From the
The functional significance of residues in the RecA protein
P-loop motif was assessed by analyzing 100 unique mutants with single
amino acid substitutions in this region. Comparison of the effects on
the LexA coprotease and recombination activities shows that Pro
The bacterial RecA protein plays at least two distinct roles
related to its function as a DNA repair enzyme. RecA catalyzes a strand
exchange activity between homologous DNAs (reviewed in Refs. 1-4)
and also, in response to DNA damage, becomes activated for a coprotease
function
(5, 6) . In this latter role RecA mediates the
autoproteolysis of the LexA repressor, which in turn increases the
expression of a number of genes including recA involved in
cell survival following DNA damage
(7, 8) . Both the
recombination and coprotease functions require a nucleoside
triphosphate (NTP) cofactor; however, coprotease activity requires only
NTP binding
(9, 10) , whereas completion of DNA strand
exchange reactions depends on NTP hydrolysis as well (reviewed in Ref.
11). The ATP binding site of the RecA protein contains a consensus
phosphate binding loop (P-loop)(
In previous
mutagenesis studies, which focused on the recombination activities of
RecA, we found that while the identity of the consensus residues in
this motif is strictly defined (Gly
We also describe the first characterization of mutants with a
rec
Cell lysis and extraction
of the RecA protein using polyethylenimine was performed as described
(24) to generate fraction II. Fraction II was dialyzed
extensively against R buffer (20 m
M Tris-HCl, pH 7.5, 5%
glycerol, 5 m
M
We have previously characterized 100 recA mutants
carrying single amino acid substitutions in the P-loop (residues
66-74) regarding their ability to perform recombinational DNA
repair and homologous genetic recombination in vivo (16) . Thirty-seven of these mutants were fully or
partially active for one or both of these functions, and the remaining
63 were completely inhibited for both
(16) . In this study we
have measured both the coprotease and DNA repair activities and
compared the effects of each mutation on these different functions. We
used two in vivo assays to measure the RecA coprotease
activity toward LexA repressor and have been able to separate all
mutants into three phenotypic categories: coprt
These coprt
A summary of all mutations and their effect on
coprotease activity is shown in Fig. 2.
In the present study we have analyzed 100 unique recA mutants, each carrying a single amino acid substitution in one of
9 residues that define the P-loop motif, and we find that Pro
We have used
the RecA/ADP crystal structure
(14, 17) as a model for
the assessment of the data presented here. In this structure the main
chain atoms of the RecA P-loop motif are arranged in a remarkably
similar orientation to those in the P-loop motif of adenylate kinase,
elongation factor Tu (EF-Tu), and p21
(14) , suggesting a
conservation of the functional properties of these residues. For RecA,
EF-Tu, and p21, the
The
crystal structure of RecA shows that the ATP binding site is near the
surface of each protein monomer such that it lies on the inner side of
the helical, oligomeric filament (Fig. 6; also see Refs. 14, 16, and
17). The side chains of 3 P-loop residues (Pro
We have found that for
positions 68 and 69 all mutations have equivalent effects on the
coprotease and recombination activities. Because DNA is a required
cofactor for the coprotease activity, mutations that alter DNA binding
may effect both functions similarly. However, at position 67 mutations
occur that differentially modify the recombination and coprotease
activities. A particularly striking example of this is seen by
comparing the Lys and Glu mutants, two substitutions that have similar
inhibitory effects on the recombination activities but precisely
opposite effects on the coprotease activity. Pro
Mutations at residues 229 (Gly
Biochemical studies of the purified Pro
The relaxed
nucleotide specificity of the Pro
We
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
is unique among these residues because only at this position did
we find substitutions that caused differential effects on these
functions. One mutant, Pro
Trp, displays high
constitutive coprotease activity and a moderate inhibitory effect on
recombination functions. Glu and Asp substitutions result in low level
constitutive coprotease activity but dramatically reduce recombination
activity. The purified Pro
Trp protein shows a
completely relaxed specificity for NTP cofactors in LexA cleavage
assays and can use shorter length oligonucleotides as cofactors for
cleavage of
cI repressor than can wild type RecA. Interestingly,
both the mutant protein and wild type RecA can use very short
oligonucleotides, e.g. (dA)
and (dT)
,
as cofactors for LexA cleavage. We have also found two mutations at
position 67, which are completely defective for LexA coprotease
activity in vivo but still maintain recombinational DNA repair
(Pro
Lys) and homologous recombination (Pro
Lys and Pro
Arg) activities. These
findings show that the recombination activities of RecA are
mutationally separable from the coprotease function and that Pro
is located in a functionally important position in the RecA
structure.
)
motif, a highly
conserved element of sequence
(12, 13) and structure
(14) that plays a central role in coupling NTP binding and
hydrolysis events to enzyme activity
(13) . The RecA P-loop
motif is defined by the sequence
GPESSGKTT
and is absolutely conserved among all eubacterial RecA proteins
for which the sequence is known
(1) , suggesting functional
and/or structural importance for each residue.
, Gly
,
Lys
, and Thr
), the other residues supported
varying levels of substitution
(15, 16) . In this study
we have compared the effects of mutations in the P-loop on the LexA
coprotease and recombination activities of RecA and find that most
mutations have similar effects on both functions. However, mutations at
Pro
cause differential effects on these activities. One of
these mutants, Pro
Trp, displays a high
constitutive coprotease activity, i.e. activity in the absence
of DNA damage. Repressor cleavage assays using the purified mutant
protein show that the catalytic properties are similar to other
constitutive RecA mutant proteins despite the fact that the amino acid
substitutions occur within different areas of the protein structure.
/coprt
phenotype,
i.e. mutants that are completely defective for LexA coprotease
activity in vivo but retain some level of recombinational DNA
repair and/or homologous recombination activity. Our results are
discussed in the context of the crystal structure of the RecA protein
(14, 17) and provide information regarding the
relationship of certain P-loop residues and mutant side chains to RecA
function.
Materials
MacConkey-lactose plates were prepared
according to the manufacturer (Difco) and contained 0.5% lactose and
100 µg/ml ampicillin. Other media (LB broth and LB agar) were
prepared as described
(18) and contained 100 µg/ml
ampicillin. Stock solutions (4 mg/ml) of ONPG
( o-nitrophenyl--
D-galactopyranoside; Sigma) were
made in Z-buffer
(19) . Mitomycin C was from Sigma or Boehringer
Mannheim. Protein concentrations in cell extract supernatants were
determined using a protein assay kit from Bio-Rad or Pierce. Purified
LexA protein was a generous gift from Donald Shepley and John Little
(Department of Biochemistry, University of Arizona) and purified
cI repressor was a generous gift from Bronwen Brown and Bob Sauer
(Department of Biology, Massachusetts Institute of Technology). NTPs,
dNTPs, and ddNTPs were from Pharmacia Biotech Inc. Radiolabeled NTPs
and dNTPs were from DuPont NEN. Single-stranded RV-1 DNA, an M13
derivative
(20) , was used for in vitro repressor
cleavage and NTPase assays and was purified as described
(21) .
Defined length (dA) and (dT) oligonucleotides were made using an
Applied Biosystems model 392 DNA/RNA synthesizer.
Strains and Plasmids
All mutant recA genes in this work are carried on plasmids derived from pTRecA103,
a pZ150-based plasmid
(21) which has the wild type recA gene under control of P(15) . Mutations were
introduced using cassette mutagenesis and have been described
previously
(16) . Plasmid pZ150
(21) was used as a
recA
control plasmid in the in vivo assays described below. Escherichia coli strain DE1663`,
which is
recA and carries the lacZ and Y genes
under control of the recA operator/promoter, was used for
in vivo assays of both DNA repair and LexA coprotease
activities of all plasmid borne recA mutants. DE1663` was
constructed by mating strain DE1663, a
( recA-srlR)
306::Tn 10
( lac-argF) U169 sulA211
malB::Tn 9 (
cI ind-1
recAo/p:: lacZY) derivative of AB1157, with strain
DE1781 which carries an F` lacI
lacPL8
lacZ4505::Tn 5 proA
B
episome. Both DE1663 and DE1781 were generous gifts from Don
Ennis (National Institutes of Health).
Cellular Levels of RecA Protein
All in vivo assays described below were performed in the absence of
isopropyl-1-thio--
D-galactopyranoside. Under these
conditions the basal level of expression of recA from
pTRecA103 is approximately 20-fold greater than from the chromosomal
recA gene in wild type E. coli (15) . This
increased basal level of expression and the inability of DNA damaging
agents to increase recA expression have little or no effect on
the ability to distinguish different classes of mutants using assays
for both the recombination and coprotease function of RecA
(15, 16, 22, 23) . Western blot analysis
showed that all mutants in this study produced levels of RecA protein
comparable to that from the wild type recA plasmid
(15) .
RecA-mediated LexA Cleavage in Vivo
Because strain
DE1663` carries the lacZ and Y genes under control of a
LexA-regulated promoter measurement of -galactosidase activity is
directly related to the extent of RecA-mediated LexA cleavage. Two
assays were used for the measurement of
-galactosidase activity. 1) Cultures carrying plasmid borne recA mutants were
grown overnight in LB-ampicillin medium at 37 °C and diluted 1:40
in the same medium, and 1.5 µl was spotted onto two
MacConkey-lactose plates, one containing 0.05 µg/ml mitomycin C and
the other containing no mitomycin C. Plates were incubated overnight at
37 °C. Colonies that express
-galactosidase activity were
red, whereas those in which lacZ remains repressed by
LexA were white. Control strains, DE1663`/pTRecA103
( recA
coprt
) and
DE1663`/pZ150 ( recA
coprt
), were included on each plate. 2)
To obtain a more quantitative measure of RecA-mediated LexA cleavage,
we determined the specific activity of
-galactosidase in the cell
extract of each recA mutant. Overnight cultures were grown in
LB-ampicillin medium, diluted 1:100 in 6 ml of the same medium, and
grown for 2 h at 37 °C. Cultures were divided in half, and to one
half mitomycin C was added to a final concentration of 0.5 µg/ml.
Incubation was continued for 40 min, cultures were chilled on ice for
20 min, and 1.5 ml of cells were pelleted by centrifugation at 4
°C. Cells were washed in 10 m
M NaCl (1 ml) and resuspended
in 1 ml Z buffer
(19) . Cells were lysed by sonication on ice
for 30s, centrifuged for 20 min, and supernatants were stored at 4
°C.
-Galactosidase activity in the supernatants was determined
using ONPG as described
(19) . Units of
-galactosidase
activity are defined as 10
mol ONPG hydrolyzed/min,
and activity is expressed as units/µg protein. Extracts from
control strains (see above) were prepared fresh along with samples for
each experiment. To determine the effect of increased time of exposure
to mitomycin C on the specific activity of
-galactosidase, samples
of control strains and selected mutants were processed at 60, 90, 120,
and 180 min as well as 40 min.
Recombination Phenotype of recA Mutants
The
ability of all mutants in this study to carry out homologous genetic
recombination and recombinational DNA repair has been described
(16) . Homologous genetic recombination was measured by
determining the plating efficiency of a
redgam
Chi
phage on lawns of recA mutants and recombinational
DNA repair was measured by observing cell survival following exposure
to 4-nitroquinoline oxide and UV light
(16) . In the present
study, time courses of UV exposure were performed and fractional
survival at 30 s relative to the positive control was calculated from
the slope of a line obtained from a plot of relative growth versus time of exposure. For certain mutants survival time courses were
performed using several different intensities of UV light. UV intensity
was measured using a UVX radiometer (UVP, Inc., San Gabriel, CA). We
note that recalibration of the radiometer showed that the UV dose in
Ref. 16 was 1.52 J/m
instead of the 0.67 J/m
indicated.
Protein Purification
Both wild type RecA and the
Pro Trp mutant RecA proteins were purified using
the following procedure. All cultures were grown in 2
LB
containing 3.8 mg/ml glucose and 100 µg/ml ampicillin. Four
1.5-liter cultures were inoculated with 50 ml of an overnight culture
grown at 37 °C. Incubation at 37 °C was continued until
A
0.8, at which time
isopropyl-1-thio-
-
D-galactopyranoside was added to a
final concentration of 5 m
M. Incubation was continued for 3.5
h, cells were harvested by centrifugation, and pellets resuspended (25
ml/1.5-liter culture) in a buffer containing 0.25
M Tris-HCl,
pH 7.5, 25% sucrose. Cell suspensions were quick frozen using liquid
nitrogen and stored at -70 °C.
-mercaptoethanol, 0.1 m
M EDTA)
containing 50 m
M NH
Cl, loaded onto a DE-52 column
(30-ml bed volume) equilibrated in the same buffer, and proteins were
eluted with a linear gradient (300 ml) of 50-500 m
M NH
Cl. Both wild type RecA and the Pro
Trp mutant proteins eluted at approximately 180-280
m
M NH
Cl. Fractions containing RecA protein were
pooled and proteins precipitated by addition of ammonium sulfate. The
resulting protein pellet was dissolved in 3 ml of R buffer, 30 m
M NH
Cl and dialyzed extensively against the same to
generate fraction III. MgCl
was then added to a final
concentration of 15 m
M, and the sample was loaded onto a
Sephacryl S-1000 gel filtration column (1.5
120 cm)
equilibrated in R buffer, 50 m
M NH
Cl, 15 m
M MgCl
. Wild type RecA and the Pro
Trp mutant proteins elute in pure form in the void volume of this
column. Fractions containing RecA were pooled and protein precipitated
as above. The precipitate was dissolved in R buffer (200-400
µl) and dialyzed extensively against the same. Glycerol was added
to a final concentration of 25%, and samples (
20 µl) were quick
frozen and stored at -70 °C. RecA proteins were judged to be
at least 95% pure in silver-stained SDS-polyacrylamide gels. The
concentration of wild type RecA protein was determined
spectrophotometrically using an extinction coefficient of
= 0.59 mg
ml
(25) . The
concentration of the Pro
Trp mutant protein was
determined using the Bio-Rad protein assay kit, the BCA protein assay
kit from Pierce and by comparing the Coomassie staining intensity of a
number of different samples of mutant with known amounts of wild type
RecA protein on SDS-polyacrylamide gels. These determinations
corresponded to an extinction coefficient for the Pro
Trp mutant of
= 1.2
mg
ml. The same
was recently
calculated for a His
Trp mutant RecA protein
(26) .
RecA-mediated Repressor Cleavage in Vitro
The
cleavage of LexA repressor by purified RecA protein (wild type and the
Pro Trp mutant) was determined using the buffer
described by Wang et al. (27) . Reaction mixtures (40
µl) contained 1 µ
M wild type or mutant RecA protein,
1.0 m
M of the indicated NTP cofactor, and 35 µ
M RV-1 single-stranded DNA or the indicated oligonucleotide cofactor
(concentration of DNA expressed as mol PO
). Alternative
nucleic acid cofactors were used as indicated. Reactions were started
with the addition of LexA protein to a final concentration of 6
µ
M. Samples (8 µl) were removed at the indicated
times, added to gel loading buffer (10 µl), heated at 95 °C for
3 min, and loaded onto 15% polyacrylamide gels containing SDS. Gels
were stained with Coomassie Brilliant Blue R, and the percentage of
LexA cleavage was determined by scanning densitometry of intact LexA
and LexA cleavage products (Biomed Instruments 1D/2D soft laser
scanning densitometer). Cleavage of
cI repressor was performed
essentially as described
(28) . Reaction mixtures (10 µl)
contained 10 µ
M wild type or mutant RecA, 35 µ
M of the indicated oligonucleotide cofactor, 0.5 m
M ATP
S, and 1 µ
M cI repressor. Following a 3.5-h
incubation at 37 °C the reaction was analyzed on SDS-polyacrylamide
gels as described for LexA cleavage.
RecA Protein NTPase Activity
Hydrolysis of
-
P-labeled ATP, dATP, GTP, dGTP, CTP, dCTP, TTP, and
UTP by purified RecA (wild type and Pro
Trp mutant)
was measured essentially as described
(29) . Reactions included
the following components: 20 m
M Tris-HCl (pH 7.5), 20 m
M KCl, 10 m
M MgCl
, 0.5 m
M EDTA, 1.0
m
M dithiothreitol, 0.5 m
M
-
P
labeled NTP (20 µCi/ml), 2.0 µ
M RecA protein (wild
type or Pro
Trp mutant), and 25 µ
M RV-1 single-stranded DNA. Percent NTP hydrolysis was measured by
scanning polyethylenimine chromotography plates using a Molecular
Dynamics PhosphorImager equipped with Imagequant software (version
5.6).
(constitutive), coprt
(inducible), and
coprt
. Mutants are defined as coprt
if
they form red colonies on MacConkey-lactose plates in the absence of
mitomycin C (non-inducing condition) and if the average non-induced
-galactosidase activity in the cell extract is at least 1.5-fold
greater than that of the positive control strain (DE1663`/pTRecA103).
Mutants are defined as coprt
if they form white
colonies on MacConkey-lactose plates in both the absence and presence
of mitomycin C and have
-galactosidase activity in the cell
extract comparable to that of the negative control strain
(DE1663`/pZ150). Mutants are defined as coprt
if they
form white colonies when grown on MacConkey-lactose plates in the
absence of mitomycin C, form red colonies when grown in the presence of
mitomycin C, and show mitomycin C induction of
-galactosidase
activity in the cell extract.
coprt
Six of the 37
recombination-proficient mutants showed constitutive coprotease
activity, and in each case the amino acid substitution occurs at either
ProMutants
or Thr
(Table I). The Pro
Trp mutant (, 67-10) is unique in that
it has high coprotease activity in the absence of mitomycin C, whereas
substitution of Pro
with Glu, Asp, or Gly and Thr
with Gly or Phe results in low level coprt
mutants.
In an extended time course of exposure to mitomycin C, coprt
mutants with substitutions at Pro
(Trp, Glu, and
Asp) show an increase in coprotease specific activity similar to wild
type RecA (Fig. 1, A and B). Because the expression
of recA is under the control of P
and,
therefore, is unaffected by exposure to DNA damaging agents, this
increase in specific activity may reflect a time-dependent activation
and/or a slower rate of degradation of the existing pool of RecA as DNA
damage accumulates.
mutations show varying
effects on recombinational DNA repair activity. For example, although
the enhanced effect on coprotease activity is similar for both a Glu
and Asp substitution at Pro
(), the Glu
mutation has a greater inhibitory effect on recombinational DNA repair
(Table II and Ref. 16). In addition, only the Pro
Trp mutant results in a high constitutive coprotease activity, but
substitution to Trp and Gly at position 67 and to Gly and Phe at
position 74 all have similar modest inhibitory effects or no measurable
effect on recombinational DNA repair ( and Ref. 16).
coprt
Twenty-nine of the
37 recombination proficient mutants scored as coprtMutants
,
and although most mutants showed levels of induction comparable to wild
type RecA, three showed low levels of inducible coprotease activity;
Pro
Phe, Pro
Tyr, and
Glu
Thr (; 67-11, 67-12,
and 68-6, respectively). The Pro
Phe mutant
is most attenuated for coprotease induction and required longer times
of exposure to mitomycin C before showing a measurable increase in
activity (Fig. 1, C and D). Inducible
coprotease activity was clearly observed for all coprt
mutants, including Pro
Phe, on
MacConkey-lactose plates showing that this method is also suitable for
detection of low levels of coprotease induction. coprt
Mutants-Two mutants previously
characterized as having partial recombination activity, Pro
Lys and Pro
Arg, showed a complete
lack of coprotease activity in vivo (, 67-14
and 67-16). Both mutants grew as white colonies on
MacConkey-lactose plates in the absence and presence of mitomycin C and
showed a level of coprotease activity in cell extracts similar to
negative control cells (). Even after prolonged exposure to
mitomycin C, neither mutant showed induction of coprotease activity
(Fig. 1, C and D). We have further tested the
DNA repair proficiency of these two mutants using a range of UV doses
(). We find that the Pro
Lys mutant
shows a steady increase in survival, up to 33% of wild type recA cells, with decreasing UV dose. In contrast, the Pro
Arg mutant fails to survive even low doses to any extent
greater than negative control cells. Interestingly, both mutants are
proficient for RecA-dependent homologous recombination as they promote
the formation of plaques by a
red
gam
Chi
phage
(16) . To our knowledge this is the first
description of recA mutants that are proficient for
recombinational DNA repair and/or homologous genetic recombination but
are completely deficient for LexA coprotease activity in vivo.
Figure 1:
RecA coprotease activity as a function
of time of cell growth and exposure to mitomycin C. -galactosidase
activity in crude cell extracts was determined as described under
``Experimental Procedures'' at 40, 60, 90, 120, and 180 min
of cell growth in the absence ( A and C) or presence
( B and D) of 0.5 µg/ml mitomycin C.
67Trp, 67Glu, and 67Asp ( A and
B) are examples of coprt
mutants. 67Phe ( C and D) shows a low level induction of
activity, whereas 67Lys and 67Arg ( C and
D) are examples of coprt
mutants that show
no induction of activity. The positive control is strain DE1663`
carrying pTRecA103 and the negative control is the same strain carrying
pZ150 (see ``Experimental Procedures''). Values are an
average of duplicate assays for which the standard error was no greater
than 18%.
All 63 of the P-loop mutants that have been previously characterized
as defective for both recombinational repair and homologous
recombination
(16) scored as coprt(data not
shown; Fig. 2).
Figure 2:
Summary of mutations and their effect on
RecA coprotease activity. The wild type RecA sequence is centered in
boldface type. Substitutions shown above allow
coprotease activity and are listed from top to bottom in decreasing
order of the level of activity seen in the absence of mitomycin C (see
Table I). Underlines indicate constitutive coprotease
activity. Substitutions shown below the wild type sequence result in
coprtmutants. This summary is derived exclusively
from single mutants.
Nucleoside Triphosphate Cofactor Specificity for LexA
Cleavage by the Pro
In order to
investigate the molecular nature of the high constitutive coprotease
activity of the Pro Trp Mutant
Trp mutation, we have begun
biochemical studies of the purified mutant protein. Studies of other
mutant RecA proteins have shown that constitutive activation of
coprotease function can be associated with a more relaxed specificity
for NTP cofactors
(27) . We, therefore, tested all of the common
NTPs, dNTPs, and ddNTPs in an in vitro LexA cleavage assay and
found that the Pro
Trp mutant protein shows a
completely relaxed specificity compared to wild type RecA. Under the
conditions of our assay ATP, dATP, and ddATP were the optimal cofactors
for both wild type RecA and the Pro
Trp mutant
protein (Fig. 3 A). In the presence of any one of these three
cofactors the initial rate of LexA cleavage was quite fast, ranging
from 60 to 78% repressor cleavage after 5 min. Both proteins also
utilized CTP and UTP equally well, although the initial rate and final
extent of LexA cleavage was somewhat less than with ATP, dATP, and
ddATP (Fig. 3, B and D). However, the mutant
protein was able to use both dCTP and ddCTP more effectively than wild
type RecA (Fig. 3 B). In sharp contrast to the above
NTPs, we found that the Pro
Trp mutant protein was
able to catalyze LexA cleavage in the presence of GTP, dGTP, ddGTP,
TTP, and ddTTP, whereas wild type RecA was completely ineffective with
these nucleotides (Fig. 3, C and D). Neither
wild type RecA nor the Pro
Trp mutant protein
showed any cleavage of LexA when ADP was used as cofactor or when ssDNA
or NTPs were omitted from the reaction mixture (data not shown).
Figure 3:
Nucleotide cofactor specificity for LexA
cleavage activity by both wild type RecA and the Pro
Trp mutant proteins. LexA cleavage assays were performed as
described under ``Experimental Procedures.'' Each panel shows
percentage of LexA cleavage as a function of time for both the wild
type and mutant proteins. NTP cofactors and symbols are identified in
each panel. Values are the average of duplicate assays for which the
standard error was no greater than 15%.
Despite the fact that the nucleotide specificity for LexA cleavage
is relaxed by the Pro Trp mutation, NTP hydrolysis
by this mutant still shows a pattern of specificity similar to wild
type RecA. We determined specific activities for the hydrolysis of
several NTPs and dNTPs. Both wild type and mutant RecA proteins
hydrolyzed dATP and ATP most efficiently (Table III). Hydrolysis of
other nucleotides (UTP, CTP, dCTP, GTP, dGTP, and TTP) by both proteins
was significantly less efficient, with GTP and TTP being the least
effective substrates. These results are in good agreement with those of
Weinstock et al. (29) regarding the nucleotide
specificity for wild type RecA NTPase activity. Hydrolysis of NTPs by
either protein required single-stranded DNA as a cofactor.
Nucleic Acid Cofactor Specificity for Repressor Cleavage
by the Pro
Previous studies have
shown that in addition to a more relaxed specificity for NTP cofactors,
coprt Trp Mutant
mutant RecA proteins may also have a relaxed
specificity for nucleic acid cofactors
(28, 30) .
McEntee and Weinstock
(28) have shown that the coprt
RecA441 protein can use shorter length (dA) and (dT)
oligonucleotides than can wild type RecA as cofactors for the cleavage
of the
cI repressor. However, no study has yet reported the
effect of defined length oligonucleotides on the LexA cleavage activity
of either wild type or any mutant RecA proteins. We, therefore,
compared the effectiveness of a series of short (dA) and (dT)
oligonucleotides as cofactors in LexA cleavage assays using both wild
type RecA and the Pro
Trp mutant protein.
Interestingly, our data show that the LexA coprotease activity of both
proteins is only minimally affected by changes in the length of the
oligonucleotide cofactor in the range from 6 to 24 bases (Fig. 4). For
example, the extent of LexA cleavage by wild type RecA in the presence
of (dA)
or (dA)
is 30% and 37%, respectively.
For the mutant protein the difference in these values is somewhat
greater, 35% versus 51%, respectively. Using (dT)
or (dT)
, the extent of LexA cleavage by wild type
RecA is 48% and 60%, while cleavage by the Pro
Trp
protein is 57% and 71%, respectively. With all lengths of (dA) and (dT)
oligonucleotides, the mutant protein catalyzes LexA cleavage to an
extent approximately 15-30% greater than wild type RecA. We also
performed time courses of LexA cleavage in which the concentration of
oligonucleotides was varied from 8 µ
M to 32 µ
M with protein held constant at 1 µ
M. In all cases both
the initial rate and extent of cleavage increased with increasing
concentration of (dA) or (dT) cofactor, and again the activity of the
mutant protein was approximately 15-30% greater than wild type
RecA (data not shown). Also apparent in Fig. 4is that (dT)
oligonucleotides are slightly more effective cofactors than the
corresponding (dA) oligonucleotide.
Figure 4:
The effect of nucleic acid cofactor length
on the cleavage of LexA repressor by wild type RecA and the
Pro
Trp mutant proteins. Percentage of LexA
cleavage was determined following a 20-min reaction containing 1 m
M dATP and 35 µ
M oligonucleotide cofactor (dA,
panel A; dT, panel B) of
the indicated length. Other reactions conditions and components are
described under ``Experimental Procedures.'' Values are the
average of triplicate assays, and the standard error is indicated in
the figure.
Because our data regarding LexA
cleavage by wild type RecA differed significantly from work that showed
a strict dependence on oligonucleotide length for cI repressor
cleavage by wild type RecA
(28) , we used our set of (dA) and
(dT) oligonucleotides in
repressor cleavage assays. We found
that, in contrast to LexA, cleavage of
repressor does indeed show
a marked dependence on oligoucleotide length (Fig. 5). Using (dA)
oligonucleotides, we observed a steady increase in the extent of
repressor cleavage by both wild type RecA (from 0% to 70% cleavage) and
the Pro
Trp protein (from 17% to 70% cleavage) as a
function of increasing length of oligonucleotide
(Fig. 5 A). With (dA)
and (dA)
,
the mutant protein showed a reproducibly higher activity than wild type
RecA, but both proteins had essentially identical activities with
(dA)
, (dA)
, and (dA)
. Using
(dT), however, we observed significant differences between the wild
type and mutant proteins (Fig. 5 B). With (dT)
,
(dT)
and (dT)
, wild type RecA was essentially
inactive, whereas the mutant protein displayed measurable activity with
(dT)
and (dT)
, which increased dramatically
with (dT)
. A significant level of activity was seen for
wild type RecA using (dT)
and was approximately 50% the
level observed for the mutant protein. Activity was equivalent for both
proteins using (dT)
. Although somewhat different in their
quantitative aspects, these results are consistent with those reported
previously for wild type RecA
(28) .
Figure 5:
The effect of nucleic acid cofactor length
on the cleavage of cI repressor by wild type RecA and the
Pro
Trp mutant proteins. Percentage of cI cleavage
was determined as described under ``Experimental
Procedures.'' Oligonucleotide cofactor was either dA or dT of the
indicated length. Values are the average of triplicate assays, and the
standard error is indicated in the figure.
Wang et al. (30) have reported that the coprtrecA1202 and recA1211 mutant proteins can use rRNA and tRNA very
effectively as cofactors for cleavage of LexA and also showed that
these mutant proteins use double-stranded DNA somewhat more effectively
than does wild type RecA. We tested the ability of the Pro
Trp mutant protein to use these alternative nucleic acid
cofactors and found that tRNA and double-stranded DNA are unable to
function in this capacity (data not shown).
is unique among them in that specific substitutions have very
different effects on the coprotease versus recombination
activities of RecA. One of these, Pro
Trp, results
in high constitutive coprotease activity and a modest effect on
recombinational DNA repair activity, whereas coprt
mutants
resulting from Glu and Asp substitutions markedly decrease the latter
activity. In contrast, Lys and Arg mutations at position 67 eliminate
in vivo coprotease function but allow for some level of
recombination activity. The Lys mutant catalyzes homologous genetic
recombination
(16) and maintains a modest level of
recombinational DNA repair activity, whereas the Arg mutant, while
capable of carrying out homologous recombination
(16) , is
completely inactivated for recombinational DNA repair. These two
represent the first description of mutants that separate the
coprotease, recombinational DNA repair and homologous recombination
functions of RecA. The identification of such mutants undoubtedly
relates to the fact that expression of recA in our system is
regulated by a non-SOS promoter, P
. Under normal SOS
regulation the in vivo recombination activity of
coprt
mutants is likely to be refractory to analysis
simply because recA expression is repressed.
- and
-phosphates of bound nucleotide are
positioned similarly but the location of the base and sugar in the
RecA-ADP complex is very different from that for the GDP of EF-Tu and
p21
(14) . Electron microscopy studies suggest that the RecA
crystal structure represents a specific inactive conformation (reviewed
in Ref. 31); however, the overall structure of the crystal filament
closely resembles the active RecA/DNA/ATP filament
(31) .
,
Glu
, and Ser
) extend inward toward the
helical axis of the protein filament and appear to be free of any
steric impedance. However, each of these positions, especially 68 and
69, shows restrictions regarding mutations that permit recombination
activity
(16) . We have previously suggested that these
restrictions reflect a constraint that is not apparent in the crystal
structure, namely their interaction with, or at least proximity to,
bound DNA
(16) . This is supported by recent work in which a
specific cross-link between poly(dT) and RecA was identified within a
peptide defined by residues Ile
to
Lys
.(
)
Glu results in a low level coprt
mutant, while Pro
Lys completely inhibits this function. These results are
consistent with the idea that position 67 is close to bound LexA
repressor and that mutations here may have a direct effect on the
interaction between RecA and LexA. This idea is supported by electron
microscopic studies of RecA/LexA/DNA complexes in which LexA is seen to
bind within the deep helical groove of the RecA filament
(32) .
Although the wild type side chain of Pro
does not extend
far from the inner surface of the protein filament, mutant side chains
such as Trp, Lys, and Glu may protrude far enough into the helical
groove to have a direct effect on LexA binding (Fig. 6). Another mutant
observed at position 67, however, suggests a more indirect effect on
RecA function. A Pro
Gly mutation results in a
modest constitutive coprotease activity, yet has little or no effect on
recombination, suggesting that activation of the coprotease function
results from increased main chain flexibility in this region of the
protein. Whether these mutations correlate with changes in LexA binding
affinities will be addressed in biochemical studies of the purified
mutant proteins.
Ser) and 243
(Arg
Leu) differentially affect cleavage of the LexA,
cI,
and
80 repressors
(33, 34) , and both the RecA
crystal structure and electron microscopic analysis of RecA/LexA/DNA
complexes support the idea that these residues occupy space within the
repressor binding site
(14, 32) . The distance between
residues 67, 229, and 243 in the RecA crystal structure (
14 Å
separation) allows for the possibility that all could be close to bound
repressor (Fig. 6).
Figure 6:
Stereo diagram of two neighboring RecA
subunits showing the position of Pro and bound ADP. An
-carbon trace is shown for two neighboring monomers in the RecA
protein filament as seen in the crystal structure (17). The thickness
of the polypeptide chain is slightly greater for the right subunit. The
side chain and backbone atoms of Pro
and bound ADP are
shown in boldface. The N-terminal residue (Asp
)
seen in the crystal structure is indicated by N in the
left subunit. 229 and 243 refer to
Gly
and Arg
and lie immediately to the
left of the side chain for each residue in both subunits. In
the left subunit, residues that flank the disordered regions
L1 and L2 are indicated by a 1 (Glu
) and 1` (Gly
) and a 2 (Ile
) and
2` (Thr
). In the right monomer, these 4 residues
are shown as a closed circle. This view shows that
the Pro
side chain extends from the inner surface of the
protein inward toward the helical axis.
Position 67 is also very near the
disordered loop 2 (L2, residues 196-209). Story et al. (17) have proposed that L2 forms part of the primary DNA binding
site of RecA and that specific conformational changes in this region
that affect DNA binding are regulated by the interaction of Glnwith the
-phosphate of bound NTP. Distance measurements
within the RecA structure show that the
-carbon of any mutant side
chain at position 67 would be within 5.0 Å of the Gln
side chain and approximately 3.5-13.0 Å from the
proximal and distal residues that flank L2, Ile
and
Thr
, respectively (Fig. 6). Therefore, the proximity of
substitutions at position 67 to Gln
or residues within L2
may contribute to their effects on the coprotease or recombination
activities of RecA.
Trp mutant protein show a complete relaxation of NTP
cofactor specificity for RecA-mediated LexA cleavage in vitro.
Although an NTP cofactor is still required for this activity, any
ribo-, deoxyribo-, or dideoxyriboNTP that we tested served in this
capacity for the mutant protein, whereas wild type RecA displayed a
significantly more stringent NTP specificity. Wang et al. (27) have previously reported that two different coprt
mutant RecA proteins, recA1202 (Gln
Lys) and recA1211 (Glu
Lys), display a
somewhat relaxed specificity for NTP cofactors in LexA cleavage assays.
These findings show that the nucleotide cofactor specificity of
coprt
mutants can be modified by mutations at very
different locations within the RecA structure.
Trp mutant
protein pertains to the coprotease activity but not to the NTPase
activity. Nucleotide cofactors serve as allosteric effectors of RecA
activity (reviewed in Ref. 11), and NTP binding and hydrolysis has been
shown to induce specific changes in the structure of the protein
(35, 36, 37) . It may be that coprt
mutations at position 67 facilitate NTP-induced structural
changes required for coprotease function that can now be achieved by
otherwise non-optimal NTP cofactors.