From the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706
Received for publication, December 18, 2002, and in revised form, February 10, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The nucleation step of Escherichia
coli RecA filament formation on single-stranded DNA (ssDNA) is
strongly inhibited by prebound E. coli ssDNA-binding
protein (SSB). The capacity of RecA protein to displace SSB is
dramatically enhanced in RecA proteins with C-terminal deletions. The
displacement of SSB by RecA protein is progressively improved when 6, 13, and 17 C-terminal amino acids are removed from the RecA protein
relative to the full-length protein. The C-terminal deletion mutants
also more readily displace yeast replication protein A than does
the full-length protein. Thus, the RecA protein has an inherent and
robust capacity to displace SSB from ssDNA. However, the displacement
function is suppressed by the RecA C terminus, providing another
example of a RecA activity with C-terminal modulation. RecA RecA protein catalyzes homologous DNA pairing and strand exchange
reactions that are at the heart of recombination processes in all
cells. Strand exchange is initiated when RecA binds to single-stranded
DNA (ssDNA)1 within a gap or
an ssDNA terminal extension. RecA protein binds DNA in at least two
steps. The first is a slow nucleation step (1-3), and this is followed
by a rapid, cooperative binding of additional monomers to lengthen the
filament uniquely in a 5' to 3' direction (4, 5). The resulting
RecA-ssDNA complex has an extended, helical conformation, with ~6
RecA monomers and 18 nucleotides (nt) of DNA per right-handed helical
turn (18.6 base pairs per turn in RecA filaments with bound ATP The ssDNA-binding protein from Escherichia coli, SSB,
affects formation of the nucleoprotein filament in several ways.
In vitro, under standard reaction conditions that generally
include 8-12 mM Mg2+, SSB stimulates filament
formation on ssDNA substrates derived from bacteriophages by binding to
and denaturing regions of secondary structure in the ssDNA that would
otherwise hinder RecA filament extension (7). SSB is then displaced by
the growing RecA filament (8). SSB thus permits the formation of a
contiguous extended filament on the DNA. However, RecA and SSB bind
ssDNA competitively in vitro, such that when SSB is prebound
to ssDNA, it inhibits the nucleation stage of RecA protein filament
formation (9, 10). Subsequent binding of RecA to ssDNA results either
when SSB transiently vacates a region of ssDNA or when the RecOR
mediator proteins facilitate RecA nucleation onto SSB-coated ssDNA (5, 11, 12).
Genetic studies also indicate that SSB inhibits RecA filament formation
and subsequent homologous recombination. Mutations in recF,
recO, or recR genes, which belong to the same
epistasis group, result in defects in the repair of stalled replication forks (13-18). These defects are probably due at least in part to the
inability of RecA protein to displace SSB from the single-stranded region of the stalled fork. In support of this model, overproduction of
SSB leads to a sensitivity to UV-inflicted DNA damage that is similar
to the phenotype of recF mutants (19). Additional evidence
supports a competition between RecA and SSB for ssDNA in
vivo. The recF, recO, or recR
phenotypes can be partially suppressed, and the suppressor mutations
map to the recA gene. These recA mutants include
recA803 (V37M) (20), recA2020 (T121I) (21, 22),
recA441 (E38K/I298V) (23-25), and recA730 (E38K)
(26, 27). These mutant RecA proteins would need to be able to
compensate in some way for the loss of the RecFOR proteins, and indeed
RecA803, RecA441, and RecA E38K proteins all exhibit an enhanced
ability to compete with SSB in vitro (8, 28). RecA E38K
protein competes best with SSB, followed by RecA441 and then RecA803
(8).
SSB protein has multiple DNA binding modes, and interconversion between
them is mediated by salt concentrations (29-31). At relatively low
divalent salt concentrations (10-100 µM), SSB binds with
a site size of 35 nt and exhibits a smooth contour in the electron
microscope. At higher salt concentrations, SSB transitions to binding
modes with site sizes of 56 (1-10 mM salt) and then 65 nt
(0.1-1 M salt). The SSB65 binding mode
is characterized by a beaded appearance in the electron
microscope. The cooperativity of SSB binding to ssDNA is
enhanced at the lower salt concentrations.
The RecA protein of Escherichia coli
(Mr 37,842) has a structure featuring three
distinct domains (32-34). The core domain (residues 31-269) includes
the ATP and DNA binding sites. The core is flanked by smaller N- and
C-terminal domains. The C-terminal domains (residues 270-352) appear
as distinct lobes on the surface of RecA filaments, which shift
position markedly in response to the presence of different bound
nucleotides (35, 36). The far C terminus of the RecA protein (defined
here as the C-terminal 25 amino acid residues) exhibits a preponderance
of negatively charged amino acids, with seven Glu or Asp residues in
the terminal 17 residues. This function of this region has been
explored with the use of C-terminal deletions (37-42). Several of
these studies documented the in vitro effects of deleting
all or most of the C-terminal 25 amino acid residues. The major effects
were an improvement of binding to dsDNA (37-39) and an evident
alteration of the conformation of the core domain (42).
To more systematically explore the function of the C terminus,
C-terminal deletions removing 6, 13, and 17 amino acids were constructed and characterized in detail (43, 44). The truncated proteins dramatically alter the pH-reaction profile for DNA strand exchange (43) and exhibit a progressive reduction in the requirement for free Mg2+ in the same reaction (44). For the RecA Proteins and Biochemicals--
E. coli SSB was
purified as described (47). SSB was stored in a buffer containing 20 mM Tris-HCl (pH 8.3), 1 mM EDTA, 50% glycerol,
1 mM DNA Substrates--
All DNA concentrations are given in terms of
total nucleotides. Poly(dT) was purchased from Amersham Biosciences,
and the approximate average length is 229 nt. The concentration of
poly(dT) was determined by UV absorption at 260 nm using an extinction coefficient of 8.73 mM ATP Hydrolysis Assays--
A coupled spectrophotometric enzyme
assay (53, 54) was used to measure the ssDNA-dependent
ATPase activities of the wild-type RecA, RecA
The reactions were carried out at 37 °C in 25 mM
Tris-OAc (80% cation), 1 mM dithiothreitol, 3 mM potassium glutamate, 5% (w/v) glycerol, an ATP
regeneration system (10 units/ml pyruvate kinase, 1.92 mM
phosphoenolpyruvate), and a coupling system (3 mM NADH and
10 units/ml lactate dehydrogenase). The concentrations of DNA (M13mp8
ssDNA or poly(dT)), RecA protein (wild-type RecA, RecA Experimental Rationale--
The goal of these experiments was to
compare the abilities of wild-type RecA and C-terminally truncated RecA
mutant proteins to compete with SSB for binding to ssDNA. RecA protein
hydrolyzes ATP when bound to DNA, and the ATPase rate is proportional
to the amount of RecA bound under most conditions (7, 53, 55). The rate
of ATP hydrolysis can be measured using a coupled enzyme assay
described previously (53, 54). Although indirect, this technique has
been used extensively to measure RecA binding to DNA (3, 5, 53, 56).
Importantly, this assay was also shown to represent a reliable and real
time method to monitor SSB displacement by RecA when SSB is prebound to
ssDNA (8). In that study, the time-dependent increase in
the rate of ATP hydrolysis was shown to correlate with the displacement
of SSB, as measured using the change in the fluorescence of SSB upon
dissociation from DNA.
SSB Competition with Wild-type RecA Protein for Binding to M13mp8
ssDNA--
The outcome of the competition between SSB and RecA for
binding to circular bacteriophage M13mp8 ssDNA strongly depends on the
relative concentrations of the proteins, on which protein is
preincubated with the ssDNA, and on the Mg2+ concentration
(7, 9). As Fig. 1 illustrates, when the
RecA protein is present at concentrations stoichiometric with its
available DNA binding sites (3 nt of ssDNA/RecA monomer), RecA
displaces prebound SSB from M13mp8 ssDNA quite slowly at 3 mM Mg2+. This Mg2+ concentration is
stoichiometric to the ATP concentration used in the experiment. At 10 mM Mg2+, the SSB is displaced much more quickly
upon the RecA addition. If, however, RecA is preincubated with the
M13mp8 ssDNA at 3 nt/monomer, some RecA protein is gradually displaced
in the presence of SSB, but the initial rates of ATP hydrolysis
indicate a high level of RecA binding. The rates decline somewhat with
time but remain much higher than the initial rates observed when SSB
was preincubated with the ssDNA. RecA protein again competes with SSB
somewhat better at 10 mM Mg2+ than at 3 mM Mg2+. The decline in rates indicates that
the DNA is not completely bound by RecA when RecA is present at these
stoichiometric levels, affording SSB significant access to the DNA.
When the RecA concentration is increased 50% (2 nt/monomer), so that
the excess RecA ensures coverage of the DNA, RecA is not displaced by
SSB when the RecA is bound to the DNA prior to the SSB (Fig. 1).
SSB Competes with Wild-type RecA Protein for Binding to Poly(dT)
ssDNA--
A complication in the study of SSB effects on wild-type
RecA binding to M13mp8 ssDNA at various Mg2+ concentrations
is that SSB can remove Mg2+-induced secondary structure in
M13mp8 ssDNA, which otherwise inhibits RecA binding (7, 57). Thus, SSB
has a stimulatory effect on RecA filament formation as well as a
competitive, inhibitory effect. In order to eliminate this stimulatory
contribution from SSB, poly(dT) ssDNA was used in place of M13mp8
ssDNA. Previously, it was shown that much more prebound RecA is
displaced from poly(dT) by SSB than from M13mp8 ssDNA (7). This occurs
to a large extent because of end-dependent disassembly of
RecA filaments (5, 56, 58), which is readily observed only on linear
DNAs, where filament extension at one end does not compensate for
disassembly at the other. Here we show that at 10 mM
Mg2+, wild-type RecA protein (3 nt/monomer) is almost
completely displaced from poly(dT) by SSB in a process that is complete
in less than 10 min (Fig. 2). At 3 mM Mg2+, the displacement of RecA by SSB is
considerably faster. RecA added to poly(dT) prebound with SSB is not
able to appreciably displace the SSB at 3 or 10 mM
Mg2+, indicating that binding of SSB to poly(dT) is highly
favored over RecA binding to the linear poly(dT). Unlike the case with M13mp8 cssDNA, the resulting steady-state rates are the same regardless of whether RecA or SSB is preincubated with the ssDNA. In these experiments, the ATP is added with the SSB, and thus ATP is not included in the RecA preincubation in the experiments where RecA is
added first. However, including ATP in the RecA preincubation had no
discernible effect on the results shown (data not shown). As previously
observed, a small net disassembly occurs in the absence of SSB, as
indicated by the slight decline in the rate of ATP hydrolysis with time
(Fig. 2, dashed lines), and disassembly and
reassembly of filaments is doubtless occurring at a steady state (5,
56, 58). The rate of hydrolysis by RecA protein in the absence of SSB
is somewhat higher at 10 mM Mg2+ than at 3 mM.
The Capacity of RecA Protein to Compete with SSB Is Enhanced with
Progressive Deletion of the C-terminal Amino Acids--
The C terminus
of RecA contains a number of acidic residues. As shown in Fig.
3A, deletions of 6, 13, or 17 amino acids from the C terminus progressively remove a total of 3, 6, or 7 glutamate and aspartate residues. We tested the ability of these
mutant proteins to compete with SSB, compared with that of wild-type RecA protein. At 3 mM Mg2+, where wild-type
RecA has the least capacity to compete with SSB for binding to
poly(dT), RecA
The ATPase reactions of each protein in the absence of SSB are also
displayed in Fig. 3B to show that at 3 mM
Mg2+, in the absence of SSB, each protein binds to poly(dT)
to approximately the same extent. The apparent
kcat for ATP hydrolysis by wild-type RecA
(calculated by assuming that all of the DNA is bound by RecA protein)
in these reactions is 11.5 min SSB Displacement by the Variant RecA Proteins Is Enhanced by
Mg2+--
All of the RecA proteins including RecA
When M13mp8 cssDNA is used, the outcome of the wild-type RecA-SSB
competition is highly dependent on which protein is preincubated with
the ssDNA (Fig. 1) (7, 9). However, when poly(dT) is used, the
resulting steady-state rates are the same no matter which protein is
preincubated with the ssDNA (Fig. 2). We find that for RecA The Capacity of RecA Protein to Compete with RPA Is Progressively
Enhanced as Deletion of the C Terminus of RecA Is Increased--
Both
the wild-type and C-terminally truncated RecA proteins are better able
to compete with SSB at 10 mM Mg2+ than at 3 mM Mg2+ (Figs. 1 and 3, B and
C). This difference could be due to effects of
Mg2+ either on the RecA proteins or on SSB.
Mg2+ is known to affect the cooperativity of the different
binding modes of SSB (59). An increase in Mg2+ could lower
the cooperativity of binding of SSB, which in turn could affect its
ability to compete with RecA protein. We therefore investigated whether
Mg2+ stimulated the competition of RecA with RPA, a ssDNA
binding protein whose binding, unpublished studies indicate, is not
affected by
Mg2+.2
Additionally, we asked whether the inhibition of wild-type RecA protein
was due to a specific protein-protein interaction with SSB mediated
through the C-terminal region of RecA. The removal of this region would
then result in the ability of C-terminally truncated RecA protein to
compete with SSB, as shown in Fig. 3, B and C.
However, as illustrated in Fig.
4A, the same result obtained with SSB is seen with RPA. At 3 and 10 mM Mg2+,
wild-type RecA has a limited capacity to compete with RPA for binding
to poly(dT). In contrast, RecA
Additional experiments were carried out with the wild-type RecA protein
and M13mp8 circular ssDNA to examine the effects of Mg2+
under conditions in which filament disassembly at filament ends would
minimally affect the results. SSB was preincubated with the ssDNA. With
this circular DNA substrate, the RecA protein can slowly displace the
SSB at 3 mM Mg2+ (Fig. 4B). The
displacement is considerably faster at 10 mM
Mg2+.
Displacement of SSB by RecA C-terminal truncation mutants is apparently
not due to a higher inherent affinity for ssDNA. The ability of RecA
protein to bind DNA in the presence of increasing NaCl concentration
correlates with its inherent DNA affinity (49). Binding to poly(dT) was
monitored indirectly with the DNA-dependent ATPase assay in
the absence of SSB and as a function of increasing NaCl concentration
(data not shown). The half-maximal binding point (taken as the NaCl
concentration where the ATPase activity is halved relative to the
maximum) is seen at ~750 mM NaCl for the wild-type
protein, decreasing slightly to about 700 mM under these
conditions for the RecA RecA Improved SSB Displacement and Increased Steady-state DNA Binding
Are Observed When a 17-Residue C-terminal Truncation Is Combined with
the RecA E38K Mutation--
Several other RecA mutations have been
shown to enhance displacement of SSB, with RecA E38K being the best of
those examined to date (8). The RecA441 double mutant (E38K/I298V) also
includes the E38K mutation, but the second mutation seems to moderate
its effects (8). We wished to compare the effects of these previously characterized mutant proteins with the RecA C-terminal deletion mutants. The results are shown in Fig. 6.
At 3 mM Mg2+, the RecA441 mutant was similar to
the RecA
The higher rates of ATP hydrolysis could reflect a higher intrinsic
rate of ATP hydrolysis by the double mutant protein. Alternatively, they could reflect a greater steady-state level of binding to the DNA.
On the linear poly(dT) DNA substrates used here, net DNA binding
reflects the balance between filament assembly and end-dependent disassembly (5, 56, 58). We evaluated the intrinsic rate of ATP hydrolysis by comparing mutants side by side with
excess protein on M13mp8 circular ssDNA and with SSB added after the
RecA protein (data not shown). Under these conditions, there is no net
filament disassembly (no ends on the circular DNA), and the DNA binding
is saturated for each mutant. The rates of ATP hydrolysis for the
double mutant and the wild-type proteins were identical (apparent
kcat = 33 min We conclude that the C-terminal region of wild-type RecA protein
negatively modulates the capacity of wild-type RecA to compete with SSB
for binding to ssDNA. Progressive removal of 6, 13, and 17 amino acids
from the C terminus of RecA results in a progressive increase in the
capacity of RecA to displace SSB. RecA The inhibition of RecA protein binding to ssDNA by SSB could be
mediated by specific protein-protein interactions, or it could reflect
a simple competition for DNA binding sites. For example, SSB could
inhibit the binding of wild-type RecA protein by means of specific
protein-protein interactions with the RecA C terminus, which would be
progressively eliminated in the C-terminally truncated RecA proteins.
Such a mechanism would imply species specificity. We examined this
possibility by substituting RPA, the ssDNA-binding protein from
S. cerevisiae, for SSB. We find that RPA competes efficiently with wild-type RecA for binding to ssDNA and that the
C-terminally truncated RecA proteins exhibit a progressively enhanced
capacity to displace RPA. This indicates that the function of the RecA
C terminus does not involve a species-specific interaction.
Excess Mg2+ (above that required to coordinate with ATP)
has a stimulatory effect on the displacement of SSB by RecA protein, over and above the enhancement conferred by the C-terminal deletions, as previously observed for wild-type RecA (7). For all of the RecA
variants and for wild-type RecA, SSB displacement is more facile in the
presence of 10 mM Mg2+ than it is at 3 mM Mg2+. This could reflect an alteration of
the binding state of SSB or an effect on RecA protein itself. SSB has
multiple salt-dependent DNA binding modes (30, 59) that
might differentially affect the ability of SSB to compete with RecA
protein for ssDNA binding sites. The experiments that substituted RPA
for SSB had a second purpose, to attempt to address the source of the
stimulation by Mg2+. Unpublished experiments suggest that
RPA does not have multiple salt-dependent DNA binding modes
of the sort observed with SSB and that Mg2+ does not
stimulate the binding of ssDNA by RPA.2 We find that the
ability of the C-terminally truncated RecA proteins and wild-type RecA
to compete with RPA is stimulated by the higher Mg2+
concentration, suggesting that the excess Mg2+ is directly
affecting the RecA protein. Mg2+ may be acting on SSB, as
well, to alter its function in this system. We hypothesized in the
previous paper (44) that a Mg2+ interaction site might
exist in the E. coli RecA C terminus, where it could be
mediated by the many glutamate and aspartate residues present there. To
the extent that Mg2+ does not affect RPA binding, the
Mg2+ effects in experiments with SSB appear to be due
largely to effects of Mg2+ on the RecA protein. Since this
is true for even the RecA Another study also found a link between the capacity of RecA to promote
strand exchange in the absence of excess Mg2+ and the
protein's ability to compete with SSB for ssDNA-binding sites. The
additions of the volume-occupying agents polyethylene glycol and
polyvinyl alcohol to RecA-mediated strand exchange reactions both
greatly reduced the excess Mg2+ requirement in strand
exchange and increased the ability of RecA to compete with SSB for
ssDNA (60). This result, combined with our similar results using
RecA It is useful to compare the properties of the C-terminally truncated
mutant RecA proteins with those of other RecA mutant proteins shown to
have an increased ability to compete with SSB for ssDNA. A primary
question is the mechanism by which RecA mutants might have an enhanced
ability to compete with SSB. Using a salt titration midpoint assay,
which reflects the equilibrium DNA affinity of RecA (61), we found that
the inherent DNA affinity of RecA In this study, we also show that the SSB displacement and steady-state
DNA binding of the 17-residue C-terminal deletion mutant protein are
improved further by the E38K mutation. The double mutant displaces SSB
with no lag that is measurable in our experiments and provides a higher
steady-state level of DNA binding on linear poly(dT) in the presence of
SSB than any mutant studied to date. Since the intrinsic ATPase
activity of the double mutant is the same as that of the individual
mutant proteins, we attribute the increase in steady-state ATP
hydrolysis to an increase in the steady-state level of DNA binding.
These results suggest that SSB displacement and/or overall DNA binding
is modulated by several different parts of the RecA protein. In the
previous paper (44), we proposed that the negative charges of the C
terminus were part of a regulatory network of protein surface salt
bridges. The increase in SSB displacement and overall DNA binding
observed when the deletion of the C-terminal 17 residues and the
replacement of an acidic residue with a basic residue at position 38 are combined could also reflect particular disruptions of an extensive
salt bridge network. We note that the double mutant appears to bind to
DNA better in the presence of SSB than in its absence. We do not
presently have an explanation for this effect.
RecA Regulation of RecA DNA binding ability and/or its ability to compete
with SSB would allow RecA to gain access to SSB-coated DNA only at the
appropriate time, such as after a replication fork stalls. The binding
of RecA to ssDNA that has been previously bound with SSB is facilitated
by the RecO and RecR mediator proteins (5, 63). These studies suggested
that these mediator proteins alter the binding of SSB to ssDNA,
creating a nucleation site for RecA filament formation. The work
presented here indicates that RecA possesses an inherent and robust
capacity to displace SSB but that this capacity is suppressed by the C
terminus. This suggests another potential mechanism of mediator protein
action. The RecO and RecR proteins might interact directly with RecA
protein during the filament nucleation process, altering RecA
conformation so that the C terminus is no longer inhibitory.
C17 also
has an enhanced capacity relative to wild-type RecA protein to bind ssDNA containing secondary structure. Added Mg2+ enhances
the ability of wild-type RecA and the RecA C-terminal deletion mutants
to compete with SSB and replication protein A. The overall binding of
RecA
C17 mutant protein to linear ssDNA is increased further by the
mutation E38K, previously shown to enhance SSB displacement from ssDNA.
The double mutant RecA
C17/E38K displaces SSB somewhat better than
either individual mutant protein under some conditions and exhibits a
higher steady-state level of binding to linear ssDNA under all conditions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S)
(6). This nucleoprotein filament can pair the bound single strand with the complementary strand of an incoming duplex, resulting in homologous recombination.
C17
mutant protein, there is no measurable requirement for Mg2+
in excess of that required to coordinate the ATP used in a given experiment (44). At Mg2+ concentrations above their
respective optima, reactions promoted by the mutant proteins are
somewhat inhibited and produce DNA species that do not migrate into the
gel. For RecA
C17, this inhibition is observed at Mg2+
concentrations (i.e. 10 mM) that are optimal for
strand exchange with wild-type RecA protein. The new DNA species are
thought to represent DNA networks that form when RecA protein is able
to bind to the outgoing strand and pair the strand again with a duplex DNA (45). This trait was previously observed in strand exchange reactions with RecA441, which has an enhanced capacity to compete with
SSB for the displaced single strand during strand exchange reactions
(46). This suggested to us that the C-terminal deletion mutants of RecA
might also have an enhanced capacity to displace SSB protein. In this
study, we demonstrate that C-terminal deletions of RecA protein greatly
enhance the capacity of the protein to compete with SSB and that the
effect increases with progressive truncation out to at least 17 amino
acid residues.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, and 500 mM NaCl. The
concentration of the purified SSB protein was determined from the
absorbance at 280 nm using the extinction coefficient of 2.83 × 104 M
1 cm
1 (48).
Saccharomyces cerevisiae replication protein A (RPA) was
purified as described (49). The concentration was determined by the
absorbance at 280 nm using the extinction coefficient of 8.8 × 104 M
1 cm
1 (50).
The wild-type RecA, RecA
C6, RecA
C13, and RecA
C17 proteins were
purified as described (43). RecA 441 was purified using the following
modification to the wild-type RecA procedure previously described (43).
The initial polyethyleneimine pellet was washed with R buffer (20 mM Tris-HCl (80% cation, pH 7.5), 1 mM
dithiothreitol, 0.1 mM EDTA, and 10% (w/v) glycerol) and
50 mM ammonium sulfate and extracted two times with R
buffer plus 150 mM ammonium sulfate. The RecA E38K (RecA
730) mutant protein was purified like the wild-type RecA protein,
except that the final fraction was subjected to an additional step. The
protein was loaded onto a PBE 94 column equilibrated with R buffer, and
the column was developed with a linear gradient from 0 to 1.0 M KCl. The RecA E38K mutant was eluted at ~600
mM KCl. The eluted protein was dialyzed extensively against
R buffer and concentrated as for the wild-type protein. The
RecA
C17/E38K double mutant protein was purified using the same
protocol as the RecA
C17 protein (43). The concentration of each RecA
and RecA variant protein was determined using the extinction
coefficient of wild-type RecA, 2.23 × 104
M
1 cm
1 (51). Unless otherwise
noted, all reagents were purchased from Fisher. Lactate dehydrogenase,
pyruvate kinase, phosphoenolpyruvate, NADH, and ATP were purchased from
Sigma. PBE 94 resin was purchased from Amersham Biosciences.
Dithiothreitol was purchased from Research Organics, Inc.
1 cm
1.
M13mp8 bacteriophage circular ssDNA was prepared as described (52). The
concentration of M13mp8 ssDNA was determined by UV absorption at 260 nm
using the extinction coefficient 9.03 mM
1
cm
1.
C6, RecA
C13, and
RecA
C17 proteins. The regeneration of ATP from phosphoenolpyruvate
and ADP was coupled to the oxidation of NADH and monitored by the
decrease in absorbance of NADH at 380 nm. The 380-nm wavelength was
used, so that the signal remained within the linear range of the
spectrophotometer for the duration of the experiment. The assays were
carried out on a Varian Cary 300 dual beam spectrophotometer equipped
with a temperature controller and a 12-position cell changer. The cell
path length and band pass were 0.5 cm and 2 nm, respectively. The NADH
extinction coefficient at 380 nm of 1.21 mM
1
cm
1 was used to calculate the rate of ATP hydrolysis.
C6,
RecA
C13, RecA
C17, RecA 441, RecA E38K, or RecA
C17/E38K), Mg(OAc)2, and SSB or RPA are indicated in the figure
legends. To initiate the assay, the ssDNA was preincubated with either a RecA protein or an ssDNA binding protein (SSB or RPA) for 10 min at
37 °C. Then SSB or an RecA protein, respectively, was added. ATP was
added to 3 mM final concentration, either with the RecA protein or with SSB or RPA, as indicated. Data collection was then
begun. In reactions in which no ssDNA-binding protein is included, SSB
storage buffer is added instead. In the salt titrations, conditions
were the same as above on poly(dT), except without SSB or SSB storage
buffer. After a steady-state rate was achieved, aliquots of
concentrated NaCl were added, allowing the reactions to come to steady
state between additions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (18K):
[in a new window]
Fig. 1.
Effects of SSB on wild-type RecA protein
binding to circular ssDNA, with either RecA or SSB preincubated with
the DNA at 3 and 10 mM Mg2+. DNA binding
was monitored indirectly by following the DNA-dependent
ATPase activity of RecA protein. Reactions were carried out as
described under "Experimental Procedures" and contained 5 µM M13mp8 ssDNA, 1.67 or 2.5 µM RecA
protein, 0.5 µM SSB protein, 3 mM ATP, and 3 or 10 mM Mg(OAc)2, as indicated. Either RecA or
SSB was preincubated with the ssDNA for 10 min before the final protein
addition (RecA first or SSB first reactions, respectively). RecA was
added to 2.5 µM only in the reaction indicated as
containing excess RecA, with RecA prebound at 10 mM
Mg2+; otherwise, the RecA was present at 1.67 µM. In each experiment, ATP was added with the SSB.
View larger version (17K):
[in a new window]
Fig. 2.
Effects of SSB on wild-type RecA protein
binding to poly(dT) ssDNA with either RecA or SSB preincubated with the
DNA at 3 and 10 mM Mg2+. DNA binding was
monitored as described in the legend to Fig. 1. Reactions contained 5 µM poly(dT) ssDNA, 1.67 µM RecA protein,
0.7 µM SSB, 3 mM ATP, and 3 or 10 mM Mg(OAc)2, as indicated in
parentheses. Either RecA or SSB was preincubated with the
ssDNA for 10 min before the final protein addition. For the controls,
denoted by dashed lines, SSB was replaced with
SSB storage buffer. In each experiment, ATP was added with the SSB or
SSB storage buffer.
C13 and, to an even greater extent, RecA
C17, bind
well to the poly(dT) that has been prebound with SSB (Fig.
3B). At 3 mM Mg2+, RecA
C6 is
unable to compete with SSB.
View larger version (20K):
[in a new window]
Fig. 3.
Displacement of SSB from poly(dT) by
wild-type RecA protein and C-terminally truncated RecA proteins
C6,
C13, and
C17 at 3 and 10 mM
Mg2+. DNA binding was monitored indirectly by
following the DNA-dependent ATPase activity of RecA
protein. A highlights the C-terminal region of RecA protein.
The core domain, which includes the P-loop (ATP binding motif), is
shown in white. The shaded and black
regions of the sequence correspond to the N-terminal and
C-terminal domains, respectively. The primary structure of the
C-terminal 17 amino acids of the RecA protein is diagrammed
below the linear sequence. The hexagons highlight
the high concentration of negatively charged amino acids in this
region. The arrows indicate points of truncation in the
deletion mutants: RecA
C6, RecA
C13, and RecA
C17. For the data
shown in B and C, reactions contained 0.7 µM SSB, 1.67 µM RecA protein, 5 µM poly(dT) ssDNA, 3 mM ATP, and either 3 mM (B) or 10 mM (C)
Mg(OAc)2. In all reactions, ATP and SSB were preincubated
with the ssDNA for 10 min before the addition of the RecA protein
indicated. The controls without SSB for each RecA protein variant are
shown as dashed lines and substituted SSB storage
buffer for the SSB. At the point where these dashed
lines are bisected by the vertical
labeling line, the listing of the proteins
(top to bottom) corresponds to the top
to bottom positioning of the dashed
lines (the top line is the reaction of
the wild-type RecA protein, etc.). WT, wild-type RecA
protein.
1, quite comparable with
rates with poly(dT) observed previously (7). However, as shown below,
this rate is lower than the nearly 30 min
1 rate observed
when RecA is carefully titrated onto circular ssDNA. The lower rate
almost certainly reflects incomplete binding of the DNA and perhaps an
equilibrium state in which filaments are undergoing steady-state
end-dependent assembly and disassembly.
C6,
compete more effectively with SSB at 10 mM
Mg2+, and at 10 mM Mg2+ it is more
evident that the ability of RecA to compete with SSB increases with
progressive deletion of the RecA C terminus (Fig. 3C). The
RecA
C17 mutant protein exhibits no detectable lag in binding,
indicating a particularly rapid SSB displacement process. Each RecA
protein binds poly(dT) efficiently in the absence of SSB at 10 mM Mg2+.
C17
protein, the steady-state rates are also the same whether RecA
C17 or
SSB is preincubated with poly(dT) (Fig. 3 and data not shown). When
RecA
C17 was preincubated with poly(dT) and ATP and then challenged
with SSB, the challenge had no measurable effect on the rate of ATP
hydrolysis at 10 mM Mg2+ and, by inference, on
the state of RecA
C17 binding. At 3 mM Mg2+,
there is a slow decline in ATPase rate occurring over ~60 min after
the challenge, after which the rate seen in Fig. 3B is
observed (data not shown). This suggests a slow displacement of the
mutant RecA protein by the SSB.
C17 does bind to poly(dT) coated with
RPA and is especially proficient at 10 mM Mg2+
(Fig. 4A). This suggests that Mg2+ has a
significant enhancing effect on RecA protein function in SSB
displacement even when the C terminus is removed.
View larger version (22K):
[in a new window]
Fig. 4.
Displacement of RPA from ssDNA by wild-type
RecA protein (WT) and C-terminally truncated RecA
proteins C6,
C13,
and
C17. DNA binding by RecA and RecA
variants was monitored indirectly by following the
DNA-dependent ATPase activity of RecA protein. Reactions in
A contained 0.6 µM RPA, 1.67 µM
RecA protein, 5 µM poly(dT) ssDNA, 3 mM ATP,
and either 3 mM or 10 mM Mg(OAc)2
as indicated by the number in parentheses. RPA
was preincubated with the ssDNA for 10 min before the addition of the
RecA protein indicated. Controls carried out in the absence of RPA
(dashed lines) are labeled as indicated in the
legend to Fig. 3. B, two reactions with wild-type RecA
carried out under the same conditions as in A but with
M13mp8 ssDNA replacing the poly(dT).
C17 mutant.
C17 Protein Binds M13mp8 ssDNA with Secondary Structure
Better than Does Wild-type RecA Protein--
RecA E38K and RecA803,
which have been demonstrated to compete with SSB for binding to ssDNA
more effectively than wild-type RecA, appear to also bind to regions of
secondary structure in ssDNA better than the wild-type protein (8). We
investigated whether RecA
C17 was more capable than wild-type RecA at
binding M13mp8 ssDNA that contains secondary structure, induced by 10 mM Mg2+, in the absence of SSB. In these
experiments, the RecA was present at 2 nt/monomer to prevent RecA
displacement in the control experiments that contained SSB. As
illustrated in Fig. 5, the rate of ATP hydrolysis of wild-type RecA protein prebound to M13mp8 ssDNA in the
presence of SSB and 10 mM Mg2+ drops
significantly when SSB is omitted, to 30% of the rate in the presence
of SSB (dashed lines in Fig. 5). This is
consistent with a considerably reduced binding to the ssDNA under these
conditions, as observed previously for RecA binding to ssDNA with
appreciable secondary structure in the absence of SSB (7). In contrast, the ATPase rate of RecA
C17 at 10 mM Mg2+
drops only a little upon omission of SSB, to 84% of the rate seen with
SSB (Fig. 5). In sum, the data indicate that RecA
C17 is better able
to bind DNA that contains secondary structure than is wild-type RecA.
View larger version (12K):
[in a new window]
Fig. 5.
Comparison of the capacity of wild-type RecA
protein (WT) to bind to secondary structure-containing
M13mp8 ssDNA with that of the RecA C17
protein. DNA binding by wild-type and C-terminally truncated RecA
protein was monitored indirectly by following the
DNA-dependent ATPase activity of RecA protein. Reactions
were carried out as described under "Experimental Procedures" and
contained 5 µM M13mp8 ssDNA, 2.5 µM RecA
protein, 3 mM ATP, and 10 mM
Mg(OAc)2. Some reactions also contained 0.5 mM
SSB as indicated (plus signs in
parentheses). Wild-type RecA protein or RecA
C17 were
preincubated the ssDNA for 10 min, before the addition of ATP and
either SSB (+) or SSB storage buffer (
). The reactions with wild-type
RecA protein are shown with dashed lines to
highlight the contrast with the reactions with the RecA
C17 mutant,
which exhibit a much reduced effect of SSB addition.
C17 mutant in its capacity to displace SSB. The RecA E38K
mutant was the best of the individual mutants in this activity. A short
but discernible lag in reaching a steady state of ATP hydrolysis was
observed for each of the individual mutants. At 10 mM
Mg2+, the activity of the RecA441 mutant was similar to
that of RecA
C13 and less than that of RecA
C17. The RecA E38K
mutant was still the best individual mutant in SSB displacement. The
lag in reaching an apparent steady state was reduced for all of the
individual mutant proteins. When the RecA
C17 and E38K mutations were
combined in a single protein, a further enhancement was observed in SSB displacement and ssDNA binding. At the low Mg2+
concentration, the double mutant protein was more effective at SSB
displacement than either single mutant. There was no discernible lag in
ATP hydrolysis with the double mutant protein under any condition
tested, and there was a substantially higher steady-state rate of ATP
hydrolysis (with an apparent kcat in excess of
20 min
1). We note that the ATP hydrolysis was higher in
the presence of SSB than in its absence, a property observed with no
other RecA variant.
View larger version (28K):
[in a new window]
Fig. 6.
Comparison of the SSB displacement and ssDNA
binding activities of various RecA mutants proteins added to
SSB-prebound poly(dT) ssDNA: RecA (wild-type; WT),
RecA C13, RecA
C17,
RecA 441, RecA E38K, and RecA
RecA
C17/E38K. DNA binding by RecA and
RecA mutant proteins was monitored indirectly by following the
DNA-dependent ATPase activity of RecA protein. Reactions
contained 0.7 µM SSB, 1.67 µM RecA or RecA
mutant protein, 5 µM poly(dT) ssDNA, 3 mM
ATP, and either 3 mM (A) or 10 mM
(B) Mg(OAc)2. In all reactions, SSB and ATP were
preincubated with the ssDNA for 10 min before the addition of the RecA
protein indicated. The controls without SSB for each RecA protein
variant are shown as dashed lines.
1) in this trial.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C17 also displaces RPA from
ssDNA much more readily than wild-type RecA. The binding of RecA
C17
to DNA containing secondary structure is enhanced relative to wild-type
RecA. This result reinforces a pattern previously established for RecA
E38K and RecA803 mutant proteins (8). The capacity of these
C-terminally truncated mutants to compete with SSB is quite high,
especially in the case of RecA
C17. The RecA
C17 truncation and the
E38K mutation work together in a double mutant to eliminate a
discernible lag in SSB displacement under some conditions and to
increase the steady-state level of DNA binding by RecA.
C17 variant, there may be a
Mg2+ interaction site or sites on RecA protein outside of
the C terminus that affect the capacity of RecA to displace SSB. In
contrast, an interaction of Mg2+ with these sites does not
appear to be required for the strand exchange reaction, since the
excess Mg2+ requirement in that reaction was largely
eliminated for RecA
C17 (44).
C17 protein, suggests that volume-occupying agents may stabilize
a conformation of the C terminus of RecA that does not inhibit these
activities. This conformation may be the same one induced by the
addition of excess Mg2+. The removal of the C terminus also
alleviates the inhibition. The addition of Mg2+, addition
of volume-occupying agents, or removal of the C terminus may expose the
RecA DNA binding site in such a way that RecA is better able to compete
with SSB and RPA or is better able to extract ssDNA from the surface of
these binding proteins.
C17 for poly(dT) is not appreciably
changed from that of wild-type RecA (data not shown). Previous studies
of RecA mutant proteins that compete better with SSB than wild-type
RecA have shown that the properties of these proteins, RecA803 V37M,
RecA441 (E38K/I298V), and RecA E38K, are also not due to an increased
ssDNA binding affinity (8, 28, 62). The capacity of these previously characterized RecA mutant proteins to compete with SSB was shown to
correlate with their rate of association with DNA, a characteristic also found with wild-type RecA protein in the presence of
volume-occupying agents (60).
C17 shares an additional property with RecA E38K and RecA803, an
increased proficiency compared with wild-type RecA of competing with
secondary structure for ssDNA binding sites (8, 28). Whereas the rates
of ATP hydrolysis for RecA E38K and RecA803 on M13mp8 ssDNA at 10 mM Mg2+ in the presence of SSB were similar to
that of wild-type protein, the rates of the mutant proteins in the
absence of SSB were much higher than that of wild-type RecA. In
addition, the RecA E38K, RecA803, and wild-type proteins all bound
equally well to etheno M13mp8 DNA, which does not contain secondary
structure. We obtained a similar result for RecA
C17. The ATPase rate
for wild-type RecA on M13mp8 ssDNA at 10 mM
Mg2+ in the absence of SSB is 30% of the rate in the
presence of SSB. In contrast, the ATPase rate of RecA
C17 at 10 mM Mg2+ drops only a moderate amount upon
omission of SSB, to 84%. In sum, the data indicate that RecA
C17,
like RecA803 and RecA E38K, is better able to bind DNA that contains
secondary structure than is wild-type RecA. Notably, this property is
not intrinsic to RecA mutant proteins that compete more efficiently
with SSB. RecA441, which is more proficient at competing with SSB than
RecA803, binds DNA with secondary structure only as well as wild-type
RecA (62).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Sergei Saveliev for purification of the E. coli SSB protein and Marc Wold (University of Iowa) for helpful discussions about the RPA protein.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM32335.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.
To whom correspondence should be addressed: Dept. of Biochemistry,
University of Wisconsin, 433 Babcock Dr., Madison, WI 53706-1544. Tel.:
608-262-1181; Fax: 608-265-2603; E-mail:
cox@biochem.wisc.edu.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M212920200
2 M. Wold, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ssDNA, single-stranded DNA;
nt, nucleotides;
ATPS, adenosine
5'-O-(thiotriphosphate);
SSB, single-stranded binding
protein;
RPA, replication protein A.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Kowalczykowski, S. C., Clow, J., and Krupp, R. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3127-3131[Abstract] |
2. | Pugh, B. F., and Cox, M. M. (1988) J. Mol. Biol. 203, 479-493[Medline] [Order article via Infotrieve] |
3. |
Pugh, B. F.,
and Cox, M. M.
(1987)
J. Biol. Chem.
262,
1326-1336 |
4. |
Register, J. C., III,
and Griffith, J.
(1985)
J. Biol. Chem.
260,
12308-12312 |
5. | Shan, Q., Bork, J. M., Webb, B. L., Inman, R. B., and Cox, M. M. (1997) J. Mol. Biol. 265, 519-540[CrossRef][Medline] [Order article via Infotrieve] |
6. | Ogawa, T., Yu, X., Shinohara, A., and Egelman, E. H. (1993) Science 259, 1896-1899[Medline] [Order article via Infotrieve] |
7. | Kowalczykowski, S. C., and Krupp, R. A. (1987) J. Mol. Biol. 193, 97-113[Medline] [Order article via Infotrieve] |
8. |
Lavery, P. E.,
and Kowalczykowski, S. C.
(1992)
J. Biol. Chem.
267,
20648-20658 |
9. | Kowalczykowski, S. C., Clow, J., Somani, R., and Varghese, A. (1987) J. Mol. Biol. 193, 81-95[Medline] [Order article via Infotrieve] |
10. |
Cox, M. M.,
and Lehman, I. R.
(1982)
J. Biol. Chem.
257,
8523-8532 |
11. | Umezu, K., Chi, N. W., and Kolodner, R. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3875-3879[Abstract] |
12. |
Bork, J. M.,
Cox, M. M.,
and Inman, R. B.
(2001)
EMBO J.
20,
7313-7322 |
13. |
Courcelle, J.,
Carswell-Crumpton, C.,
and Hanawalt, P.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3714-3719 |
14. | Smith, K. C., and Sharma, R. C. (1987) Mutat. Res. 183, 1-9[Medline] [Order article via Infotrieve] |
15. |
Galitski, T.,
and Roth, J. R.
(1997)
Genetics
146,
751-767 |
16. | Asai, T., and Kogoma, T. (1994) J. Bacteriol. 176, 7113-7114[Abstract] |
17. |
Steiner, W. W.,
and Kuempel, P. L.
(1998)
J. Bacteriol.
180,
6269-6275 |
18. |
Cox, M. M.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
8173-8180 |
19. | Moreau, P. L. (1988) J. Bacteriol. 170, 2493-2500[Medline] [Order article via Infotrieve] |
20. | Madiraju, M. V., Templin, A., and Clark, A. J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6592-6596[Abstract] |
21. | Wang, T. C., and Smith, K. C. (1986) J. Bacteriol. 168, 940-946[Medline] [Order article via Infotrieve] |
22. | Wang, T. C. V., Madiraju, M. V. V. S., Templin, A., and Clark, A. J. (1991) Biochimie (Paris) 73, 335-340 |
23. | Thomas, A., and Lloyd, R. G. (1983) J. Gen. Microbiol. 129, 681-686[Medline] [Order article via Infotrieve] |
24. | Volkert, M. R., Margossian, L. J., and Clark, A. J. (1984) J. Bacteriol. 160, 702-705[Medline] [Order article via Infotrieve] |
25. |
Knight, K. L.,
Aoki, K. H.,
Ujita, E. L.,
and McEntee, K.
(1984)
J. Biol. Chem.
259,
11279-11283 |
26. | Wang, T. C., Chang, H. Y., and Hung, J. L. (1993) Mutat. Res. 294, 157-166[Medline] [Order article via Infotrieve] |
27. | Ennis, D. G., Levine, A. S., Koch, W. H., and Woodgate, R. (1995) Mutat. Res. 336, 39-48[Medline] [Order article via Infotrieve] |
28. | Madiraju, M. V., Lavery, P. E., Kowalczykowski, S. C., and Clark, A. J. (1992) Biochemistry 31, 10529-10535[Medline] [Order article via Infotrieve] |
29. | Lohman, T. M., Bujalowski, W., and Overman, L. B. (1988) Trends Biochem. Sci. 13, 250-255[Medline] [Order article via Infotrieve] |
30. |
Bujalowski, W.,
Overman, L. B.,
and Lohman, T. M.
(1988)
J. Biol. Chem.
263,
4629-4640 |
31. | Lohman, T. M., and Ferrari, M. E. (1994) Annu. Rev. Biochem. 63, 527-570[CrossRef][Medline] [Order article via Infotrieve] |
32. | Story, R. M., Weber, I. T., and Steitz, T. A. (1992) Nature 355, 318-325[CrossRef][Medline] [Order article via Infotrieve] |
33. | Roca, A. I., and Cox, M. M. (1997) Prog. Nucleic Acid Res. Mol. Biol. 56, 129-223[Medline] [Order article via Infotrieve] |
34. | Lusetti, S. L., and Cox, M. M. (2002) Annu. Rev. Biochem. 71, 71-100[CrossRef][Medline] [Order article via Infotrieve] |
35. | VanLoock, M. S., Yu, X., Yang, S., Lai, A. L., Low, C., Campbell, M. J., and Egelman, E. H. (2003) Structure 11, 1-20[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Yu, X.,
Jacobs, S. A.,
West, S. C.,
Ogawa, T.,
and Egelman, E. H.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
8419-8424 |
37. |
Benedict, R. C.,
and Kowalczykowski, S. C.
(1988)
J. Biol. Chem.
263,
15513-15520 |
38. | Tateishi, S., Horii, T., Ogawa, T., and Ogawa, H. (1992) J. Mol. Biol. 223, 115-129[Medline] [Order article via Infotrieve] |
39. |
Rusche, J. R.,
Konigsberg, W.,
and Howard-Flanders, P.
(1985)
J. Biol. Chem.
260,
949-955 |
40. | Yarranton, G. T., and Sedgwick, S. G. (1982) Mol. Gen. Genet. 185, 99-104[Medline] [Order article via Infotrieve] |
41. | Larminat, F., and Defais, M. (1989) Mol. Gen. Genet. 216, 106-112[Medline] [Order article via Infotrieve] |
42. | Yu, X., and Egelman, E. H. (1991) J. Struct. Biol. 106, 243-254[Medline] [Order article via Infotrieve] |
43. |
Lusetti, S. L.,
Wood, E. A.,
Fleming, C. D.,
Modica, M. J.,
Korth, J.,
Abbott, L.,
Dwyer, D. W.,
Roca, A. I.,
Inman, R. B.,
and Cox, M. M.
(2003)
J. Biol. Chem.
278,
16372-16380 |
44. |
Lusetti, S. L.,
Shaw, J. J.,
and Cox, M. M.
(2003)
J. Biol. Chem.
278,
16381-16388 |
45. |
Chow, S. A.,
Rao, B. J.,
and Radding, C. M.
(1988)
J. Biol. Chem.
263,
200-209 |
46. |
Lavery, P. E.,
and Kowalczykowski, S. C.
(1990)
J. Biol. Chem.
265,
4004-4010 |
47. |
Shan, Q.,
Cox, M. M.,
and Inman, R. B.
(1996)
J. Biol. Chem.
271,
5712-5724 |
48. | Lohman, T. M., and Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603[Abstract] |
49. |
Eggler, A. L.,
Inman, R. B.,
and Cox, M. M.
(2002)
J. Biol. Chem.
277,
39280-39288 |
50. |
Sugiyama, T.,
Zaitseva, E. M.,
and Kowalczykowski, S. C.
(1997)
J. Biol. Chem.
272,
7940-7945 |
51. |
Craig, N. L.,
and Roberts, J. W.
(1981)
J. Biol. Chem.
256,
8039-8044 |
52. |
Neuendorf, S. K.,
and Cox, M. M.
(1986)
J. Biol. Chem.
261,
8276-8282 |
53. |
Lindsley, J. E.,
and Cox, M. M.
(1990)
J. Biol. Chem.
265,
9043-9054 |
54. | Morrical, S. W., Lee, J., and Cox, M. M. (1986) Biochemistry 25, 1482-1494[Medline] [Order article via Infotrieve] |
55. | Shan, Q., and Cox, M. M. (1996) J. Mol. Biol. 257, 756-774[CrossRef][Medline] [Order article via Infotrieve] |
56. | Arenson, T. A., Tsodikov, O. V., and Cox, M. M. (1999) J. Mol. Biol. 288, 391-401[CrossRef][Medline] [Order article via Infotrieve] |
57. | Muniyappa, K., Shaner, S. L., Tsang, S. S., and Radding, C. M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 2757-2761[Abstract] |
58. |
Bork, J. M.,
Cox, M. M.,
and Inman, R. B.
(2001)
J. Biol. Chem.
276,
45740-45743 |
59. | Bujalowski, W., and Lohman, T. M. (1986) Biochemistry 25, 7799-7802[Medline] [Order article via Infotrieve] |
60. |
Lavery, P. E.,
and Kowalczykowski, S. C.
(1992)
J. Biol. Chem.
267,
9307-9314 |
61. | Menetski, J. P., and Kowalczykowski, S. C. (1985) J. Mol. Biol. 181, 281-295[Medline] [Order article via Infotrieve] |
62. | Lavery, P. E., and Kowalczykowski, S. C. (1988) J. Mol. Biol. 203, 861-874[Medline] [Order article via Infotrieve] |
63. |
Umezu, K.,
and Kolodner, R. D.
(1994)
J. Biol. Chem.
269,
30005-30013 |