From the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706
Received for publication, December 18, 2002, and in revised form, February 10, 2003
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ABSTRACT |
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A set of C-terminal deletion mutants
of the RecA protein of Escherichia coli, progressively
removing 6, 13, 17, and 25 amino acid residues, has been generated,
expressed, and purified. In vivo, the deletion of 13 to 17 C-terminal residues results in increased sensitivity to mitomycin C. In vitro, the deletions enhance binding to duplex DNA as
previously observed. We demonstrate that much of this enhancement
involves the deletion of residues between positions 339 and 346. In
addition, the C-terminal deletions cause a substantial upward shift in
the pH-reaction profile of DNA strand exchange reactions. The
C-terminal deletions of more than 13 amino acid residues result in
strong inhibition of DNA strand exchange below pH 7, where the
wild-type protein promotes a proficient reaction. However, at the same
time, the deletion of 13-17 C-terminal residues eliminates the
reduction in DNA strand exchange seen with the wild-type protein at pH
values between 7.5 and 9. The results suggest the existence of
extensive interactions, possibly involving multiple salt bridges,
between the C terminus and other parts of the protein. These
interactions affect the pKa of key groups involved
in DNA strand exchange as well as the direct binding of RecA protein to
duplex DNA.
The bacterial RecA protein plays a central role in the
processes of homologous DNA recombination and DNA repair. RecA is a DNA-dependent ATPase that catalyzes an in vitro
strand exchange reaction between single-stranded DNA and homologous
double-stranded DNA molecules. The RecA protein of Escherichia
coli consists of 352 amino acid residues. The three-dimensional
structures of both the RecA protein alone and complexed with inhibitory
ADP cofactor have been determined (1, 2). There is a central core
domain and two smaller N- and C-terminal domains. The core domain
contains the nucleotide binding site and the putative DNA binding
site(s). This domain is highly conserved among bacterial and eukaryotic RecA homologs (3-6) and exhibits sequence and/or structural homology with a range of proteins including DNA helicases (7), DNA pumps (8),
the F1-ATPase (9), and adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (10). The amino-terminal domain of RecA
is involved in monomer-monomer interactions (1, 3). The
carboxyl-terminal domain of RecA protein consists of residues 270-352,
the last 24 of which are disordered in the apoenzyme crystal structure
(1, 2).
The present study focuses on the C-terminal 25 amino acid residues,
which we will refer to as the C terminus (as opposed to the entire
domain). Over half of these terminal 25 residues have side chains that
are either negatively charged (7 of the last 17 are Glu or Asp
residues) or contain hydroxyl groups (six Ser or Thr residues) (Fig.
1). Positively charged amino acid side chains are absent. Other
ssDNA1-binding proteins such
as single-stranded binding protein (SSB) of E. coli and the
gene 32 protein of phage T4 also have highly negatively charged
C-terminal regions. Upon C-terminal deletion to remove these negative
charges, SSB and the gene 32 protein show increased dsDNA affinities
relative to the intact proteins (11, 12).
Primary structure provides few clues to the function of the RecA C
terminus. Sequence conservation in this part of the protein is quite
limited even when comparisons are limited to other bacterial RecA
proteins. The major feature of the primary structure in the E. coli protein is the preponderance of negatively charged residues in this region. This feature is found in most but not all other bacterial RecA sequences. A few RecA proteins, notably from
Bacteroides and Mycoplasma species, lack this
protein segment altogether (4). In a few other species, particularly
Streptomyces, the C terminus is lengthened and exhibits a
preponderance of positively charged residues (4).
In addition to a general lack of structural information about the RecA
C terminus, there has been little indication that this part of the
protein has functional significance. Several C-terminal deletion
mutants of the E. coli RecA protein have been characterized. Ogawa and colleagues (13) characterized RecA Somewhat shorter C-terminal deletions of RecA protein have also been
constructed and characterized. A 17-residue C-terminal deletion mutant
was shown not to affect UV resistance, induction of the SOS response,
or Weigle reactivation (17). The same study showed that this mutant had
a minimal effect on recombination when expressed by itself. A small
effect on conjugational recombination was observed only when the
wild-type and mutant proteins were both present in vivo
(17). The removal of about 18 residues from the C terminus resulted in
a substantial conformational difference in RecA filaments bound to
dsDNA, as observed in three-dimensional reconstructions of electron
microscopy images (18). The biochemistry of these C-terminal deletion
mutants was not explored.
We have initiated an effort to explore the function of the C-terminal
25 amino acid residues of RecA protein in more detail. To better define
what parts of this segment are involved in activity changes, we have
constructed a set of C-terminal deletion mutations, removing 6, 13, and
17 amino acid residues. These remove 3, 6, and 7 of the negatively
charged amino acid residues, respectively (Fig.
1). The RecA protein mutant with 25 amino
acids removed from the C terminus was also included in part to provide
a reference point with which to compare our results with those
previously published by Ogawa and co-workers (13). The first study
examines the effects of these mutations on cell survival and on
fundamental in vitro properties of RecA protein.
Enzymes and Biochemicals--
E. coli SSB was
purified as described (19). The concentration of the purified SSB
protein was determined from the absorbance at 280 nm using the
extinction coefficient of 2.83 × 104
M Buffers and Media--
P buffer contained 20 mM
potassium phosphate (pH 6.8), 1 mM DTT, 0.1 mM
EDTA, and 10% (w/v) glycerol. R buffer contained 20 mM
Tris-HCl (80% cation, pH 7.5), 1 mM DTT, 0.1 mM EDTA, and 10% (w/v) glycerol. TAE buffer contained 40 mM Tris-OAc (80% cation) and 1 mM EDTA.
Luria-Burtani medium (LB broth) is 10 g/liter trypone, 5 g/liter yeast
extract, and 10 g/liter NaCl, with pH adjusted to 7.0. M9 minimal
medium is 12.8 g/liter
Na2HPO4·7H2O, 3 g/liter KH2PO4, 0.5 g/liter NaCl, 1.0 g/liter
NH4Cl, and 4% (w/v) glucose.
DNA Substrates--
Bacteriophage Cloning and Overexpressing RecA Proteins--
A plasmid was
constructed to express the wild-type E. coli RecA protein
under the control of the bacteriophage T7 RNA polymerase promoter. The
wild-type recA open reading frame (ORF) was cloned in two
parts. The N-terminal coding region came from the
NcoI-PstI fragment of plasmid pTRecA103, which
was a gift from Kendall Knight (25). PCR was used to generate the
remaining coding region of the recA ORF using plasmid pGE226
as a template, kindly given by George Weinstock (26). Primers were
designed to amplify from the PstI site to the stop codon of
the recA ORF with the introduction of a HindIII
site just downstream of the stop codon. The full-length recA
ORF (NcoI-HindIII fragment) was cloned downstream
of the T7 RNA polymerase promoter in the AmpR plasmid
pET21d(+) from Novagen to generate plasmid pAIR79. Both strands of the
recA ORF were manually sequenced to ensure the integrity of
the coding region.
To overexpress the wild-type RecA protein, pAIR79 was co-transformed
with pT7POL26 (KanR) into the multiply nuclease-deficient
strain STL327 (exoI
The recA Preparation of E. coli Strains for in Vivo Experiments--
The
genes encoding the wild-type RecA, RecA Sensitivity of E. coli Strains Expressing Wild-type RecA,
RecA Purification of the RecA Protein--
Wild-type RecA protein was
purified using modifications to previously described protocols (31,
32). All steps were carried out at 4 °C. Cell paste (20 g) was
thawed and fully resuspended in 80 ml of a solution of 25% (w/v)
sucrose and 250 mM Tris-HCl (80% cation, pH 7.5). Cells
were lysed by a 60-min incubation with 40 ml of a 5 mg/ml solution of
lysozyme in 250 mM Tris-HCl (80% cation, pH 7.5), followed
by the addition of 50 ml of 25 mM EDTA, sonication, and
centrifugation. The lysate was precipitated with 22 ml of 5% (w/v)
polyethyleneimine, pH 7.5 (0.5% final concentration), and centrifuged.
The pellet was washed with 50 ml of R buffer plus 150 mM
ammonium sulfate and extracted two times with 25 ml of R buffer plus
300 mM ammonium sulfate. The protein solution was
precipitated by the addition of 0.28 g of solid ammonium sulfate per ml of solution (48% saturation). The resulting pellet was washed
two times with R buffer plus 2.1 M ammonium sulfate,
resuspended in 50 ml of R buffer plus 50 mM KCl, and
dialyzed versus the same. The protein was loaded onto a
DEAE-Sepharose column and washed with two column volumes of R buffer
plus 50 mM KCl. Flow-through peak fractions were identified
by SDS-PAGE analysis, pooled, and dialyzed versus P buffer.
Protein was then loaded onto a hydroxyapatite column, washed with two
column volumes of P buffer, and eluted with a linear gradient from
20-350 mM phosphate buffer (pH 6.8) over 10 column
volumes. Peak fractions were identified by SDS-PAGE analysis, pooled,
concentrated with Centricon Plus-20, 10,000-dalton molecular
mass cut-off concentrators (Amicon) or by ammonium sulfate precipitation and resuspension in R buffer, dialyzed into R buffer, flash-frozen in liquid N2, and stored at Purification of the RecA C-terminal Deletion Mutant
Proteins--
The deletion mutants RecA Electron Microscopy of Circular ssDNA-RecA
Samples for electron microscopy analysis were prepared as follows. All
incubations were carried out at 37 °C. wild-type RecA or RecA ATPase Assay--
A coupled spectrophotometric enzyme assay (35,
36) was used to measure the DNA-dependent ATPase activities
of the wild-type RecA, RecA
The reactions were carried out at 37 °C in 25 mM
Tris-OAc (80% cation), MES (33% anion), or Tris-OAc (56% cation) for
a reaction pH of 7.3, 6.0, or 8.0, respectively, 1 mM DTT,
3 mM potassium glutamate, 10 mM
Mg(OAc)2, 5% (w/v) glycerol, an ATP regeneration system
(10 units/ml pyruvate kinase, 2.2 mM PEP for reactions with
single-stranded DNA or 3.0 mM PEP for reactions with duplex DNA), a coupling system (3 mM NADH and 10 units/ml lactate
dehydrogenase), and the concentration of DNA and wild-type RecA,
RecA DNA Three-strand Exchange Reactions Promoted by the Wild-type and
Deletion Mutant Proteins--
Three-strand exchange reactions were
carried out in 25 mM buffer (varied as indicated to alter
pH), 1 mM DTT, 5% (w/v) glycerol, 3 mM
potassium glutamate, and 10 mM Mg(OAc)2. The
buffers used were MES (33% anion, pH 6.0), MES (44% anion, pH 6.2),
MES (55% anion, pH 6.4), MES (61% anion, pH 6.5), MES (76% anion, pH
6.9), HEPES (24% cation, pH 7.0), Tris-OAc (80% cation, pH 7.3),
HEPES (39% cation, pH 7.3), HEPES (56% cation, pH 7.6), Tris-OAc
(56% cation, pH 8.0), Tris-OAc (39% cation, pH 8.3), TAPS (44%
anion, pH 8.4), CHES (17% anion, pH 8.5), Tris-OAc (20% cation, pH
8.7), TAPS (72% anion, pH 8.8), CHES (33% anion, pH 8.9) for the
indicated reaction pH. In each case, the reported pH is the expected
final pH of the reaction mixture, obtained by determining the pH of a
model reaction mixture containing all reaction components but substituting TE buffer for the DNA additions and the appropriate protein storage buffers for each protein addition. Reactions also contained an ATP regeneration system of 10 units/ml pyruvate kinase and
3.2 mM PEP. All incubations were carried out at 37 °C.
The following are final concentrations. The wild-type RecA, RecA Experimental Design--
A set of C-terminal deletion proteins was
constructed to systematically test the role of groups of acidic
residues in DNA binding and DNA strand exchange reactions (Fig. 1). The
RecA E. coli Strains Expressing RecA C-terminal Deletion Mutants Are Not
UV- or Ionizing Radiation-sensitive but Are Mitomycin
C-sensitive--
We tested whether strains containing the C-terminal
deletion mutants on the chromosome were more sensitive to DNA damage
than a strain containing the full-length RecA protein. Ogawa and
colleagues (37) previously determined that a strain expressing
RecA
We also wished to determine whether a more significant challenge to
cellular DNA metabolism might reveal an in vivo defect in
the strains harboring the truncated RecA proteins. We thus examined the
cells' sensitivity to the cross-linking agent mitomycin C and to
ionizing radiation. The strain expressing RecA The RecA C-terminal Deletion Mutant RecA The ssDNA-dependent ATPase Activity of the RecA
C-terminal Deletion Mutants--
ATP hydrolysis by the RecA protein
has been used in many studies as an indirect measure of DNA binding.
This activity is almost entirely DNA-dependent under most
reaction conditions and generally correlates well with other measures
of DNA binding (16). The measured kcat for ATP
hydrolysis by RecA protein bound to ssDNA is ~30 min
We measured the rates of ssDNA-dependent ATP hydrolysis
using M13mp8 circular ssDNA cofactor for each C-terminal deletion mutant at pH 7.3, 6.0, and 8.0 (Table I).
The rate of ATP hydrolysis was first examined as a function of
wild-type and RecA The C Terminus Affects the Lag in dsDNA Binding Exhibited by the
Wild-type RecA Protein--
The wild-type RecA protein exhibits a long
lag in binding to dsDNA, reflecting a slow nucleation step, at pH
values close to and above physiological pH (15, 16). In order to
determine which group or groups of negative charges in the C terminus
contribute to this effect, we measured and compared the nicked circular
dsDNA-dependent ATP hydrolysis rates of the wild-type RecA,
RecA
As expected, the wild-type RecA protein exhibited a long lag in DNA
binding at pH 7.3,
The lag exhibited in dsDNA binding by the wild-type RecA protein can be
alleviated by lowering the pH of the reaction (15, 16). We repeated the
dsDNA-dependent ATPase experiments at pH 6.05. As expected,
the wild-type RecA protein bound rapidly to dsDNA at this lower pH
(Fig. 6B). The lag in dsDNA binding was also reduced for all
of the other proteins tested.
The RecA C-terminal Deletion Mutants Promote DNA Strand Exchange of
Bacteriophage C-terminal Deletions of RecA Protein Dramatically Alter the
pH-Reaction Profiles of RecA Protein-mediated DNA Strand Exchange
Reactions--
We repeated DNA strand exchange reactions at pH 6.0 (Fig. 8). The DNA strand exchange
facilitated by the RecA
The result shown in Fig. 8 triggered a broader investigation of the
effects of the C-terminal deletions on the pH-reaction profile of DNA
strand exchange. In an attempt to reduce the level of complex
aggregates produced by RecA
The pH-reaction profile for the wild-type protein exhibits more up and
down fluctuations around the smooth curve drawn
in the bottom panel of Fig. 9 than is evident in
the curves for the C-terminal deletion mutant proteins. Significant
scatter is often seen in results obtained in DNA strand exchange
experiments. However, much of the variation seen here for the wild-type
protein is not attributable to experimental scatter but instead
represents reproducible variation (experiments were repeated four
times, with S.D. not exceeding 6%), reflecting the use of different
buffers in adjacent points (e.g. HEPES versus
Tris). The fluctuations are most evident between pH 7 and 8.5. At a
given pH, the particular buffer employed in the experiment can have a
significant effect on the yield of strand exchange products, if all
other reactants and parameters are held constant. However, notably,
this effect is evident only with the wild-type protein. The deletion
mutants exhibit no significant difference in strand exchange product
yield with the different buffers of similar pH used here.
This study was carried out to more precisely define the effects of
RecA protein C-terminal deletions on fundamental RecA protein functions
both in vivo and in vitro. The C-terminal
deletions of the E. coli RecA protein have much more
significant and complex effects on the activity of the protein than
previously appreciated, signaling a more robust role of the C-terminal
domain in RecA function. There are three major conclusions. First, we
have identified the first in vivo deficiency of C-terminal
RecA deletion mutants. Whereas strains harboring C-terminal deletion
mutants of RecA are not more UV-sensitive than normal, as previously
reported (17, 37), and are not more sensitive to ionizing radiation, they do exhibit a significant increase in sensitivity to the DNA cross-linker mitomycin C. Second, we can attribute much of the in
vitro enhancement of binding to dsDNA, previously observed in
RecA The difference in the in vivo sensitivities to DNA damage
caused by UV or ionizing irradiation and mitomycin C observed for the
RecA C-terminal deletion mutants suggests that the C terminus of RecA
protein is critical to RecA function only when the cell faces
particular DNA metabolic stresses. The C-terminal deletion mutants do
not confer greater sensitivity to UV or ionizing radiation challenges.
However, strains expressing the RecA This study was designed not only to examine the role of the C terminus
of the RecA protein but also to determine more precisely which group or
groups of negative charges located at the C terminus of the RecA
protein are responsible for activity changes. The most significant
change in activity previously reported for C-terminal deletion mutants
is the improvement in the rate of nucleation onto dsDNA at
physiological pH (13, 14). The major improvement in binding to dsDNA
occurs in the transition from RecA Some of the most dramatic effects of the C-terminal deletions are
seen in the pH-reaction profile for DNA strand exchange. The wild-type
RecA protein promotes a fairly facile strand exchange reaction between
pH 6 and 7.5, with a maximum near pH 7. The yield of strand exchange
products then declines gradually until reaction is largely eliminated
above pH 8.6. Progressive deletion of 13 or 17 amino acid residues from
the C terminus leads to a marked upward shift of the pH-reaction
profile (RecA The C-terminal deletions and the groups affected by them could be
altering any of several steps of DNA strand exchange. The lack of
strand exchange at high (wild-type and RecA The patterns evident in Fig. 9 are complex. However, the results
suggest that the C terminus interacts with the rest of the protein and
affects conformational transitions that must occur during RecA
protein-mediated DNA strand exchange. We propose that there are
multiple salt bridges that exist at least part of the time between Glu
and Asp residues in the C terminus and positively charged groups
elsewhere in the protein. The pKa values of
additional groups involved in DNA strand exchange may be affected by
the changes in conformation that occur or do not occur as C-terminal residues are deleted. Multiple scenarios are possible, but a minimal working hypothesis might involve the ionization state of two different residues (both outside the C terminus), such that both the ascending and descending legs of the pH-reaction profile are shifted upward by
multiple pH units. It is possible, of course, that the observed pH-reaction effects reflect the ionization of more than two groups. Recent work from the Record laboratory (51) has documented the importance of salt bridges in mediating the extensive binding of DNA to
a protein surface. A similar phenomenon may mediate the binding of the
duplex DNA to the RecA nucleoprotein filament during DNA strand exchange.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C25 (RecA5327), and
Benedict and Kowalczykowski (14) described a RecA mutant in which a
fragment of the protein, about 15% of the of the RecA polypeptide, was
missing from the C terminus. The primary reported effect of these
deletions was a faster nucleation, leading to filament formation on
dsDNA, reducing the telltale lag in dsDNA-dependent ATP
hydrolysis that is well documented with wild-type RecA (15, 16). It was
proposed that the negatively charged C terminus of RecA regulates the
binding of RecA to dsDNA by electrostatically repelling the phosphate
backbone of the DNA (13, 14). Both C-terminal deletion mutants were
shown to be proficient in the key RecA protein reaction of DNA pairing,
and the RecA
C25 protein promoted significant levels of final product
formation during DNA strand exchange under at least two sets of conditions.
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Fig. 1.
The C terminus of the E. coli
RecA protein. The core domain (amino acids 33-270), highly
conserved among bacterial RecA proteins, is shown in white.
This region includes the P-loop motif for ATP binding. 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 25 amino acids of
the RecA protein (residues 328-352) is diagrammed below the linear
sequence. These residues are disordered in the crystal structure of
Story et al. (1). 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, RecA
C17, and RecA
C25.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 cm
1 (20). SSB was also
purchased from Sigma. Unless otherwise noted, all reagents were
purchased from Fisher and were of the highest grade available.
EcoRI and HindIII restriction endonucleases were purchased from New England Biolabs. ATP
S was purchased from Roche Molecular Biochemicals. DTT and TAPS were obtained from Research Organics. CHES, MES, HEPES, lysozyme, phosphoenolpyruvate (PEP), pyruvate kinase, ATP, polyethyleneimine, bromphenol blue, mitomycin C,
and NADH were purchased from Sigma. XhoI restriction
endonuclease and DEAE-Sepharose Fast Flow resin were purchased from
Amersham Biosciences. Hydroxyapatite resin was obtained from Bio-Rad.
Isopropyl-1-thio-
-D-galactopyranoside was purchased from BioVectra.
X174 circular
single-stranded DNA (virion) was purchased from New England Biolabs.
X174 RF I supercoiled circular duplex DNA was purchased from
Invitrogen. Full-length linear duplex DNA was generated by the
digestion of
X174 RF I DNA (5386 bp) with the XhoI
restriction endonuclease, using conditions suggested by the enzyme
supplier. The digested DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1), followed by ethanol precipitation. Circular single-stranded and supercoiled circular duplex DNAs from bacteriophage M13mp8 (7229 nucleotides) was prepared using previously described methods (21-23). Circular M13mp8 duplex DNA containing a single nick
(nicked circular dsDNA) was generated using a previously described
method (24). The concentrations of ssDNA and dsDNA were determined by
absorbance at 260 nm, using 36 and 50 µg ml
1
A260
1, respectively, as conversion
factors. All DNA concentrations are given in µM nucleotides.
exoIII
endoI
recJ
) provided by Susan Lovett. The strain is
also named RDK1896 (27). Plasmid pT7POL26 codes for T7 RNA polymerase
under the control of a lac promoter (28). Eight liters of
culture were grown in LB broth to an A600
of 0.5, and RecA protein expression was induced by the addition of
isopropyl-1-thio-
-D-galactopyranoside to 0.4 mM. Following a 3-h incubation at 37 °C, ~20 g of
cells were harvested by centrifugation, flash-frozen in liquid
N2, and stored at
80 °C.
C6, recA
C13, and
recA
C17 genes were constructed by
oligonucleotide-directed mutagenesis using the Kunkel selection method
(29). An EcoRI-HindIII fragment from pAIR79
coding for the C terminus region of the recA ORF was cloned
into the AmpR plasmid pGEM3Zf(
) from Promega to create
plasmid pAIR34. This plasmid was used as the template for the in
vitro mutagenesis. Oligonucleotides encoding stop codons were used
to introduce new translational stop sites at the appropriate positions
in the C-terminal coding region. Both strands of the subcloned region
were manually sequenced to ensure that no other mutations were
introduced during mutagenesis. Cloning was used to swap these
C-terminal coding regions into plasmid pAIR79. The plasmid containing
the recA
C25 gene (pTH5327) was a gift from Tomoko Ogawa
(13). The plasmids containing the mutant genes recA
C6,
recA
C13, and recA
C17, designated pAIR64,
-45, and -46, respectively, were each co-transformed with pT7POL26
(KanR) (28) into the nuclease deficient strain STL2669
(
recA-srlR)306::Tn10 TetR, xonA2 (exoI
) in
an AB1157-derived genotypic background; a gift from Susan Lovett).
Culture growth and protein induction conditions were the same as for
the wild-type RecA protein.
C6, RecA
C13, and
RecA
C17 proteins were each integrated into the identical site on the
chromosome (base 1899 of the rmlD gene of MG1655
recA tetS 127fRT#1/pLH29 (30) using the FLIRT
system (30). FLIRT makes use of the Flp site-specific recombination
system to efficiently introduce cloned DNA onto the bacterial
chromosome. In each case, the proteins were expressed from the
wild-type recA promoter. The plasmids used to introduce wild-type
recA, recA
C6, recA
C13, and
recA
C17 to the chromosome are designated pEAW 222, 232, 230, and 229, respectively. The wild-type recA promoter was
added to the start of each recA gene by including the 200 base pairs of the DNA sequence upstream of the recA gene in
MG1655 (a gift from George Weinstock). Multiple attempts to clone the
recA
C25 gene onto the chromosome in the same manner as
the others were unsuccessful.
C6, RecA
C13, or RecA
C17 Proteins to UV, Mitomycin C, and
Ionizing Radiation--
10 ml of LB broth was inoculated with 100 µl
of an overnight culture of the
recA MG1655 strain
containing the wild-type recA, recA
C6, recA
13, or
recA
C17 genes described above and grown for
1.5 h at 37 °C. Cells were pelleted in a clinical centrifuge and washed with 5 ml of M9 minimal medium. Cells were pelleted again
and resuspended in 5 ml of M9 minimal medium. Cells were diluted
further in M9 to an A600 of 0.06 in a volume of
5 ml. Further serial 1:10 dilutions were made in M9 to optimize cell density to obtain ~30-100 colonies per experiment. The various dilutions (50 µl of each) were plated on LB agar plates. Uncovered plates were exposed to UV using a Stratalinker UV cross-linker, model
1800 (Stratagene) in a darkened room at the J/m2 indicated
in the figure. The plates were incubated overnight at 37 °C in the
dark. Colonies were counted using GelExpert software (Nucleotech) and
adjusted for dilutions. Mitomycin C and ionizing radiation sensitivity
tests were performed as with the UV tests above except that diluted
cells were plated on LB agar plates containing the indicated amount of
mitomycin C or exposed to ionizing radiation in a model 30 Mark I 137 cesium irradiator (J. L. Shepard and Associates) for times necessary
to achieve the indicated dose and incubated overnight in the dark.
80 °C. The
concentration of the wild-type RecA protein (37,842 Da) was determined
from the absorbance at 280 nm using the extinction coefficient
2.23 × 104 M
1
cm
1 (33). The protein was free of detectable nuclease activities.
C6, RecA
C13, RecA
C17,
and RecA
C25 were purified using the following modifications to the
wild-type RecA procedure from above. (a) The initial
polyethyleneimine pellet was washed with R buffer plus 50 mM ammonium sulfate and extracted two times with R buffer
plus 150 mM ammonium sulfate. The RecA
C6, RecA
C13,
and RecA
C17 proteins were precipitated with 0.31 g of solid
ammonium sulfate per ml of solution (48% saturation). The RecA
C25
protein solution was precipitated with 0.28 g of solid ammonium
sulfate per ml of solution. The RecA
C25 protein was precipitated
from the supernatant fraction after centrifugation with an additional
0.113 g of solid ammonium sulfate per ml of solution (to 65%
saturation). Each deletion mutant protein was resuspended in R buffer
plus 300 mM KCl, dialyzed one time versus the
same buffer and then two times versus R + 100 mM
KCl for the RecA
C6, RecA
C13, and RecA
C17 proteins and
versus R buffer for the RecA
C25 protein. (b)
All deletion mutants were eluted from the DEAE-Sepharose column with a
linear gradient of KCl, from 100 to 500 mM for the
RecA
C6, RecA
C13, and RecA
C17 proteins and from 0 to 500 mM for the RecA
C25 protein. All proteins eluted from the
DEAE-Sepharose column at ~250 mM KCl. (c) The
calculated molecular masses of the RecA
C6, RecA
C13, RecA
C17,
and RecA
C25 proteins are 36,999, 36,200, 35,680 and 34,783 Da,
respectively. Each mutant protein was determined to be greater than
95% pure by SDS-PAGE (Fig. 2) and free
of detectable nuclease activities. The wild-type RecA extinction
coefficient was used to calculate the concentration of each mutant
protein.
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Fig. 2.
SDS-polyacrylamide gel of purified wild type
and the four C-terminal deletion RecA proteins. The purification
protocols and the calculated molecular weights of each mutant can be
found under "Experimental Procedures."
C17 Nucleoprotein
Filaments--
A modified Alcian method was used to visualize RecA
filaments and unreacted DNA if present. Activated grids were prepared as follows. 0.2% Alcian in 3% acetic acid was left for many days to
dissolve and then centrifuged in a Millipore 0.22-µm filter unit at
5000 rpm for 40 min. 25 µl of the clear solution was diluted with 500 µl of distilled water. A carbon film (attached to an electron
microscope grid) was floated on a 70-µl drop of this solution (placed
on a Teflon block) for 10 min. The film was then washed by touching to
a drop of water and finally floated on two 3-ml drops of water, each
for 5 min. After the final wash, an empty 5-µl glass pipette was used
to draw off remaining liquid from the edge of the grid by capillary
action. The activated grid was then dried under a heat lamp.
C17
protein (6.7 µM) was preincubated with 20 µM M13mp8 circular ssDNA, 25 mM Tris-OAc
(80% cation) buffer, 1 mM DTT, 5% (w/v) glycerol, 3 mM potassium glutamate, and 10 mM
Mg(OAc)2 for 10 min. An ATP regeneration system of 10 units/ml pyruvate kinase and 5 mM PEP was also included in
the preincubation. ATP and SSB protein were added to 3 mM
and 2 µM, respectively, and the reaction was incubated
for 10 min longer, after which ATP
S was added to 3 mM to
stabilize the filaments and incubated for 5 min. An 8-µl sample of
the reaction mixture described above was diluted 17-fold with 200 mM ammonium acetate, 10 mM Hepes (pH 7.0), and
10% glycerol and adsorbed to the activated carbon film for 3 min. The
grid was then touched to a drop of the above buffer followed by
floating on a drop of the same buffer for 1 min. The sample was stained
by touching to a drop of 5% uranyl acetate followed by floating on a
fresh drop of the same solution. Finally, the grid was washed by
touching to a drop of water followed by immersion in two 10-ml beakers
of water and one beaker of ethyl alcohol. After the sample was dried,
it was rotary-shadowed with platinum. This protocol is designed for
visualization of complete reaction mixtures, and no attempt was made to
remove unreacted material. Although this approach should yield results
that give a true insight into reaction components, it does lead to
samples with a high background of unreacted proteins. Photography and measurement of filament and DNA length were performed as described previously (34).
C6, RecA
C13, RecA
C17, and
RecA
C25 proteins. The regeneration of ATP from PEP and ADP was
coupled to the oxidation of NADH and followed by the decrease in
absorbance of NADH at 380 nm (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, or RecA
C25 proteins indicated in
the table or figure legends. Unless otherwise noted, ATPase assays that
included RecA
C25 protein also included an additional 40 mM potassium glutamate. The aforementioned components were
incubated for 10 min. The assay was initiated by the addition of the
SSB protein (to 0.8 µM) and the concentration of ATP
indicated in the figure legends. SSB protein was omitted from reactions
with nicked circular dsDNA.
C6, RecA
C13, RecA
C17, or RecA
C25 protein (6.7 µM)
was preincubated with 20 µM
X174 circular ssDNA for 10 min. SSB protein (2 µM) and ATP (3 mM) were
then added, followed by another 10-min incubation. The reactions were
initiated by the addition of
X174 linear dsDNA to 20 µM. A 10-µl aliquot was removed to use for a zero time
point, The reaction was incubated, and at the indicated time points, 10-µl aliquots were removed and the reaction was stopped by the addition of 5 µl of a solution containing 60 mM EDTA, 6%
SDS, 25% (w/v) glycerol, and 0.2% bromphenol blue. Samples were
subjected to electrophoresis in 0.8% agarose gels with 1× TAE buffer,
stained with ethidium bromide, and exposed to ultraviolet light. Gel
images were captured with a digital CCD camera utilizing GelExpert
software (Nucleotech). When indicated, the intensity of DNA bands was
quantitated with the software package TotalLab version 1.10 from Phoretix.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C6 deletion removes 6 amino acid residues from the C terminus, 3 of which are negatively charged: Glu347,
Glu350, and Asp351. The RecA
C13 deletion
removes an additional 7 residues, 3 of which contribute negative
charges: Glu343, Asp341, and
Asp340. The RecA
C17 deletion removes an additional 4 residues, 1 of which is negatively charged: Asp336. The
RecA
C25 deletion removes an additional 8 residues but does not
remove any further negative charges. Because the RecA
C25 mutant
(RecA5327) was previously characterized by Ogawa and co-workers (13),
this construct provided a good basis with which to compare our results
with published work. The RecA
C25 protein exhibited a tendency to
precipitate with loss of activity under standard reaction conditions.
To stabilize the protein, 40 mM potassium glutamate was
added to reactions with this mutant except where noted.
C25 protein from a plasmid is not more UV-sensitive than a
strain expressing the wild-type RecA protein. Fig.
3A shows that strains expressing the RecA
C6, RecA
C13, and RecA
C17 proteins from the chromosome are also as resistant to UV irradiation as the strain expressing the wild-type protein.
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Fig. 3.
Survival curves for E. coli
strains harboring the wild-type recA
gene (closed triangles), no
recA gene (closed
circles), the recA C6
gene (open circles), the
recA
C13 gene (open
triangles), or the
recA
C17 gene (open
squares) as a function of UV dose
(top), mitomycin C concentration
(middle), or ionizing radiation dose
(bottom). See "Experimental Procedures" for
conditions. The
recA strain was extremely sensitive to
these agents; therefore, cell survival could not be monitored above 15 J/m2 UV, 1.0 µg/ml mitomycin C, or 25 krads of ionizing
radiation.
C6 was as resistant to
mitomycin C as the strain expressing wild-type RecA protein. However,
strains expressing the RecA
C13 or RecA
C17 proteins from the
chromosome exhibited an enhanced sensitivity to this reagent. The
strains harboring the RecA
C13 or RecA
C17 protein are ~1000-fold
more sensitive to 2 µg/ml mitomycin C than the strain expressing
wild-type RecA protein (Fig. 3B). This experiment was
repeated twice with consistent results. It was not possible to test the
RecA
C25 mutant in this experiment, since it proved refractory to
cloning on the chromosome using the protocol used for the other mutants
(see "Experimental Procedures"). In contrast to the results
obtained with mitomycin C, the presence of the C-terminal deletions had
no evident effect on the sensitivity of cells to ionizing radiation
(Fig. 3C).
C17 Forms Full and
Extended Filaments on Circular ssDNA--
In order to confirm that the
RecA deletion mutants were forming complete, extended filaments, we
analyzed RecA
C17-ssDNA nucleoprotein filaments by electron
microscopy and compared them with wild-type RecA-ssDNA filaments (Fig.
4A). Measurements indicate
that both the RecA
C17 and the wild-type RecA filaments are well
extended by 1.66- and 1.67-fold, respectively, relative to
double-stranded M13mp8 DNA (Fig. 4, B and
C).
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Fig. 4.
RecA C17-ssDNA
nucleoprotein filaments analyzed by electron microscopy.
A, an electron micrograph showing the RecA
C17 protein
coating circular ssDNA to form a nucleoprotein filament. The
top inset shows an enlargement of a portion of
the RecA
C17 full filament; the bottom inset
shows an enlargement of a wild-type RecA filament (full filament not
shown) formed under the same conditions as the mutant (see
"Experimental Procedures"). Measured lengths of these filaments are
shown as a histogram comparing the length of the wild-type RecA
nucleoprotein filament (B) and the RecA
C17 filament
(C) with the length of duplex DNA (7.25 kilobase
pairs).
1
and is reduced to under 20 min
1 when the RecA is bound to
dsDNA or is promoting DNA strand exchange (3, 38, 39).
C17 protein concentration in Fig.
5. The shape of the titration curves is similar for both proteins, although the somewhat lower rate of ATP
hydrolysis seen at each point for the C-terminal mutant might be
interpreted as reflecting a somewhat reduced intrinsic affinity for
ssDNA. This type of data was obtained for all of the proteins, verifying that the concentration of each protein needed to measure the
maximal rate of circular ssDNA-dependent ATP hydrolysis is approximately the same (data not shown). The apparent turnover number
(kcat, the number of ATP molecules hydrolyzed
per RecA molecule per unit of time) given for each of the proteins in
Table I was determined using Vmax
(µM ATP/min) rates at saturating RecA protein
concentrations and dividing by the concentration of RecA binding sites
in the DNA (assuming that all are bound by RecA). All of these RecA
variants appear to have similar intrinsic rates of
ssDNA-dependent ATP hydrolysis under these conditions.
ATP hydrolysis turnover numbers (kcat) for the wild-type and
C-terminal deletion mutant proteins
1 deviation. The values reported are
the average of the two measurements. ND, not determined.
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Fig. 5.
The rate of ATP hydrolyzed as a function of
wild-type RecA (circles) or
RecA C17 (squares) protein
concentration. These data are representative of the protein
titration data collected for all mutants investigated in the current
study. The reactions were carried out as described under
"Experimental Procedures" at pH 7.3 and included 8 µM
circular ssDNA, 3 mM ATP, 0.8 µM SSB protein,
and the indicated concentrations of the wild-type and RecA
C17
proteins. Each point on the titration curve is an average of three
(wild type) and seven (
C17) independently collected data points.
Error bars for the wild type and RecA
C17
averaged data point are shown on the right and
left, respectively.
C6, RecA
C13, RecA
C17, and RecA
C25 proteins (Fig.
6). The reactions were carried out at pH
7.3 (Fig. 6A) or at pH 6.0 (Fig. 6B). Potassium
glutamate was not included in the RecA
C25 reaction at pH 7.3, since
the instability of the protein was less evident at this pH, and in this
one case the rate of the reaction was greater in the absence of the
added salt. The rates given in Fig. 6C were calculated using
linear regression on the amount of ATP hydrolyzed during the
steady-state achieved after binding appeared to be complete. The
apparent kcat was determined by assuming that
all DNA binding sites on the DNA were occupied by RecA. The binding to
dsDNA by the wild-type and RecA
C6 was so slow as to make it unlikely
that a saturated binding state had been achieved during the time of the
experiment, and those rates are not included in Table I.
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Fig. 6.
The effect of C-terminal deletions on the
binding to dsDNA at pH 7.3 and 6.0. The DNA cofactor is nicked
circular duplex M13mp8 DNA. The reactions were performed under
conditions described under "Experimental Procedures" at pH 7.3 (A) or pH 6.0 (B) and included 2 µM
wild-type or mutant RecA protein, 4.2 µM nicked circular
dsDNA, and 3 mM ATP. The inset to A
is an expansion of the early times in the assay and is intended to
highlight the difference in lag times between the RecA C25,
RecA
C17, and RecA
C13 proteins. The dotted
lines are extrapolations back to the x axis from
the linear segments (reflecting the steady state) of the curved
lines achieved at late reaction times. The maximum rates of
ATP hydrolysis at pH 7.3 and 6.0 are plotted in the histogram shown in
C. The rates indicated are averaged rates recorded over six
(pH 7.3) or three (pH 6.0) independent experimental runs (one
representative experiment for each pH is shown in A and
B).
170 min. The RecA
C6 exhibited a long lag as
well,
100 min. The major increase in the rate of binding was observed
when 13 amino acid residues were deleted from the C terminus. The
RecA
C13 and RecA
C17 mutants exhibited binding lags of about 24 and 20 min, respectively (Fig. 6A, inset). The RecA
C25 protein bound faster than any of the others, exhibiting a
lag of only about 7 min.
X174 Substrates--
We determined the effect of the
C-terminal deletion mutations on the fundamental RecA protein-promoted
DNA strand exchange reaction (Fig. 7).
The mutants are able to promote homologous pairing, indicated by the
formation of joint molecules as evident in the agarose gels.
Furthermore, the mutants were able to promote a complete DNA strand
exchange reaction, as indicated by the production of nicked circular
product. Under these conditions (pH 7.3), the effect that these
C-terminal deletions appear to have on the RecA-facilitated DNA strand
exchange reaction is in the kinetics and the extent of final product
formation. In the wild-type RecA protein reaction, final nicked
circular products appear in the 15-min time point. Significant amounts
of these products first appear in the 30-min time point for RecA
C6,
RecA
C13, and RecA
C17 reactions and in the 60-min time point for
the RecA
C25 reactions. Furthermore, reactions promoted by the
RecA
C17 and RecA
C25 proteins do not reach the same levels of
final nicked circular product as the other proteins in 90 min, and the
RecA
C25 was consistently quite weak.
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Fig. 7.
DNA strand exchange reactions promoted by the
wild-type, C6,
C13,
C17, and
C25 RecA proteins. Reactions were carried out
as described under "Experimental Procedures" at pH 7.3 and included
3 mM ATP. Aliquots of the reaction were removed and stopped
at the times indicated above the individual lanes
and subjected to agarose gel electrophoresis. The joint molecule
intermediates (I) and nicked circular products
(P) are distinguishable from the circular ssDNA
(S2) and the linear duplex DNA
(S1) substrates of the reaction by their relative
mobilities. Lane M, nicked circular and
supercoiled dsDNA marker.
C6 proteins was increased, somewhat stronger
than the wild-type RecA reaction at this pH. Conversely, the
RecA
C13, RecA
C17, and RecA
C25 proteins were not able to
promote a complete DNA strand exchange (evidenced by the lack of
formation of final nicked circular product) at this lower pH, and even
the formation of joint molecule intermediates was reduced.
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Fig. 8.
DNA strand exchange reactions promoted by the
wild-type and mutant RecA proteins at pH 6.0. The DNA strand
exchange conditions are described under "Experimental Procedures"
at pH 6.0 and included 3 mM ATP. The labels are detailed in
the legend to Fig. 7. Lane M, nicked circular
(P) and supercoiled dsDNA marker.
C13 and RecA
C17, the ATP concentration
was reduced to 400 µM in the experiment shown in Fig.
9. RecA
C25 was not included in this
analysis. The top four panels of Fig.
9 show the nicked circular product formation promoted in 90 min by the
wild-type and variant proteins as a function of reaction pH. The final
product yield is quantified for all four proteins in the
bottom panel of Fig. 9. The wild-type protein exhibits a reaction optimum near pH 7, trailing off gradually at higher
pH values until the reaction is eliminated at pH values above 8.6. The
reactions seen with RecA
C6 are similar although reproducibly
stronger than wild-type reactions at low pH values. The entire
pH-reaction profile is markedly shifted to higher pH values for the
RecA
C13 and RecA
C17 mutant proteins (Fig. 9). Similar results
were obtained in a set of trials at 3 mM ATP, although the
RecA
C6-promoted reaction did not fall off quite as abruptly at pH
values above 7.5 (data not shown).
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Fig. 9.
Dependence of the wild type- and mutant RecA
protein-promoted DNA strand exchange reaction on pH. DNA strand
exchange reactions were performed under the conditions described under
"Experimental Procedures" at the pH indicated and included 400 µM ATP. The labels are detailed in the legend
to Fig. 7. Lane M, substrate linear dsDNA
(S1). In the bottom panel, the
product formation by the wild type and the C-terminal deletion mutants,
C6,
C13, and
C17 is quantified as a function of pH.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C25 (13), to deletion of residues between positions 339 (RecA
C13) and 346 (RecA
C6). A smaller but significant enhancement of binding is also observed when the residues between positions 327 (RecA
C25) and 335 (RecA
C17) are deleted. Third and finally, short
C-terminal deletions have dramatic effects on the pH-reaction profile
of the DNA strand exchange reaction. This result indicates that the C
terminus of the wild-type protein affects the pKa of
at least two groups in other parts of the protein that are important in
the DNA strand exchange reaction and suggests a potentially extensive
and complex interaction between the C terminus and the rest of the protein.
C13 and RecA
C17 proteins are
1000-fold more sensitive to 2 µg/ml of mitomycin C. The DNA-damaging
agent mitomycin C is known to introduce interstrand cross-links into
duplex DNA (40). The sensitivity observed in strains expressing the
RecA
C13 and RecA
C17 mutant proteins is not evident until a
threshold of 1 µg/ml of mitomycin C in the growth medium is reached.
This suggests that the mutants have a deleterious effect on repair and
cell survival only when the load of DNA cross-links exceeds a
particular level. The repair of such cross-links is thought to require
the recombinase function of RecA, the excinuclease function of
the UvrABC complex and the strand synthesis function of the DNA
polymerase I protein (41-43). It is possible that the direct
action of RecA protein at the site of interstrand cross-link repair
involves the C terminus of the protein, perhaps as an interaction site
for other proteins involved in the repair process. The result
reinforces the overall hypothesis that some RecA functions are quite
specialized and needed only in certain circumstances (44).
C6 to RecA
C13, which involves
deletion of the negatively charged residues Glu343,
Asp341, and Asp340. However, a smaller but
significant improvement in binding is seen when the RecA deletion is
increased from 17 to 25 amino acid residues (residues 328-335), and
there are no additional negatively charges removed from the polypeptide
in this region. Thus, the slow binding to dsDNA by wild-type RecA
protein may be ascribed in some measure to the removal of a protein
segment that imparts some other property besides negative charge.
C13 and RecA
C17) such that the strand exchange
reaction is eliminated at low pH and enhanced at high pH.
C6) or low (RecA
C13
and RecA
C17) pH could reflect a disruption of RecA filament
formation. However, over much of the pH range investigated, DNA pairing
intermediates suggesting the presence of minimally functional filaments
were present even when complete product formation was not observed.
Alternatively, the C-terminal deletions might affect a conformational
change to a form active in strand exchange, or ATP hydrolysis during
DNA strand exchange. When RecA protein is bound to ssDNA, it is in a
conformation that exhibits limited cooperativity and little
exchange of RecA monomers between free and bound forms. When homologous
duplex DNA is introduced, the conformation changes to a form that is
more cooperative and more dynamic (38, 45), and the rate of ATP
hydrolysis drops about one-third (39). ATP hydrolysis also becomes
coupled to DNA strand exchange in this mode (32, 46-50). The
C-terminal deletions could be affecting the ionization properties of
groups critical to the conformation change, and/or to the hydrolysis of
ATP in the new conformation.
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ACKNOWLEDGEMENTS |
---|
We thank Ruth Saecker and Tom Record for useful discussions; Kendall Knight, Tomoko Ogawa, and George Weinstock for plasmids; and Susan Lovett and Steven Sandler for strains and for helpful discussions.
![]() |
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.
Present address: Dept. of Biochemistry MS 140, Rice University,
6100 Main St., Houston, TX 77005-1892.
§ 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.M212917200
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ABBREVIATIONS |
---|
The abbreviations used are:
ssDNA, single-stranded DNA;
SSB, single-stranded binding protein;
dsDNA, double-stranded DNA;
ATPS, adenosine
5'-O-(thiotriphosphate);
DTT, dithiothreitol;
TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic
acid;
CHES, 2-(cyclohexylamino)ethanesulfonic acid;
MES, 4-morpholineethanesulfonic acid;
PEP, phosphoenolpyruvate;
ORF, open
reading frame;
WT, wild type.
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REFERENCES |
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