether a RecA nucleoprotein filament was formed in
the absence of SSB, we assayed strand exchange between the gapped
plasmid and a homologous DNA fragment. We found that RecA promoted
strand exchange in the absence of SSB under the same conditions in
which no lesion bypass was
observed.3 This indicates
that a functional RecA nucleoprotein filament was formed in the absence
of SSB. Therefore, it seems that facilitating the formation of a RecA
nucleoprotein filament is not the only role for SSB in lesion bypass.
View larger version (21K):
[in a new window]
Fig. 8.
Titration of UmuD' required to saturate
lesion bypass on DNA with large gaps. Reactions were performed on
gap-lesion plasmids with a 350-nucleotide gap (GP21) or a
850-nucleotide gap (GP21XL) with 100 nM pol V and the
indicated concentrations of UmuD' for 8 min. For GP21, 2 µM RecA and 50 nM SSB were added, whereas for
GP21XL 6 µM RecA and 150 nM SSB were added.
, GP21XL;
, GP21.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3' direction (41). It can
then continue to assemble on the double-stranded portion of the DNA
(28-30). Based on the amounts of DNA and RecA, there is enough RecA to
coat the DNA entirely. This is indeed seen in the electron micrograph
in the presence of ATP
S (Fig. 5E). However, under
standard reaction conditions, the ssDNA region is covered with RecA
whereas the dsDNA region remains largely uncoated. This is not because
of the presence of UmuC and UmuD', because the same results were
obtained in their absence. Glutaraldehyde effectively cross-links
proteins to proteins and to ssDNA, but not to dsDNA. However,
glutaraldehyde treatment does fix the RecA filament on dsDNA, probably
because of the special structure of the protein-DNA complex (42). Thus,
it seems that the electron micrograph results showing the dsDNA largely
uncoated, reflect the situation in solution under reaction conditions.
S, where the entire DNA molecule was covered
with RecA, there was a strong inhibition of the activity of pol V,
primarily because of a 10-fold reduction in initiation of synthesis.
This inhibition of initiation by pol V may have been caused by the
tight and extended binding of RecA to DNA in the presence of ATP
S,
leading to limited accessibility of pol V to the primer terminus. In
addition, the inhibition may have been caused, at least in part, by the
altered conformation of RecA filaments in the presence of ATP
S. The
ATP
S experiments provide some hints on the dynamics of RecA during
the action of pol V. A priori it is possible that the
dissociation of RecA takes place only after the polymerization/bypass
step. RecA stretches and unwinds the DNA and upon this the ssDNA in the
RecA nucleoprotein filament is partially exposed (4, 5). Thus, the
access of the polymerase to ssDNA might not be hindered in the presence of RecA. Nevertheless, the inhibitory effect of ATP
S on bypass is
consistent with a model in which polymerization requires dissociation of RecA from DNA (Fig. 6).
-subunit and the five subunit
complex) were also required (10).
This is not likely to be caused by a contamination of the 6 proteins in
our protein preparations, which were highly purified. The extra
requirements stem, in our view, from the difficulty in assembling a
stable and functional RecA nucleoprotein filament on the particular
substrate used by Goodman and his co-workers (10). They used a linear
DNA substrate, with the lesion located on a single-stranded region, 50 nucleotides from the 5'-end of the DNA. Because RecA filaments assemble
on DNA in a polar 5'
3' direction (41), this relatively short stretch of ssDNA 5' to the lesion may be incompletely covered with RecA, or
else the complex may be unstable at the primer terminus. Addition of
the processivity proteins presumably stabilized the protein-DNA complex, enabling bypass. This interpretation is supported by their
observation that the requirement for processivity proteins was
alleviated when ATP
S was used instead of ATP (12). In that case, the
presence of ATP
S presumably promoted formation of a stable RecA
filament, which could enable bypass to a low extent. Nevertheless,
whereas not absolutely required for lesion bypass, the processivity
proteins might have a stimulatory effect on pol V.
S on bypass described above seems to
contradict the report of Goodman and co-workers (12), who found that
ATP
S stimulated bypass by pol V. This difference stems, most likely,
from the difference in the absolute extents of bypass in the two
systems. Goodman and co-workers (12) report a stimulation of bypass
from a state of no bypass to low bypass, whereas we observed an
inhibition from high bypass to low bypass (Fig. 6). Because no
quantification was given in their paper for bypass in the presence of
ATP
S, it is difficult to make a precise comparison. However, in both
laboratories the bypass in the presence of ATP
S was low.
View larger version (24K):
[in a new window]
Fig. 9.
A model for translesion replication by pol
V. The gray rectangle represents the DNA lesion, and
M is an incorrect nucleotide inserted opposite the lesion by
pol V. See text for details.
![]() |
FOOTNOTES |
---|
* This research was supported by Grants 96-00448 and 1999141 from the United States-Israel Binational Science Foundation. Grant 31-58841.99 from the Swiss National Science Foundation, and a grant from the Human Science Frontiers Program (to A. S. and A. Z. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Incumbent of The Maxwell Ellis Professorial Chair in Biomedical Research. To whom all correspondence should be addressed: Dept. of Biological Chemistry, Weizmann Inst. of Science, Rehovot 76100, Israel. Tel.: 972-8-934-3203; Fax: 972-8-934-4169; E-mail: zvi.livneh@weizmann.ac.il.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M006828200
2 We define UmuC as pol V, whereas Goodman and co-workers (10, 12) define the UmuD'2C as pol V. Until the function of UmuD' is elucidated, we prefer to avoid including UmuD' as part of the polymerase. Notice that UmuD' has no effect on the intrinsic polymerase activity of UmuC (in the absence of RecA and SSB, Ref. 11).
3 A. Berdichevsky and Z. Livneh, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ssDNA, single-stranded DNA;
SSB, single-strand DNA-binding protein;
MBP, maltose-binding protein;
ATPS, adenosine
5'-O-thiotriphosphate;
PAGE, polyacrylamide gel
electrophoresis.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Friedberg, E. C., Walker, G. C., and Siede, W. (1995) DNA Repair and Mutagenesis , ASM Press, Washington, D. C. |
2. | Livneh, Z., Cohen-Fix, O., Skaliter, R., and Elizur, T. (1993) Crit. Rev. Biochem. Mol. Biol. 28, 465-513[Abstract] |
3. | Little, J. W., and Mount, D. W. (1982) Cell 29, 11-22[Medline] [Order article via Infotrieve] |
4. | Eggleston, A. K., and West, S. C. (1996) Trends Genet. 12, 20-26[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Cox, M. M.
(1998)
Genes Cells
3,
65-78 |
6. | Walker, G. C. (1995) Trends Biochem. Sci. 20, 416-420[CrossRef][Medline] [Order article via Infotrieve] |
7. | Rajagopalan, M., Lu, C., Woodgate, R., O'Donnell, M., Goodman, M., and Echols, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10777-10781[Abstract] |
8. | Reuven, N. B., Tomer, G., and Livneh, Z. (1998) Mol. Cell 2, 191-199[Medline] [Order article via Infotrieve] |
9. |
Tang, M.,
Bruck, I.,
Eritja, R.,
Turner, J.,
Frank, E. G.,
Woodgate, R.,
O'Donnell, M.,
and Goodman, M. F.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9755-9760 |
10. |
Tang, M.,
Shen, X.,
Frank, E. G.,
O'Donnell, M.,
Woodgate, R.,
and Goodman, M. F.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
8919-8924 |
11. |
Reuven, N. B.,
Arad, G.,
Maor-Shoshani, A.,
and Livneh, Z.
(1999)
J. Biol. Chem.
274,
31763-31766 |
12. | Tang, M., Pham, P., Shen, X., Taylor, J. S., O'Donnell, M., Woodgate, R., and Goodman, M. F. (2000) Nature 404, 1014-1018[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Maor-Shoshani, A.,
Reuven, N. B.,
Tomer, G.,
and Livneh, Z.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
565-570 |
14. | Wagner, J., Gruz, P., Kim, S. R., Yamada, M., Matsui, K., Fuchs, R. P. P., and Nohmi, T. (1999) Mol. Cell 4, 281-286[Medline] [Order article via Infotrieve] |
15. | Nelson, J. R., Lawrence, C. W., and Hinkle, D. C. (1996) Nature 382, 729-731[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Johnson, R. E.,
Prakash, S.,
and Prakash, L.
(1999)
Science
283,
1001-1004 |
17. |
Johnson, R. E.,
Washington, M. T.,
Prakash, S.,
and Prakash, L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12224-12226 |
18. |
Woodgate, R.
(1999)
Genes Dev.
13,
2191-2195 |
19. |
Friedberg, E. C.,
Feaver, W. J.,
and Gerlach, V. L.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5681-5683 |
20. |
Goldsmith, M.,
Sarov-Blat, L.,
and Livneh, Z.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
11227-11231 |
21. | Lohman, T. M., and Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603[Abstract] |
22. |
Cox, M. M.,
McEntee, K.,
and Lehman, I. R.
(1981)
J. Biol. Chem.
256,
4676-4678 |
23. |
Tomer, G.,
Reuven, N. B.,
and Livneh, Z.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14106-14111 |
24. | Tomer, G., and Livneh, Z. (1999) Biochemistry 38, 5948-5958[CrossRef][Medline] [Order article via Infotrieve] |
25. | Stasiak, A. Z., Larquet, E., Stasiak, A., Muller, S., Engel, A., Van Dyck, E., West, S. C., and Egelman, E. H. (2000) Curr. Biol. 10, 337-340[CrossRef][Medline] [Order article via Infotrieve] |
26. | Roca, A. I., and Cox, M. M. (1990) Crit. Rev. Biochem. Mol. Biol. 25, 415-456[Medline] [Order article via Infotrieve] |
27. | Kowalczykowski, S. C., Dixon, D. A., Eggleston, A. K., Lauder, S. D., and Rehrauer, W. M. (1994) Microbiol. Rev. 58, 401-465[Abstract] |
28. |
Shaner, S. L.,
and Radding, C. M.
(1987)
J. Biol. Chem.
262,
9211-9219 |
29. |
Shaner, S. L.,
Flory, J.,
and Radding, C. M.
(1987)
J. Biol. Chem.
262,
9220-9230 |
30. | Lindsley, J. E., and Cox, M. M. (1989) J. Mol. Biol. 205, 695-711[Medline] [Order article via Infotrieve] |
31. | Flory, J., and Radding, C. M. (1982) Cell 28, 747-756[Medline] [Order article via Infotrieve] |
32. |
Cox, M. M.,
Soltis, D. A.,
Livneh, Z.,
and Lehman, I. R.
(1983)
J. Biol. Chem.
258,
2577-2585 |
33. | Kowalczykowski, S. C., and Krupp, R. A. (1987) J. Mol. Biol. 193, 97-113[Medline] [Order article via Infotrieve] |
34. | Tsang, S. S., Muniyappa, K., Azhderian, E., Gonda, D. K., Radding, C. M., Flory, J., and Chase, J. W. (1985) J. Mol. Biol. 185, 295-309[Medline] [Order article via Infotrieve] |
35. |
Rehrauer, W. M.,
Bruck, I.,
Woodgate, R.,
Goodman, M. F.,
and Kowalczykowski, S. C.
(1998)
J. Biol. Chem.
273,
32384-32387 |
36. |
Frank, E. G.,
Hauser, J.,
Levine, A. S.,
and Woodgate, R.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8169-8173 |
37. | Freitag, N., and McEntee, K. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8363-8367[Abstract] |
38. | Peat, T. S., Frank, E. G., McDonald, J. P., Levine, A. S., Woodgate, R., and Hendrickson, W. A. (1996) Nature 380, 727-730[CrossRef][Medline] [Order article via Infotrieve] |
39. | Peat, T. S., Frank, E. G., McDonald, J. P., Levine, A. S., Woodgate, R., and Hendrickson, W. A. (1996) Structure 4, 1401-1412[Medline] [Order article via Infotrieve] |
40. | Roca, A. I., and Cox, M. M. (1997) Prog. Nucleic Acid Res. Mol. Biol. 56, 129-223[Medline] [Order article via Infotrieve] |
41. |
Register, J., III,
and Griffith, J.
(1985)
J. Biol. Chem.
260,
12308-12312 |
42. | Stasiak, A., Stasiak, A. Z., and Koller, T. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 745-749[Medline] [Order article via Infotrieve] |
43. | Griffith, J. D., Harris, L. D., and Register, J., III (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 553-559[Medline] [Order article via Infotrieve] |
44. | Lohman, T. M., and Ferrari, M. E. (1994) Annu. Rev. Biochem. 63, 527-570[CrossRef][Medline] [Order article via Infotrieve] |
45. | Muniyappa, K., Williams, K., Chase, J. W., and Radding, C. M. (1990) Nucleic Acids Res. 18, 3967-3973[Abstract] |
46. |
Williams, J. G.,
Shibata, T.,
and Radding, C. M.
(1981)
J. Biol. Chem.
256,
7573-7582 |
47. | Sommer, S., Bailone, A., and Devoret, R. (1993) Mol. Microbiol. 10, 963-971[Medline] [Order article via Infotrieve] |
48. | Boudsocq, F., Campbell, M., Devoret, R., and Bailone, A. (1997) J. Mol. Biol. 270, 201-211[CrossRef][Medline] [Order article via Infotrieve] |