Biotechnology Laboratory, Takasaki Radiation
Chemistry Research Establishment, Japan Atomic Energy Research
Institute, Takasaki 370-1292,1 United
Graduate School of Agricultural Science, Tokyo University of
Agriculture and Technology, Fuchuu 183-8509,2
Faculty of Agriculture, Utsunomiya University, Utsunomiya
321-8505,3 and Department of
Biomolecular Sciences, Graduate School of Life Sciences, Tohoku
University, Sendai 980-8578,4 Japan
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TEXT |
Deinococcus radiodurans
is characterized by its extraordinary radiation resistance phenotype,
which is considered to be due to a highly proficient DNA repair
capacity (3, 25, 34). The most striking feature of
D. radiodurans is that it can mend over 100 double-strand
breaks (DSBs) of genomic DNA during postirradiation incubation
(2, 18). As the rejoining of DSBs can be prevented by
adding chloramphenicol to the incubation mixture, proteins induced by
irradiation are necessary for the rejoining of DNA breakages
(18). Several DNA damage-inducible proteins that may be
required for DNA repair have been detected in the cell extract of
D. radiodurans by two-dimensional polyacrylamide gel
electrophoresis (PAGE) (12, 35). These observations
suggest that D. radiodurans possesses a DNA damage response
mechanism. However, little is known about the molecular basis for the
control of the inducible proteins.
In Escherichia coli, the inducible DNA repair system (the
SOS system) is regulated by two key proteins; RecA and LexA (8, 38). E. coli RecA is activated by DNA damage to
mediate the proteolytic cleavage of the E. coli LexA
repressor, resulting in derepression of the SOS regulon. The SOS
response in Bacillus subtilis progresses in a similar
manner, with B. subtilis RecA having an identical role in
controlling the SOS regulon together with a cellular repressor protein
that is functionally homologous to the E. coli LexA
repressor (42). The B. subtilis repressor (termed DinR) binds the promoter regions of several din
genes and B. subtilis recA (20, 23, 40, 41) and
undergoes autodigestion under alkaline conditions and RecA-mediated
cleavage under more physiological conditions (23, 41). It
has also been shown that the intracellular level of intact DinR is
significantly reduced following DNA damage (23). Thus, the
basic mechanism of the SOS response seems to be conserved between
E. coli and B. subtilis. Deinococcus
species form a coherent phylogenetic cluster related to the
Thermus-Meiothermus lineage (30),
indicating that the Deinococcus lineage is distinct from the
lineages of both proteobacteria and gram-positive bacteria. Although
SOS-like processes have been documented in a wide variety of
eubacterial species (24, 32), the involvement of RecA and
LexA in the SOS response is poorly understood in Deinococcus
and closely related bacterial species.
As expression of the deinococcal recA gene is enhanced after
irradiation (4), the recA gene seems to be
a member of a DNA damage response regulon in D. radiodurans.
In the present study, D. radiodurans LexA was purified from
E. coli cells and its ability to cleave itself was examined.
The changes in intracellular levels of the LexA and RecA proteins
following irradiation were also investigated by using
lexA and recA disruptant strains to gain insight
into the DNA damage response mechanism in D. radiodurans. Our results indicated that deinococcal LexA undergoes RecA-mediated cleavage but is not involved in the regulation of deinococcal RecA.
Expression plasmid construction.
pDC144 is a cosmid clone in a
genomic library of D. radiodurans strain KD8301 (Table
1). pZA8 (16) contains
D. radiodurans lexA in a 6,005-bp
SalI-SacI fragment from pDC144. The nucleotide sequence of this region was confirmed to be perfectly consistent with
the corresponding region of D. radiodurans
(39). To isolate the lexA coding region, PCR
was carried out by using pZA8 DNA with the specific oligonucleotides
5'-GGCAAACTGCGCGCATATGCCGCCTGAACTG-3' and
5'-GTCGGGATCCTACTCGGTCACGCGGTGGCTCACG-3',
containing restriction sites (NdeI and BamHI,
which are underlined). PCR products were then digested with
NdeI and BamHI to adapt the termini for the in-frame insertion of lexA into the
NdeI-BamHI sites in the pET3a vector. The
resultant expression plasmid was designated pET3lexAwt (Table 1). The
DNA sequence of the expression plasmid was checked to confirm the lack
of introduction of errors by PCR.
Protein purification.
D. radiodurans LexA was induced by
isopropyl-
-D-thiogalactopyranoside (IPTG) in E.
coli strain BL21(DE3) carrying pLysS and pET3lexAwt. Cells were
harvested, washed, and resuspended in a buffer containing 20 mM sodium
phosphate (pH 7.4), 1 mM phenylmethylsulfonyl fluoride, and 0.1%
(wt/vol) protease inhibitor cocktail. The suspension was sonicated for
10 min, and debris was removed by centrifugation. Ammonium sulfate was
added to the supernatant to give 30% saturation. The suspension was
stirred for 1 h and then centrifuged for 30 min. The pellets were
resuspended in a buffer containing 20 mM sodium phosphate (pH 7.4) and
0.1 mM EDTA, and the suspension was dialyzed for 18 h. The protein
was further purified to apparent homogeneity by column chromatography
on HiTrap Heparin HP and Resource S (Fig.
1). The N-terminal amino acid sequence of
the purified protein was found to be
Pro-Pro-Glu-Leu-Thr-Pro-Thr-Arg-Arg-Ser-Ile-Leu-Gln-Ala-. This was
completely consistent with the sequence from residue 2 to residue 15 of
the primary structure predicted from the DNA sequence data. Thus, the
purified protein was confirmed to be D. radiodurans
LexA.
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FIG. 1.
Purification of D. radiodurans LexA
protein. Samples were subjected to sodium dodecyl sulfate-15% PAGE
and stained with Coomassie brilliant blue. Lanes: 1, 10-kDa protein
ladder (Invitrogen); 2, total cellular proteins from E.
coli BL21(DE3)/pLysS/pET3lexAwt induced by IPTG; 3, resuspension from 30% ammonium sulfate precipitation; 4, pooled LexA
fractions from a HiTrap Heparin HP column; 5, pooled LexA fractions
from a Resource S column. The position of the 25-kDa band of LexA is
indicated on the right.
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Autodigestion and RecA-mediated cleavage.
First, whether the
purified protein has autodigestion and RecA-mediated cleavage activity
was investigated. For this purpose, antiserum raised against the
purified D. radiodurans LexA protein was generated. The
autodigestion reaction was assayed by incubating 0.4 µM LexA in 50 mM
Tris-HCl (pH 10) at 37°C and monitored by Western analysis. As shown
in Fig. 2, LexA was autodigested to yield
two breakdown products. For RecA-mediated cleavage, reactions were
carried out in a buffer consisting of 20 mM Tris-HCl (pH 7.4) and 10 mM
MgCl2 with 6 µM oligonucleotide (35-mer) and 1 mM adenosine-5'-O-[-thio]triphosphate (ATPS). LexA
(0.4 µM) was incubated with D. radiodurans RecA (4.2 µM)
at 37°C for 1 h and sampled for Western analysis. The
purification of the RecA protein will be reported elsewhere. LexA was
cleaved by incubation with RecA to yield two breakdown products the
sizes of which were identical to those observed in autodigestion. When
the RecA, oligonucleotide, or ATPS was omitted from the reaction
mixture, no breakdown product was observed (Fig. 2). From these
results, we concluded that D. radiodurans LexA maintains
proteolytic activity and that D. radiodurans RecA can
promote the proteolytic activity of LexA.
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FIG. 2.
Autodigestion and RecA-mediated cleavage of purified
LexA protein visualized by Western analysis with D.
radiodurans LexA antiserum (diluted 1:10,000). LexA (0.4 µM)
was incubated in 50 mM Tris-HCl (pH 10) for 0 h (lane 1) and
8 h (lane 2). For RecA-mediated cleavage, LexA was incubated with
D. radiodurans RecA under the conditions described in
the text. Lanes: 3, RecA omitted; 4, oligonucleotide omitted; 5, ATPS omitted; 6, complete reaction mixture. The arrows on the right
indicate the positions of breakdown products of LexA.
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Construction of gene disruptant strains.
To test our
assumption mentioned above, we investigated the in vivo interaction of
LexA and RecA by detecting changes in intracellular protein levels
following irradiation. For this purpose, we generated a
lexA disruptant strain and a recA disruptant
strain by the direct insertional mutagenesis technique
(9). The disruptant strains were isolated on TGY
plates (28) containing chloramphenicol at 3 µg/ml and
designated XE1 (carrying lexA166::cat)
and RN201 (carrying recA229::cat),
respectively. Disruption of the genes was confirmed by amplifying the
targeted allele by PCR. The DNA damage sensitivity phenotype of strain
RN201 was confirmed by measuring cell survival following irradiation (Fig. 3). RN201 exhibited
extreme ray sensitivity, as observed in the recA
disruptant strain 1R1A constructed previously (10) and
mutant strain rec30 (10, 26, 35) carrying a
recA670 mutation (29). In addition, strain
RN201 had a slow growth rate compared with that of its parental strain.
On the other hand, the growth rate of strain XE1 carrying the
lexA166::cat mutation was almost the
same as that of the parental strain. Although XE1 cells showed a
slightly higher rate of cell death than the parental strain at high
doses (6 and 8 kGy) of rays, the disruption of lexA did
not severely affect ray resistance (Fig. 3).
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FIG. 3.
Sensitivity of lexA and
recA disruptant strains to rays. Cells grown to
early stationary phase were resuspended in 10 mM sodium phosphate
buffer (pH 7), challenged with 60Co irradiation, spread
on TGY plates, and incubated at 30°C. After 3 days, surviving
colonies were counted. Each point represents the average result
of three independent experiments. Symbols: open squares, strain
KD8301; filled squares, strain XE1
(lexA166::cat); open circles,
strain KR1; filled circles, strain RN201
(recA229::cat).
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Changes in intracellular LexA and RecA levels following
irradiation.
The changes in LexA and RecA levels following irradiation were compared among the disruptants and their parental
strains. Early stationary phase cells were resuspended in 10 mM sodium phosphate buffer (pH 7) and divided into two fractions. One fraction was irradiated at a dose of 2 kGy, and the other fraction was not
irradiated. The cells were then incubated in fresh TGY broth for 2 h at 30°C with agitation. The protein extracts were subjected to
Western analysis with D. radiodurans LexA antiserum (diluted 1:10,000) and E. coli RecA antiserum (1:500). The E. coli RecA antiserum was raised against purified E. coli
RecA protein (Promega). As a control, D. radiodurans GroEL
(22) was detected by using E. coli GroEL
antiserum (diluted 1:2,000) (StressGen Biotechnologies Corp.) (Fig.
4).
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FIG. 4.
Changes in intracellular LexA, RecA, and GroEL levels
following irradiation. Each sample contained 10 µg of protein. Lanes:
1 and 2, strain KR1; 3 and 4, strain RN201
(recA229::cat); 5 and 6, strain
KD8301; 7 and 8, strain XE1
(lexA166::cat). Odd- and
even-numbered lanes contained nonirradiated samples and those
irradiated with 2 kGy, respectively.
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In wild-type strain KR1, the level of LexA was
decreased (2.7-fold) and the level of RecA was, in contrast, increased
after irradiation (2.5-fold) (Fig. 4, lanes 1 and 2). These results suggested that RecA is activated during postirradiation incubation and
promotes the proteolytic cleavage of LexA. In strain RN201, the RecA
signal completely disappeared (Fig. 4, lanes 3 and 4), confirming the
disruption of recA. Importantly, irradiation did not
affect the level of LexA in RN201. These results supported our
observation of in vitro RecA-mediated LexA cleavage and further suggested that RecA is the sole protein required for LexA cleavage. As
in strain KR1, a decrease in LexA (2.5-fold) and
an increase in RecA (2.5-fold) were observed in strain KD8301 after
irradiation (Fig. 4, lanes 5 and 6). In strain XE1, a derivative of
strain KD8301, the LexA signal disappeared because of disruption of
lexA (Fig. 4, lanes 7 and 8). If RecA represses the
expression of recA, constitutive production of LexA at an
elevated level can be seen in unirradiated lexA disruptant
cells. However, this was not the case in strain XE1. The level of RecA
in unirradiated XE1 cells was comparable to those in unirradiated
KR1 and KD8301 cells, and RecA induction
following irradiation (2.0-fold) was observed in XE1 as in
KR1 and KD8301. The level of GroEL was constant
irrespective of irradiation in all of the strains tested. Thus, the
results of our experiments did not support the involvement of LexA in the induction of RecA in D. radiodurans.
Discussion.
The results obtained in this study indicate that
D. radiodurans LexA undergoes RecA-mediated cleavage (Fig.
2) and RecA is the sole protein responsible for cleavage of LexA in
vivo (Fig. 4). E. coli RecA mediates the proteolytic
cleavage of the bond between Ala-84 and Gly-85 of LexA
(13). Hydrolysis of the LexA Ala-Gly bond proceeds
similarly to that of serine proteases, with Ser-119 acting as a
nucleophile and Lys-156 acting as an activator (33).
Alignment of the amino acid sequences of E. coli LexA, B. subtilis DinR, and D. radiodurans LexA (Fig.
5) revealed that the amino acid residues
involved in the cleavage reaction are also conserved in D. radiodurans LexA (Ala-83, Gly-84, Ser-119, and Lys-158).
Therefore, we assumed that the two breakdown products observed in the
in vitro cleavage assays (Fig. 2) were the N-terminal and C-terminal
fragments of LexA cleaved between Ala-83 and Gly-84.
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FIG. 5.
Multiple amino acid sequence alignment of E.
coli RecA (14), B. subtilis DinR
(31), and D. radiodurans LexA. Multiple
alignment was achieved with the CLUSTAL W program (37).
Dashes indicate gaps in the alignment. Numbers on the right are the
coordinates of the proteins. Asterisks indicate identical residues. The
conserved Ala and Gly residues in the cleavage site and the Ser and Lys
residues required for cleavage are indicated by filled triangles. The
locations of three  helices (H1, H2, and H3) found in E.
coli LexA are indicated according to the description of Fogh et
al. (7).
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The N-terminal domain of E. coli LexA is involved in DNA
binding (15), and the LexA DNA binding domain contains
three helices, of which helices 2 and 3 form a variant
helix-turn-helix DNA binding motif (7). The N-terminal
region of the D. radiodurans LexA sequence, however, showed
very limited similarity to analogous regions in E. coli LexA
and B. subtilis DinR (Fig. 5). If the N-terminal region of
D. radiodurans LexA serves as a DNA binding domain, D. radiodurans possesses a distinct LexA binding motif that is
different from the E. coli SOS box (38) and the
B. subtilis DinR box (40). For the purification
of D. radiodurans LexA, we found a heparin column to be very
effective (Fig. 1), suggesting that the LexA protein retains DNA
binding ability. In the preliminary gel mobility shift assay, we also
found that the purified LexA protein could bind to the upstream region
of its own gene (data not shown). A detailed analysis of the D. radiodurans lexA operator is ongoing in our laboratory.
Under our experimental conditions (irradiation with 2 kGy, followed by
2 h of incubation), the RecA induction ratio was 2.0- to 2.5-fold
(Fig. 4). This induction ratio was much smaller than that observed in a
previous study (50- to 100-fold; irradiation with 5 kGy, followed by
2 h of incubation) (4). It has been shown that, after
irradiation at 3 kGy, a cell's recovery from DSBs is complete within
3 h (2). Therefore, one explanation for the induction
ratio difference is the different ray doses used in the two
experiments. It is conceivable that most DSB repair is completed under
our experimental conditions; thereby, the induction ratio was smaller
than that in the previous study. However, the 50- to 100-fold RecA
induction ratio seems to be an overestimate because such
radiation-induced protein was not detected by two-dimensional PAGE
analysis in which cells were irradiated at 6 kGy and incubated for
2 h (35).
It has been shown that several eubacterial species lack a
lexA gene (1, 6). A lack of inducibility of
RecA has also been demonstrated in some genera (24). Thus,
while the recA gene is clearly conserved in a wide variety
of eubacterial species, the control mechanism of its expression is not.
Our results indicated that the recA gene is part of a DNA
damage response regulon in D. radiodurans. However, our
findings did not support the suggestion that D. radiodurans
LexA is involved in the induction of RecA. This, in turn, led us to
speculate that D. radiodurans has an alternative DNA damage
response mechanism with which to control recA expression. In
E. coli, the sulA gene, whose products inhibit cell division, is under the direct control of E. coli LexA
(38). Consequently, E. coli lexA (Def) mutants
are viable only if they contain an additional mutation on the
sulA gene. In addition, it has been shown that B. subtilis lexA (Def) mutants exhibit a strong filamentation
phenotype, accompanied by significant loss of viability
(11). On the other hand, the D. radiodurans
lexA disruptant generated in this study was viable. This different behavior has been reported previously for lexA disruptants
of Xanthomonas campestris (43) and
Rhodobacter sphaeroides (36) in which LexA
functions as a repressor of recA expression. The D. radiodurans genome does not encode a homolog of the E. coli sulA gene (39). However, it has been shown that the
DNA damage-induced delay in D. radiodurans chromosomal DNA
replication is dose dependent and that the length of the delay always
exceeds the time required for repair of the DNA damage that caused the
inhibition (5, 19, 27). Based on these observations, it
has been proposed that there is a regulatory mechanism in D. radiodurans that controls chromosome replication and, as a
consequence, controls cell division (2, 3). We suggest
that D. radiodurans LexA is probably not involved in such a
regulatory mechanism because of the behavior of LexA disruptant cells.
Interestingly, it has recently been shown that D. radiodurans encodes a second but diverged copy of LexA (DRA0074)
that retains the potential DNA binding domain and the autocleavage
domain (21). It would be interesting to determine whether
DRA0074 is involved in the control of the DNA damage response in
D. radiodurans. Recently, we succeeded in overproducing
DRA0074 in E. coli. Characterization of this recombinant
protein will provide useful information about control of the DNA damage response.
Nucleotide sequence accession number.
The nucleotide
sequence reported here (the Dienococcus radiodurans
gene for aldehyde dehydrogenase, succinic semialdehyde dehydrogenase,
partial and complete cds) has been deposited in the DDBJ/EMBL/GenBank
databases under accession no. AB003475.
We thank Xiang-Rong Kong for determining preliminary conditions for
the purification of LexA. We are also grateful to Rieko Nakano for
constructing strain RN201.
This work was performed as part of an Atomic Energy Crossover Project
of the Ministry of Education, Culture, Sports, Science and Technology
(MEXT), Japan, and supported by a Grant-in-Aid for Scientific Research
from MEXT.
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