Biochem. J. (2010) 431, 149–157 (Printed in Great Britain)
149
doi:10.1042/BJ20100446
Characterization of an ATP-regulated DNA-processing enzyme and
thermotolerant phosphoesterase in the radioresistant bacterium
Deinococcus radiodurans
Swathi KOTA, C. Vijaya KUMAR and Hari S. MISRA1
Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400 085, India
A multiprotein DNA-processing complex identified from
Deinococcus radiodurans exhibits uncharacterized ATP-sensitive
nuclease functions. DR0505 was one of the 24 polypeptides
identified from the complex. It contains two 5 nucleotidase
motifs, one is at the C-terminal end of the N-terminal CPDD
(calcineurin phosphodiesterase domain), with the second at the
C-terminal end of the protein. Recombinant DR0505 showed
both phosphomonoesterase and phosphodiesterase activities with
chromogenic substrates, showing higher affinity for bis-(pnitrophenyl) phosphate than for p-nitrophenyl phosphate. The
enzyme exhibited pH optima ranging from 8.0 to 9.0 and
metal-ion-dependent thermotolerance of esterase functions. Both
mono- and di-esterase activities were stable at temperatures
up to 50 ◦C in the presence of Mg2+ , whereas monoesterase
activity was observed at temperatures up to 80 ◦C in the
presence of Mn2+ and up to 50 ◦C with Ca2+ . The purified
enzyme showed 5 nucleotidase activity on a wide range
of natural mononucleotides including cyclic mononucleotides
INTRODUCTION
The nucleoside analogues used in medicine as drugs, are
phosphorylated by nucleotide kinases before they undergo
metabolic degradation by the combined action of deaminases
and nucleotidase family proteins [1]. Two types of nucleotidases,
5 nucleotidases (EC 3.1.3.5) and 3 nucleotidases (EC 3.1.3.6),
have been reported from different organisms. The 5 nucleotidases
are the main enzymes involved in regulating intracellular
deoxynucleotide pools [2–4]. Several 5 nucleotidases, differing
in their subcellular localization, protein sequence and substrate
affinities, have been identified from both mammalian and bacterial
systems. The roles of these enzymes have been greatly emphasized
in nucleoside drug metabolism in eukaryotes and in purine
metabolism in bacteria [5]. The pathogenic bacteria use this
process as a defence mechanism to escape from the human
immune system [6]. Bacteria exposed to abiotic stresses, such
as radiation, desiccation and oxidants, suffer a wide range of
base changes in their genome. The proficient excision repair
mechanisms involve the excision of damaged bases followed
by re-synthesis of new DNA strands. The excised nucleotides
are released into the cellular pool of nucleotides and recycled
by indigenous mechanisms. Recycling of chemically altered
nucleotides is required to avoid their re-incorporation during
DNA synthesis and thus prevent mutagenesis. The spontaneous
mutation rates differ in different bacteria conferring different
and 8-oxo-GMP. DR0505 showed a nearly 7-fold higher
activity on ADP than AMP, but this activity was inhibited
with ATP. Interestingly, DR0505 also showed single-stranded
endonuclease and 3 → 5 exonuclease activities on both singlestranded and double-stranded DNA-substrates. Unlike for
the exonuclease activity, the single-stranded endonuclease
activities observed on stem-loop substrates and at the single
strand–double-strand junction in forked-hairpin substrates were
not inhibited with ATP. These results suggested that DR0505 is
an ATP-regulated DNA-processing enzyme and a thermotolerant
esterase in vitro. We therefore suggest possible roles of this
enzyme in nucleotide recycling and DNA processing, which is
required for efficient double-strand break repair and the high
radiation tolerance observed in D. radiodurans.
Key words: 5 -nucleotidase, Deinococcus radiodurans, DNAend-processing, DR0505, dual-function enzyme, thermotolerant
esterase.
levels of abiotic stress tolerance [7,8]. Mutation rates are
significantly lower in bacteria exhibiting extraordinary tolerance
to oxidative stress produced by ionizing radiation, desiccation and
chemical oxidants.
Deinococcus radiodurans strain R1 has an extraordinary
tolerance to various abiotic stresses, including radiation,
desiccation and chemical oxidants [9,10]. Its extreme phenotype is
attributed to an efficient DNA DSB (double-strand break) repair
[11,12] and strong oxidative stress tolerance mechanisms. This
bacterium contains all the components of base excision repair,
like a DNA repair polymerase [13], AP (apurinic/apyrimidinic)endonuclease-type activity [14] and various types of DNA
glycosylases [15]. In addition, the bacterium also encodes
enzymes responsible for both the pathways of nucleotide excision
repair [16,17] and mismatch repair [18,19]. Through these
excision repair pathways both damaged and normal nucleotides
are released into the cellular pool of nucleotides. Similarly, D.
radiodurans, exposed to high doses of γ -radiation, showed a
burst of in vivo DNA degradation [20,21] before DNA synthesis
is resumed. These processes would eventually generate a large
pool of natural, as well as damaged, deoxynucleotides in the
cell. Therefore it has been suggested that there are mechanisms
that help to recycle these mononucleotides and ‘clean’ the
radiation-damaged, potentially mutagenic, nucleotide species
from dividing cells in D. radiodurans [10]. The genome of this
bacterium encodes a large number of NUDIX (for nucleoside
Abbreviations used: AP, apurinic/apyrimidinic; BNPP, bis-(p -nitrophenyl) phosphate; ds, double-stranded; DIG, digoxigenin; DSB, double-strand
break; DTT, dithiothreitol; Ni-NTA, Ni2+ -nitrilotriacetate; NUDIX, nucleoside diphosphate linked to some other moiety x; ORF, open reading frame; PDE,
phosphodiesterase; PME, phosphomonoesterase; PNPP, p -nitrophenyl phosphate; ss, single-stranded.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2010 Biochemical Society
150
S. Kota, C. V. Kumar and H. S. Misra
diphosphate linked to some other moiety x) family enzymes
and uncharacterized nucleotide metabolic enzymes and hence
the roles of these proteins in recycling and detoxification of
mononucleotides are worth investigating.
In the present study we functionally characterized DR0505,
a 5 nucleotidase family protein, from D. radiodurans. The
recombinant DR0505 showed both PME (phosphomonoesterase)
and PDE (phosphodiesterase) activities and DNA-processing
activities in vitro. The PME activity of the enzyme was resistant
to increasing temperature, up to 50 ◦C with Mg2+ and 80 ◦C with
Mn2+ , with PDE function resistant up to 50 ◦C with both of these
metal ions. The enzyme dephosphorylated normal nucleotides,
8-oxo-GMP and 2 ,3 -cyclic nucleotides at different rates. The
enzyme showed ATP-sensitive 3 → 5 exonuclease activities on
both ss (single-stranded) DNA and ds (double-stranded) DNA
substrates; however, the ssDNA endonuclease activities on stemloop and forked-hairpin substrates were not inhibited with ATP.
These results suggest that DR0505 is a bifunctional enzyme
exhibiting temperature-resistant esterase functions and ATPregulated DNA-processing activities that are possibly required
for recycling of nucleotides and DSB repair during the postirradiation recovery of D. radiodurans.
MATERIALS AND METHODS
Bacterial strains and materials
The D. radiodurans strain ATCC13939 was a gift from
Dr M. Schaefer (Division of Epigenetics, Deutsches
Krebsforschungszentrum, Heidelberg, Germany) [22]. Wildtype bacteria and their derivatives were grown aerobically in
TGY (0.5 % Bacto tryptone, 0.3 % Bacto yeast extract and
0.1 % glucose) broth or on agar plates as required at 32 ◦C.
Escherichia coli expression vector pET28a+ and its derivatives
were maintained in E. coli strain HB101 in the presence of
25 μg/ml kanamycin. Other recombinant techniques used were
as described in [23]. All the molecular biology grade chemicals
including restriction enzymes and DNA-modifying enzymes
were purchased from Sigma–Aldrich, Roche Biochemicals, New
England Biolabs or Bangalore Genei.
Construction of DR0505 expression plasmid
Genomic DNA of D. radiodurans R1 was prepared as
described previously [24]. The 1.7 kb coding sequence
of DR0505 was PCR-amplified from the genomic DNA of
D. radiodurans using the gene-specific forward primer (5 GGAATTCCATATGAAGAAAAACCTGCTGCT-3 ) and reverse primer (5 -CGGAATTCCTCATTACTTCAGTTTGACGAT3 ). The PCR product was cloned between the NdeI and EcoRI
sites in pET28a+. Restriction analysis and nucleotide sequencing
ascertained the identity and correctness of the gene cloned
in expression vector. The recombinant plasmid pET0505 was
transformed into E. coli BL21 (DE3) pLysS for expression of
recombinant proteins.
Expression and purification of recombinant protein
The recombinant DR0505 was purified from transgenic E. coli
BL21 (DE3) pLysS, harbouring pET0505, by Ni-NTA (Ni2+ nitrilotriacetate) affinity chromatography as described previously
[25]. In brief, the IPTG (isopropyl β-D-thiogalactopyranoside)induced cell pellet was dissolved in lysis buffer containing 20 mM
Tris/HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA and 1 mm PMSF,
c The Authors Journal compilation c 2010 Biochemical Society
and was incubated with 200 μg/ml lysozyme for 30 min. The
mixture was treated with 0.25 % Triton X-100 for 30 min at
37 ◦C and sonicated using a 4 μm tip at 40 % duty cycle by
giving 10 s pulses with intermittent cooling on ice for 2 min. The
majority of the recombinant protein was separated from clear
supernatant by centrifugation at 12 000 g for 30 min. The pellet
was dissolved in buffer B (10 mM Tris/HCl, pH 8.0, 100 mM
NaH2 PO4 and 8M urea). The mixture was incubated at room
temperature (25 ◦C) under shaking for 1 h and centrifuged at
12 000 g for 20 min. The Ni-NTA matrix (Qiagen) was directly
added to this supernatant and the protein was allowed to bind
to the resin under gentle shaking conditions for 2 h. The resin,
with protein bound, was passed through a 3 kDa cut-off filter
column and washed thoroughly with buffer C (buffer B at pH 6.3).
The proteins were eluted with buffer D (buffer B at pH 5.9) and
subsequently with buffer E (buffer B at pH 4.5) and analysed
via SDS/PAGE. The fractions eluted with buffer E, containing
nearly 99 % pure protein, were pooled and dialysed sequentially
in a buffer containing 10 mM Tris/HCl, pH 7.5, 50 mM NaCl,
1 mM DTT (dithiothreitol) and 1 mM EDTA with decreasing
concentrations of urea (from 8 M to zero). Finally the pooled
fractions were dialysed in a buffer containing 20 mM Tris/HCl,
50 mM NaCl, 1 mM DTT, 1 mM EDTA, 1 mM PMSF and
50 % (v/v) glycerol. The protein was stored in small aliquots
at −20 ◦C for subsequent use. The MS analysis of purified protein
and peptide mass fingerprints generated from MALDI (matrixassisted laser-desorption ionization) MS confirmed both the purity
and identity of the recombinant protein as a DR0505.
Measurements of enzyme activity
The PME activity was measured with the disodium salt of PNPP
(p-nitrophenyl phosphate), using a standard method as described
previously [26]. In brief, the activity assay was carried out at
37 ◦C in a 0.5 ml reaction volume containing 500 ng of enzyme
in 50 mM Tris/HCl, pH 8.0, 5 mM of MnCl2 , MgCl2 or CaCl2 ,
and 10 mM PNPP. After an 20 min incubation the reaction was
stopped with 6 M NaOH and the concentration of p-nitrophenol
was determined from the absorbance at 410 nm against substrate
blank. Enzyme activity was expressed as nmol or μmol of
p-nitrophenol formed/min per mg of protein. Similarly, the total
PDE activity was assayed with 500 ng of purified protein in a
0.5 ml reaction volume containing 500 ng of enzyme in 50 mM
Tris/HCl, pH 8.0, 5 mM of MnCl2 , MgCl2 or CaCl2 , and 1 mM
BNPP [bis-(p-nitrophenyl) phosphate] and incubated at 37 ◦C for
20 min. The reaction was stopped with 3 M NaOH and release of
p-nitrophenol was measured at 410 nm as described previously
[27]. Subsequently, all enzyme assays were carried out in the
presence of 5 mM MnCl2 , unless mentioned.
The pH optima of enzyme were determined as described
previously [28]. In brief, the activities at different pH were
assayed in different buffers: 50 mM sodium citrate buffer for
the pH range from 3 to 6; 50 mM Tris/HCl for the pH range
from 7 to 9; and 100 mM carbonate/bicarbonate for the pH range
from pH 10 to 11. For temperature optima determination, the
esterase activities were measured at different temperatures and at
pH 8.0 in the presence of 5 mM MnCl2 , 5 mM MgCl2 or 5 mM
CaCl2 . Similarly, to determine the ATP effect and the various
DNA-processing activities, 500 ng of protein was pre-incubated
with different concentrations of ATP in a 0.5 ml reaction volume
containing 50 mM Tris/HCl, pH 8.0, and 5 mM MnCl2 for 20 min
at room temperature, followed by an esterase assay with 10 mM
PNPP and different types of DNA substrates.
Deinococcus thermotolerant esterase with DNA-processing functions
The nucleotidase activity on natural nucleotide substrates was
assayed as described previously [28]. In brief, a 0.1 ml reaction
mixture, containing 50 mM Tris/HCl, pH 8.0, 5 mM MnCl2 ,
0.2 mM 8-oxo-dGMP (or 0.5 mM for other nucleotides) and
0.2 μg of protein sample, was incubated at 37 ◦C for 20 min.
The reaction was stopped by the addition of a 1/4 volume of
Malachite Green reagent. The mixture was incubated further for
20 min and then levels of Pi were measured at 630 nm. Similarly,
the PDE activity with 2 ,3 -cyclic nucleoside monophosphates was
measured essentially as described in [29]. The reaction
was performed in 80 μl reaction volumes containing 50 mM
Tris/HCl, pH 8.0, 5 mM MnCl2 , 0.5 mM 2 ,3 -cAMP or 2 ,3 cGMP and 0.2 μg of protein and incubated for 40 min at 37 ◦C.
The reaction was stopped by the addition of Malachite Green
reagent and the amount of Pi released was measured after a
20 min incubation at 630 nm. For the K m and V max determination,
the enzyme assay was performed with 500 ng of purified protein
with different concentrations of substrates (1–10 μM for PNPP;
0.1–10 μM for BNPP; 10–200 nM for AMP; and 2–100 nM for
cAMP) and the Pi released was measured with Malachite Green
as described above.
DNA-binding and nuclease activity assay
The DNA-binding assay was performed in 50 μl reaction mixture
volumes containing 50 mM Tris/HCl, pH 8.0, 5 mM MnCl2 ,
75 mM NaCl, 0.1 mM DTT, 200 ng of dsDNA substrates and
200 ng of protein at 37 ◦C for 5 min. Products were analysed
on a 0.8 % agarose gel. The nuclease activity was checked on
dsDNA and circular ssDNA (M13Mp18) substrates. The 200bp PCR-amplified linear dsDNA was labelled at the 3 ends
with DIG (digoxigenin)–dUTP using Klenow enzyme and DIGlabelling kit protocols (Roche Biochemicals). An approx. 50 ng
equivalent of DIG–dUTP linear dsDNA and 500 ng of unlabelled
circular M13mp18 ssDNA were incubated with 500 ng of purified
protein in a reaction mixture containing 50 mM Tris/HCl, pH 8.0,
75 mM NaCl, 0.1 mM DTT and 5 mM MnCl2 (or other bivalent
metal ions) at 37 ◦C for a defined time period. Products from the
DIG–dUTP-labelled dsDNA substrate were separated by native
PAGE (5 % gels), transferred on to nylon membranes (Hybond;
GE Healthcare), blotted with an anti-DIG–AP antibody (Roche
Molecular Biochemicals) and chemiluminescent signals were
detected as described in the manufacturer’s protocol. For the
temperature effect on DNA-binding activity and the effect of
ATP on nuclease activity, 200 ng of protein was pre-incubated
in reaction buffer containing 50 mM Tris/HCl, pH 8.0, 5 mM
MnCl2 , 75 mM NaCl and 0.1 mM DTT for 20 min at different
temperatures and at different ATP concentrations respectively.
Treated samples were mixed with DNA substrates and incubated
at 37 ◦C for 5 min, in the case of the DNA-binding assay, and for
different time periods for the nuclease activity assay. The mixture
was heated at 65 ◦C with 95 % formamide containing 25 mM
EDTA to dissociate the nucleoprotein complex as described
previously [30]. Products from unlabelled linear dsDNA, circular
ssDNA and superhelical plasmid DNA (pBluescript SK+) were
analysed on agarose gels and detected by ethidium bromide
staining.
151
labelled at the 3 end with [α-32 P]dCTP using Klenow enzyme
and at the 5 end with [γ -32 P]ATP using polynucleotide kinase.
The unincorporated nucleotides were removed using G-25 spin
columns (GE Healthcare).
DNA-end-processing activities were assayed as described
previously [31,32]. In brief, 160 nM enzyme was mixed
with ∼10 nM each of the labelled homopolymer of dA40
(40 × deoxyadenosine phosphate substrate), HP2 (hairpinforked substrate, 5 -AAAAAAGACCTGGCACGTAGGACAGCAGCTGCTGTCCTACGTGCCAGGTCAAAAAA-3 ) [33]
or HP78 (stem-loop substrate, 5 -GTTTCTATTCAGCCCTTTGACGTAATCCAGCCCCGGGTTTTCCCGGGGCTGGATTACGTCAAAGGGCTGAATAGAAAC-3 ) [34]. The reaction mixture was incubated at 37 ◦C for 1 h with HP78, for 2 h with
HP2 and for various times with the dA40 substrate. The ss
products of HP2, HP78 and dA40 were separated by urea/PAGE
(8 M urea/12 % acrylamide gels). Radiolabelled products were
detected by autoradiography.
Statistics
All the experiments were repeated at least three times. Results
presented without an S.D. are from a representative experiment.
RESULTS AND DISCUSSION
A multiprotein DNA-processing complex isolated from D.
radiodurans R1 has been shown previously to be an ATPsensitive exonuclease and to have an ATP-stimulated DNA endjoining activity, along with bivalent ion and ATP-independent
topoisomerase functions [35]. DR0690 (topoisomerase 1B type),
DRB0100 (an ATP-type DNA ligase) and a few uncharacterized
ORFs (open reading frames) including DR0505 (a putative 5
nucleotidase family enzyme) were among the 24 polypeptides
identified from the multiprotein complex. Hence a further study
on the uncharacterized ORFs was performed to identify the ATPsensitive nuclease functions of the complex.
DR0505 showed structural similarities with 5 nucleotidase
enzyme of Thermus species
All of the uncharacterized proteins detected in the complex
were checked for the presence of putative functional domains
in silico. Among them, DR0505 was found to have three distinct
functional signatures: (i) a leader peptide in the first 27 amino
acids at the N-terminus; (ii) a calcineurin-type PDE domain
from amino acids 32–297; and (iii) two 5 nucleotidase domains,
one from 225–297 and a second from 363–557 towards the Cterminal end of the polypeptide (Supplementary Figure S1 at
http://www.BiochemJ.org/bj/431/bj4310149add.htm). Structure
prediction using I-TASSER (iterative threading assembly
refinement) tools indicated that this protein was likely to form
a structure resembling the 5 nucleotidase family protein from
Thermus species at the most stable energy minimization state
(TM score of 0.63 +
− 0.13 and root mean square deviation values
of 9.2 +
− 4.6 Å) (results not shown). This indicated the possibility
that DR0505 conferred both nuclease and esterase activities and
hence this protein was characterized subsequently.
DNA-end-labelling and processing activity assay
The DNA-processing activity of the protein was assayed on DNA
substrates with different topologies and structures of DNA ends.
These substrates were labelled at either 3 or 5 ends using standard
protocols as described in [23]. In brief, the substrates were
Recombinant DR0505 was characterized as a dual esterase
The recombinant DR0505 protein was purified to near
homogeneity with > 99 % purity (Supplementary Figure S2
c The Authors Journal compilation c 2010 Biochemical Society
152
S. Kota, C. V. Kumar and H. S. Misra
(Figure 1B) at 37 ◦C. ATP inhibited the esterase function of this
enzyme in the presence of both Mg2+ and Mn2+ ions (Figure 1C);
DR0505 did not show an ADP-related effect on its esterase activity
(results not shown). A differential response of nucleotidases to
ATP and ADP has been reported from mammalian systems with
the distinctions between the ADP and ATP effect on nucleotidase
activity primarily depending upon the cellular location of the
enzyme in multicellular organisms [5].
The K m and V max for PME and PDE activities were
measured using double-reciprocal plots (Supplementary Figure
S3 at http://www.BiochemJ.org/bj/431/bj4310149add.htm). The
enzyme had a K m and V max of 2.52 +
− 0.245 μM and
5.558 +
− 0.845 μmol/min per mg of protein with PNPP,
and 0.423 +
− 0.053 μM and 35.037 +
− 2.873 μmol/min per mg
of protein with BNPP respectively at pH 8.0 and 37 ◦C. These
results indicated that the DR0505 was a better PDE than PME
with its pH optimum in alkaline conditions (pH 9) at 37 ◦C.
Although the ATP inhibition of esterase function was found to
be a unique feature of this enzyme, the functional significance of
this regulation in the radioresistance of D. radiodurans is unclear.
Recently, we showed that in D. radiodurans ATP levels increased
within 1 h post-irradiation [36], with the levels subsequently
dropping with a concurrent increase in DNA synthesis and
reassembly of the shattered genome. The level of expression of
DR0505 gene is also highest at 1 h post-irradiation [37], although
they remained high for 4 h. These results indicate that
although the level of DR0505 was high, ATP might serve to
regulate the enzyme’s activity during early in the initial postirradiation response in D. radiodurans.
DR0505 protein can be characterized as a thermotolerant
mesophilic enzyme
Figure 1
in vitro
Phosphoesterase activity characterization of DR0505 protein
(A) The phosphoesterase activities of purified recombinant DR0505 was assayed on chromogenic
substrates PNPP and BNPP in the presence of 5 mM of each MnCl2 (1), CaCl2 (2) or MgCl2 (3).
(B) The PME (with PNNP) and PDE (with BNNP) activities of DR0505 was assayed at different
pH in the presence of 5 mM MnCl2 . (C) The effect of ATP on PDE activity of DR0505 was
measured at 0.5 mM (2), 1.0 mM (3) and 2.0 mM (4) ATP and compared with the control (1)
without ATP. All the experiments were repeated at least three times in triplicate and the results
are means +
− S.D.
at http://www.BiochemJ.org/bj/431/bj4310149add.htm) from
transgenic E. coli BL21 (DE3) pLysS cells harbouring the
pET0505 plasmid. The recombinant protein showed both PME
and PDE activities on PNPP and BNPP respectively. These
substrates were then used for the determination of the bivalent ion
requirement, the pH and temperature optima, and the catalytic
efficiency. The enzyme showed a bivalent ion preference for
its both PME and PDE activities (Figure 1); the activity was
higher in the presence of Mn2+ , as compared with Mg2+ and
Ca2+ ions (Figure 1A). The esterase activities of the protein were
characterized for temperature and pH optima, and the catalytic
efficiency in terms of K m and V max using PNPP and BNPP. The
enzyme showed highest activity in the pH range from 8.0 to 9.0
c The Authors Journal compilation c 2010 Biochemical Society
While determining the temperature optimum for PME activity
using PNPP, we observed that DR0505 showed a continuous
increase in its activity at temperature up to 45 ◦C. Hence,
both the esterase activities of DR0505 were measured as a
function of temperature at pH 8.0. Interestingly, the enzyme
showed a bivalent ion preference in temperature tolerance. The
PME activity of the enzyme was significant at 37 ◦C, and the
activity increases continued at temperatures up to 80 ◦C at
pH 8.0 in the presence of Mn2+ ions (Figure 2A). PME
activity measured in presence of both Mg2+ and Ca2+ ions
separately was less but showed a showed similar pattern of
thermotolerance. However, the PDE activity showed maximum
temperature tolerance only up to 50 ◦C in presence of Mn2+
ions (Figure 2B). The K m and V max for both PME and PDE
activities were determined at 80 ◦C and 50 ◦C. The K m and V max
were 3.652 +
− 1.137 μM and 20.46 +
− 3.215 μmol/min per mg of
protein for PME activity, and 0.739 +
− 0.213 μM and 69.86 +
−
7.512 μmol/min per mg of protein for PDE activity
respectively. Although the K m values measured at higher temperatures (50 ◦ C for PDE and 80 ◦ C for PME) remained nearly
similar to that measured at 37 ◦C, the V max was nearly 2-fold
higher on both the substrates in presence of Mn2+ ion at higher
temperatures. This suggested that DR0505 was a mesophilic
thermotolerant esterase. Although, the thermotolerant nature
of DR0505 in D. radiodurans, a mesophilic bacterium, may
be a vestigial property of an enzyme that evolved to operate
under primordial conditions, it might provide a higher stability
in order to perform functions under conditions of high γ irradiation, when the cellular temperature would increase. Other
thermotolerant enzymes isolated from mesophilic bacteria have
been reported with similar temperature characteristics, i.e. a wide
Deinococcus thermotolerant esterase with DNA-processing functions
153
Table 1 Nucleotidase activity characterization of DR0505 on different
types of nucleotide substrates
Figure 2
Effect of temperature on phosphoesterase activities of DR0505
(A) PME activity was measured in the presence of 5 mM MnCl2 (Mn), 5 mM MgCl2 (Mg) and
5 mM CaCl2 (Ca) and (B) PDE activity was measured in the presence of 5 mM MnCl2 (Mn)
and 5 mM MgCl2 (Mg) ions at different temperatures. All the experiments were repeated three
times and the results are means +
− S.D.
temperature optima from 37 ◦C to higher temperature [38]. In
addition, thermotolerant enzymes from thermophilic bacteria,
when produced in mesophilic hosts like E. coli, have continued
to be thermotolerant [39]. These results suggest that DR0505
was a thermotolerant mesophilic enzyme, and its expression in
E. coli therefore, did not affect the structural requirement for its
thermotolerance.
DR0505 was characterized as 5 nucleotidase in vitro
D. radiodurans has a very high tolerance to γ -irradiation and
oxidative stress, which induce different types of DNA base
damage that are repaired by efficient excision/incision repair
pathways [10,40]. As a result, the levels of both damaged
and undamaged nucleotides are bound to increase in the cell.
These nucleotides are subsequently recycled in fresh nucleotide
biosynthesis and/or are exported by NUDIX family enzymes [41].
This helps the cells by avoiding re-incorporation of chemically
altered (mutagenic) bases in DNA during replication. How this
process operates in Deinococcus is not understood; however,
the mutation rate following irradiation is extremely low in
this bacterium [11] and the possibility of an efficient system
for recycling of non-toxic nucleotides and detoxification of
mutagenic nucleotides cannot be ruled out. Nucleotidases are
known for their involvement in nucleotide recycling in living cells.
Therefore the 5 nucleotidase activity of DR0505 was assessed
with different nucleotides including 8-oxo-GMP, a biomarker of
oxidative damage of DNA and a potent mutagenic agent. The
enzyme showed differential activity on different mononucleotides
and cyclic-nucleotides (Table 1). The enzyme dephosphorylated
dCMP and dGMP ∼1.5–2.0-fold faster than CMP and GMP
respectively. This activity on cAMP, cGMP and 8-oxo-GMP
Substrates
Specific activity (nmol of phosphate released/min
per mg of protein ( +
−S.D.)
dCMP
CMP
GMP
dGMP
AMP
ADP
cAMP
cGMP
8-Oxo-GMP
607.42 +
− 57.12
321.44 +
− 28.31
428.15 +
− 48.12
614.31 +
− 31.56
553.78 +
− 41.37
3500.92 +
− 178.23
808.05 +
− 33.19
715.14 +
− 72.36
643.34 +
− 46.12
substrates was ∼1.5-fold higher than AMP and GMP respectively.
The enzyme showed a nearly 7-fold faster ADP degradation
than AMP degradation, indicating the possible activation of this
enzyme by ADP (an ADP-mediated activation of nucleotidases
has been reported in mammalian systems [5]).
As the level of cAMP changes in response to γ -irradiation and
it has been shown to be an important regulator of stress-response
enzymes, the affinity of DR0505 toward cAMP was checked and
compared with AMP. The K m and V max of this enzyme were determined for AMP and cAMP. For AMP, the K m and V max were
56.948 +
− 12.345 nM and 70.375 +
− 9.143 nmol/min per mg of
protein, for cAMP, these parameters were 7.875 +
− 2.0156 nM,
and V max and 497.742 +
− 72.021 nmol/min per mg of protein
respectively. This indicated the higher affinity of DR0505 enzyme
toward cAMP. Recently, we demonstrated the kinetic change
in the levels of ATP and cAMP during the post-irradiation
response of D. radiodurans [36] and argued that the higher cAMP
levels post-irradiation is the net effect of low cyclic PDE and
higher adenylate cyclase activities. The cyclic PDE activity starts
increasing after 2 h post-irradiation and has reached a maximum
in 4 h post-irradiation. The transcriptome analysis also shows
γ -irradiation-induced expression of DR0505 1 h post-irradiation
and onwards [37]. DR0505, which gets induced during this period,
was incidentally found to have cyclic PDE activity indicating the
possible role of this enzyme in maintaining cellular levels of
cAMP during the post-irradiation reponse. The identification and
characterization of another calcineurin-like phosphoesterase [42]
and structure–function studies of a phosphotriesterase homologue
[43] have been reported from Deinococcus. These findings
indicate that esterases have a significant role in this bacterium
and suggest the possible role of DR0505 in recycling of both
natural and chemically altered nucleotides in vivo.
DR0505 was characterized as an ATP-sensitive exonuclease
In order to study the significance of the PDE activity of DR0505
in DNA metabolism, the DNA-binding activity of recombinant
DR0505 protein was first checked with both circular and linear
dsDNA substrates. The enzyme showed DNA-binding activity
with both linear dsDNA (Figure 3A) and superhelical plasmid
DNA (Figure 3B) at 37 ◦C. However, the pre-incubation of purified
DR0505 at higher temperature abolished its interaction with DNA
at 37 ◦C (Figure 3). These results suggest that DR0505 is a nonspecific DNA-binding protein. Unlike the esterase activities, the
DNA–protein interaction function of this protein was destroyed
when at higher temperatures.
On longer incubations, the enzyme showed metal ion preference
for nucleolytic degradation of both linear dsDNA and circular
ssDNA substrates. Both of these activities were inhibited by ATP
c The Authors Journal compilation c 2010 Biochemical Society
154
Figure 3
S. Kota, C. V. Kumar and H. S. Misra
Effect of temperature on DNA-binding activity of DR0505
The 200 bp (A) linear dsDNA and (B) superhelical (CCC) plasmid DNA pBluescript SK+
substrates were incubated at 37 ◦C for 5 min with recombinant DR0505 pretreated at different
temperatures for 20 min. Products were analysed on 0.8 % agarose gels and DNA bands were
visualized with ethidium bromide. Products were compared with EcoRI-linearized plasmid DNA
(linear) to rule out the possibility that superhelical dsDNA is converted into linear dsDNA by
DR0505.
in vitro (Figures 4 and 5). The enzyme showed nuclease activity on
circular M13mp18 ssDNA in the presence of both Mg2+ and Mn2+
ions but these activities were higher in the presence of Mn2+ as
compared with Mg2+ (Figures 4A and 4B). As the endonucleolytic
cleavage of circular DNA is a prerequisite of exonuclease activity,
the degradation of the circular ssDNA clearly indicated the
presence of endonuclease activity in DR0505. A time course assay
of DR0505 on circular ssDNA showed the processive degradation
of ssDNA in the absence of ATP, which was completely inhibited
in presence of ATP (Figure 4C).
The nuclease activity on linear dsDNA labelled at 3 termini
was highest in the presence of Mn2+ and the majority of the
3 label was rapidly hydrolysed in less than 40 min incubation
at 37 ◦C (Figure 5A). This activity was significantly lower in
the presence of other bivalent metal ions, such as Mg2+ , Ca2+ ,
Zn2+ and Co2+ . ATP inhibition was observed with both Mn2+ (Figure 5) and Mg2+ - (results not shown) dependent nuclease
activities. Incubation of the enzyme with 3 -labelled dsDNA for
various times at 37 ◦C showed a rapid removal of the 3 label on
dsDNA and more than 60 % of the total 3 labels were removed in
a 10 min incubation (Figures 5B and 5C). These results indicated
that DR0505 is an ATP-sensitive Mn2+ -dependent nuclease.
Absence of endonucleolytic products on dsDNA substrate and
faster removal of label from 3 -labelled dsDNA indicated that
DR0505 was possibly a 3 → 5 dsDNA exonuclease. Although
the distinction of exo- from endo-nuclease activity could not be
made at this stage, the enzyme showed time-dependent hydrolysis
of circular ssDNA, which is the characteristic of an exonucleolytic
degradation of DNA. ATP treatment of DR0505 quickly inhibited
both ssDNA and dsDNA exonuclease activities. After a longer
incubation of ATP-treated DR0505 with circular ssDNA there
were detectable levels of smaller size DNA products as obtained
from density scanning of agarose gels (Figure 4C and results not
shown) suggesting a low level of ssDNA endonuclease activity.
This result might argue that DR0505 was a non-specific nuclease,
and ATP inhibited its exonuclease activities on both ssDNA and
c The Authors Journal compilation c 2010 Biochemical Society
Figure 4 Nucleolytic activity assay of DR0505 on cicular ssDNA substrates
in presence of different bivalent ions
(A) M13mp18 circular ssDNA substrate was incubated at 37 ◦C with (2–5) or without (1 and 6)
recombinant DR0505 for 40 min, in the presence of 5 mM MnCl2 (1, 2 and 3) or 5 mM MgCl2
(4, 5 and 6), and with (2 and 5) or without (3 and 4) 2 mM ATP. The mixture was heated at 65 ◦C
in presence of 95 % formamide and 25 mM EDTA for 15 min as detailed in the Materials and
methods section. Products were analysed on 0.8 % agarose gels. (B) The fluorescence intensity
of DNA substrates (light bars) and products (dark bars) in (A) were obtained by density scanning
of the intact DNA band and the smear in the running track respectively. (C) M13mp18 circular
ssDNA substrate was incubated at 37 ◦C with DR0505 protein for 10, 20, 30 and 40 min in the
absence and presence of 2 mM ATP. The products were analysed on 0.8 % agarose gel and
compared with DNA without protein.
dsDNA in vitro. Further distinction of both the endonuclease
and exonuclease activities were next obtained with different
structured DNA substrates.
DR0505 showed ssDNA endonuclease and ATP-sensitive 3 → 5
exonuclease
DR0505 showed ATP-mediated regulation of ssDNA/dsDNA
exonuclease activity and circular ssDNA endonuclease. It did not
show dsDNA endonuclease activity (Figure 5). Since M13mp18
ssDNA forms numerous islands of dsDNA resulting in the
formation of ssDNA–dsDNA (ss–ds) junctions, the possibility
of DR0505 cutting at the ss–ds DNA junction has not been ruled
out. For this, the DNA processing activity of DR0505 was checked
on synthetic oligonucleotides forming a linear ss homopolymer
(dA40), stem-loop (HP78) or forked-hairpin (HP2) substrates
with homopolymeric forks and heteropolymeric dsDNA stems.
These 5 32 P-labelled DNA substrates were incubated with protein
and products were analysed by urea/PAGE. Results indicated
a successive although slow degradation of dA40 incubated for
Deinococcus thermotolerant esterase with DNA-processing functions
Figure 5 Nucleolytic activity assay of DR0505 on dsDNA substrates in the
presence of different bivalent ions
The 200 bp PCR product was labelled at the 3 end with DIG–dUTP and incubated with
recombinant DR0505 at 37 ◦C for 20 min in (A) the presence of 5 mM each of MnCl2 (Mn),
MgCl2 (Mg) CoCl2 (Co), CaCl2 (Ca) or ZnCl2 (Zn) and (B) for different time periods in presence
of 5 mM MnCl2 with and without ATP. Reaction mixtures were heated at 65 ◦C in the presence
of 95 % formamide and 25 mM EDTA for 15 min as detailed in the Material and methods
section. Products were analysed on a 0.8 % agarose gel. (C) The Mn2+ -dependent nucleolytic
degradation of dsDNA in the absence (−ATP) and presence (+ATP) of ATP was quantified by
density scanning of DIG-labelled DNA bands and plotted as a function of time.
different time periods, and both sizes and amount of substrate
(dA40) decreased concurrently with the increase in levels of
intermediate products and 5 terminal nucleotide ([32 P]dAMP)
(Figure 6A). This activity was completely inhibited in the
presence of ATP. The enzyme showed successive degradation of
5 -labelled HP78 in the absence of ATP, producing intermediate
products and terminal mononucleotide ([32 P]dGMP). This activity
was attenuated in the presence of ATP and three prominent
products, which presumably had arisen by cleaving either the
ss loop region, or the ss–ds DNA junction in HP78, were
generated (Figure 6B). Similarly, the 5 -labelled HP2, which
contains a 7 nt homopolymeric fork and forms a ss–ds DNA
junction was processed gradually into intermediate products in
the absence of ATP. In the presence of ATP, it gave a nearly
7 nt (fork size) 32 P-labelled product, which could have arose by
cleavage at ss–ds junctions in HP2 and complete attenuation
of exonuclease activity (Figure 6C). Enzyme incubated with
3 -labelled HP78 generated exclusively deoxymononucleotide
(dCMP[32 P]), the 3 terminal base, and no intermediate products
were seen (Figure 6D). The release of low amounts of [32 P]dGMP
from 5 -labelled HP78 and faster release of dCMP[32 P] from 3 labelled HP78 confirmed the initiation of HP78 degradation from
155
3 ends towards 5 ends. Similarly, the successive degradation
of 5 -labelled dA40 and release of [32 P]dAMP and 37–39-mer
endonucleolytic products from 5 -labelled HP78 and the nearly
7 nt (fork size) product from 5 -labelled HP2 (Figure 6), suggested
that DR0505 contained both 3 → 5 exonuclease activity and
ss–ds DNA junction endonuclease activity in vitro. Differential
regulation of these activities by ATP could characterize this
protein as a novel Mn2+ -dependent dual-function nuclease in the
bacterial system. It is noteworthy that purified DR0505 showed
relatively low activity on both homopolymeric linear ssDNA
(dA40) and heteropolymeric circular ssDNA as compared with
linear dsDNA. This could be due to the nature of this enzyme
or because its higher activity requires interaction with other
proteins of the multiprotein complex, the source from which it was
identified. Similar results have been found for another protein of
the complex DRB0100 as it requires some of the complex proteins
for its higher activity in vitro [44].
The present study has brought forth some interesting findings
to suggest that DR0505 is a dual-function enzyme having both
esterase and nuclease activities, and provided indirect evidence for
its activity regulation by ATP in vivo. ATP inhibition of nuclease
activity and thermotolerance of esterase functions are two novel
features of this enzyme. ATP inhibition of nuclease activity
has not been shown for bacterial nucleases to date. In contrast,
the stimulation of exonuclease activity with ATP (exonuclease
V) and a Mn2+ -dependent Mre11/Rad50-type function of the
SbcCD complex have been shown in both E. coli [31,45] and
in D. radiodurans [32]. Purified DR0505 showed differential
regulation of its exonuclease and endonuclease activities by ATP
(compare Figures 4, 5 and 6) in vitro, indicating the significance of ATP regulation in DR0505 functions during the postirradiation response in D. radiodurans. These functions might
have a direct relevance in recycling of nucleotide pools and
DNA processing to order to achieve efficient DSB repair in D.
radiodurans. As both the ssDNA endonuclease and the hairpin
endonuclease activities of this protein were insensitive to ATP,
the functional significance of these activities, irrespective of
ATP status, during the post-irradiation response can be speculated
upon. The radiation-induced synthesis of DR0505 [37] and
increased ATP levels [36] coincides with the early-phase of the
post-irradiation response in D. radiodurans. This might mean
that attenuation of the exonuclease activity of DR0505, which
could lead to the degradation of genetic materials, is required
due to the higher ATP levels at 1 h post-irradiation. Subsquently,
from 1 h onwards when ATP is low, the enzyme contributes to
DNA end-processing for efficient recombination repair. Since,
γ -irradiation induces different types of DNA breaks and their
processing is required for efficient reassembly, it is therefore
possible that the ss–ds DNA junction endonuclease activity of
this protein is active during the post-irradiation response. It
is noteworthy that a deletion mutant of dr0505 did not show
any significant change in tolerance to γ -irradiation compared
with the wild-type strain (results not shown) suggesting possible
functional redundancy for this protein in vivo and that DR0505
seems to be one of the members of a protein family doing similar
or related functions in this bacterium. In previous studies, the
phosphoesterases [42,43] and DNA-end-processing activity of an
X family DNA polymerase [46] and SbcCD proteins [32,47] were
reported in D. radiodurans. Thus DR0505, characterized in the
present study, represents a novel protein conferring two important
functions, the thermotolerant esterase and endo- and exo-nuclease
activities in vitro, reponses which are differentially regulated by
ATP. Further studies are required to understand the nature of
micro-environment responsible for the differential temperature
responses in the esterase and nuclease activities in same protein
c The Authors Journal compilation c 2010 Biochemical Society
156
Figure 6
S. Kota, C. V. Kumar and H. S. Misra
DNA-processing activity assay of DR0505 on DNA substrates with different structures in solution
(A) The ss 40-mer poly dA (dA40), (B) stem-loop 78-mer (HP78) and (C) hairpin-forked 56-mer (HP2) were labelled at the 5 end with (D) HP78 also labelled at the 3 end. Purified substrates (S)
were incubated with purified protein for the indicated times with dA40, 1 h with HP78 labelled at the 5 end, 2 h with HP2 and 2 h with HP78 labelled at the 3 end both in the presence and absence
of ATP. Products were analysed by urea/PAGE and radiolabelled DNA bands were visualized by autoradiography. Arrows indicate the position of different substrates and the corresponding products
generated from both endonucleolytic and exonucleolytic cleavage of respective substrate as shown in the autoradiogram. Labelled deoxymononucleotides are released by either the terminal cleavage
of the (D) 3 -labelled base (dCMP) or 3 → 5 processive degradation of 5 -labelled (A) ssDNA and (B and C) dsDNA substrates.
molecule and the exact role of this protein in DNA metabolism
and radiation tolerance of D. radiodurans.
AUTHOR CONTRIBUTION
Swathi Kota identified DR0505 from the multiprotein complex, proposed possible functions
for this protein, conducted experiments, analysed the results, and discussed and wrote
paper. Vijay Kumar obtained and analysed results. Hari Misra, conceived the idea, planned
experiments, wrote the paper and served as the principal investigator for the project.
ACKNOWLEDGEMENTS
We thank Dr S.K. Apte for his valuable suggestions and criticisms during the pursuance
of this work.
FUNDING
This work was support by the Department of Atomic Energy (a fellowship to C.V.K.).
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Received 30 March 2010/20 July 2010; accepted 26 July 2010
Published as BJ Immediate Publication 26 July 2010, doi:10.1042/BJ20100446
c The Authors Journal compilation c 2010 Biochemical Society
Biochem. J. (2010) 431, 149–157 (Printed in Great Britain)
doi:10.1042/BJ20100446
SUPPLEMENTARY ONLINE DATA
Characterization of an ATP-regulated DNA-processing enzyme and
thermotolerant phosphoesterase in the radioresistant bacterium
Deinococcus radiodurans
Swathi KOTA, C. Vijay KUMAR and Hari S. MISRA1
Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400 085, India
Figure S1 Schematic representation of different functional domains in
DR0505 polypeptide
DR0505 contains a leader peptide (LP) followed by a well-defined calcineurin phosphodiesterase
domain (CPDD) at the N-terminus and 5 nucleotidase domain at the C-terminus of the protein.
Figure S2
E. coli
Expression and purification of recombinant DR0505 protein from
The dr0505 coding sequences were PCR amplified and cloned at the NdeI and EcoRI sites
in pET28a+ to yield pET505. Transgenic E. coli harbouring pET505 was grown to exponential
-phase and induced with IPTG (isopropyl β-D-thiogalactopyranoside). (A) Total proteins from
uninduced (1) and induced (2) cells were analysed via SDS/PAGE. (B) The recombinant protein
was purified (P) from transgenic E. coli expressing DR0505 protein on pET505 and size was
ascertained. Lane M shows standard size protein markers.
Figure S3 Lineweaver–Burk plots of enzyme activity as a function of
substrate concentrations
Enzyme activity of DR0505 was determined at different concentrations of PNNP and BNPP in the
presence of 5 mM MnCl2 as described in the Materials and methods section in the main paper.
Reciprocals of both enzyme activity (1/V) and substrate concentration (1/[S]) were plotted for
determination of K m and V max values. All the experiments were repeated three times and the
results are means +
− S.D.
Received 30 March 2010/20 July 2010; accepted 26 July 2010
Published as BJ Immediate Publication 26 July 2010, doi:10.1042/BJ20100446
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2010 Biochemical Society