From the Istituto di Scienze Biochimiche, Università di Parma, I-43100 Parma, Italy
Received for publication, November 26, 2000, and in revised form, February 19, 2001
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ABSTRACT |
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Two novel, structurally and functionally
distinct phosphatases have been identified through the functional
complementation, by maize cDNAs, of an Escherichia coli
diphosphonucleoside phosphatase mutant strain. The first, ZmDP1,
is a classical Mg2+-dependent and
Li+-sensitive diphosphonucleoside phosphatase that
dephosphorylates both 3'-phosphoadenosine 5'-phosphate (3'-PAP) and
2'-PAP without any discrimination between the 3'- and 2'-positions. The
other, ZmDP2, is a distinct phosphatase that also catalyzes
diphosphonucleoside dephosphorylation, but with a 12-fold lower
Li+ sensitivity, a strong preference for 3'-PAP, and the
unique ability to utilize double-stranded DNA molecules with
3'-phosphate- or 3'-phosphoglycolate-blocking groups as substrates.
Importantly, ZmDP2, but not ZmDP1, conferred resistance to a DNA
repairdeficient E. coli strain against oxidative
DNA-damaging agents generating 3'-phosphate- or
3'-phosphoglycolate-blocked single strand breaks. ZmDP2 shares a
partial amino acid sequence similarity with a recently identified human
polynucleotide kinase 3'-phosphatase that is thought to be involved in
DNA repair, but is devoid of 5'-kinase activity. ZmDP2 is the first DNA
3'-phosphoesterase thus far identified in plants capable of converting
3'-blocked termini into priming sites for reparative DNA polymerization.
Diphosphonucleoside phosphatases
(DPNPases)1 catalyze the
conversion of diphosphonucleosides such as 3'-phosphoadenosine
5'-phosphate (3'-PAP) into their 5'-monophosphorylated derivatives
(5'-AMP). They are ubiquitous among prokaryotes and eukaryotes and
belong to a superfamily of Mg2+-dependent,
lithium-sensitive phosphohydrolases, which also includes fructose-1,6-bisphosphate 1-phosphatase and various
inositol-polyphosphate phosphatases. The prototype of prokaryotic
DPNPases is the product of the Escherichia coli gene
cysQ, which, if mutated, abolishes the capacity of bacterial
cells to grow on sulfate as the sole source of sulfur (1). The first
eukaryotic DPNPase to be isolated was the Li+- and
Na+-sensitive enzyme encoded by the Saccharomyces
cerevisiae gene HAL2 (2). By preventing the
accumulation of 3'-PAP, an inhibitory side product generated upon
reduction of 3'-phosphoadenosine 5'-phosphosulfate (3'-PAPS) to
sulfite, the Hal2p phosphatase controls the flux of sulfate
along the sulfur assimilation pathway (3). Because of the blockage of
sulfur assimilation and the concomitant methionine auxotrophy caused by
PAP accumulation under conditions of salt-inhibited Hal2p, this enzyme
is considered a specific target of salt toxicity (4). During the last
few years, various Li+-sensitive phosphatases, all capable
of restoring the ability of a yeast hal2/met22 mutant to
grow on sulfate as the sole source of sulfur, have been identified in
fungi, plants, and mammals (5-11). Despite the widespread occurrence
of PAPS as an activated sulfate derivative for assimilatory sulfate
reduction or sulfation reactions, the main metabolic scope of PAP
hydrolysis by microbial or higher eukaryotic DPNPases is different. In
fact, reductive sulfate assimilation leading to de novo
cysteine or methionine biosynthesis does not take place in mammals, and
it mainly proceeds through a PAPS-independent pathway in plants (12).
The major role of DPNPases in these organisms is thus to act in concert with sulfotransferases to drive sulfate ester production through the
removal of the inhibitory by-product PAP (13, 14). An even more general
role of DPNPases has been revealed by the recent demonstration that PAP
accumulation inhibits RNA-processing 5' No DNA 3'-phosphatase activity has thus far been identified in plants,
organisms that, besides being subjected to oxidative DNA damage
caused by endogenously produced activated oxygen species, are also
particularly exposed to (and have a strong capacity to cope with)
oxidative agents such as ozone, redox-cycling herbicides, and other
environmental pollutants. Based on the assumption that a
diphosphonucleoside such as 3'-PAP might be acted upon by both standard
DPNPases and DNA 3'-phosphatases, we took advantage of the high
sensitivity and lack of sequence bias of in vivo functional complementation to search for a DNA 3'-phosphatase activity in maize.
This approach led to the isolation of ZmDP1
(Zea mays diphosphonucleoside
phosphatase-1), a new
Mg2+-dependent, Li+-sensitive
DPNPase, and ZmDP2, the first plant 3'-phosphoesterase thus far
identified with the unique ability to catalyze the removal of
3'-phosphate- or 3'-phosphoglycolate-blocking groups from
double-stranded DNA molecules and to confer resistance to oxidative DNA
damage in a repair-deficient bacterial strain.
Functional Complementation of Bacterial and Yeast DPNPase
Mutants--
A plasmid-borne (pBluescript KS+, Stratagene)
cDNA expression library prepared from the roots of sulfate-deprived
maize seedlings (19) was used to search for clones conferring cysteine
prototrophy to E. coli cysQ mutant cells (strain
MC4100, a gift of D. Berg, Washington University Medical School, St.
Louis, MO). DNA transformation was carried out by electroporation, and
transformants were selected on M9 minimal medium containing 100 µg/ml
ampicillin and 0.15 mM
isopropyl-
For yeast complementation assays, the ZmDP1 and
ZmDP2 cDNAs were subcloned into the yeast expression
vector pFL61 (a gift of M. Minet, CNRS, Gif-sur-Yvette, France).
Properly oriented pFL61-ZmDP constructs, identified by
restriction analysis, were transformed into electrocompetent, S. cerevisiae met22 mutant cells (strain CD108, kindly
provided by Y. Surdin-Kerjan, CNRS). Yeast transformants were first
selected for their ability to grow on synthetic dextrose-agar
medium supplemented with methionine, but lacking uracil, and
subsequently selected on synthetic dextrose medium lacking both uracil
and methionine.
DNA and RNA Analyses--
Genomic DNA for gel blot analysis was
extracted following a previously described procedure (20). DNA samples
(20 µg each) were digested with EcoRI and
BamHI, followed by electrophoresis on a 0.8% agarose gel,
which was subsequently denatured and neutralized by standard procedures
(21). A random priming labeling kit (Amersham Pharmacia Biotech) was
used to prepare 32P-labeled hybridization probes from the
ZmDP1 (1453 bp) and ZmDP2 (992 bp) cDNAs.
Blotting onto Hybond-N nylon membranes (Amersham Pharmacia Biotech),
prehybridization, hybridization, and washing were conducted according
to the manufacturer's instructions.
Total RNA for RNase protection and primer extension analyses was
isolated as described previously (19). 32P-Labeled
antisense riboprobes for RNase protection assays were prepared by
in vitro SP6 RNA polymerase (Promega) transcription of pCRII
plasmids (Invitrogen) carrying a 461-bp fragment of ZmDP1 (positions 675-875) or a 240-bp fragment of ZmDP2
(positions 109-348), previously digested with either PstI
or EcoRV, respectively. The resulting riboprobes were 540 nucleotides (ZmDP1) and 313 nucleotides (ZmDP2)
in length, and both included 80 nucleotides of vector-derived sequence.
Saturating amounts of a 210-nucleotide maize glyceraldehyde-3-phosphate dehydrogenase (GAPDH) riboprobe (including 9 nucleotides of
vector-derived sequence) were added to all reactions and used as an
internal reference; hybridization (15 µg of total RNA/assay), RNase
A/T1 digestion, and sample processing prior to fractionation on
denaturing 5% polyacrylamide gels were carried out as described (19).
Primer extension analysis was conducted according to a previously
described protocol (21). Briefly, a 32P-labeled antisense
28-mer, annealing between positions 79 and 106 of the ZmDP2
cDNA, was hybridized overnight at 42 °C with 25 µg
of total RNA, followed by extension with Moloney murine leukemia virus
reverse transcriptase (Superscript II, Life Technologies, Inc.)
according to the manufacturer's instructions. Extended products were
ethanol-precipitated and analyzed on sequencing gels. The ZmDP2 cDNA was sequenced in parallel with the same
antisense primer, and sequencing reaction products (run on the same
gel) were utilized as size markers.
Expression and Purification of Recombinant ZmDP1 and ZmDP2
Proteins--
The coding region of ZmDP1, starting from
position 107 and including 279 bp of 3'-untranslated region plus 20 bp
of vector sequence for a total of 1367 bp, was polymerase chain
reaction-amplified (25 cycles) using the ZmDP1 cDNA as
template (20 ng), a high-fidelity thermophilic DNA polymerase (Vent,
New England Biolabs Inc.), and a pair of primers consisting of a
sequence-specific NdeI-tailed upstream primer
(5'-CCATATGGCTTCGGGGAACC) and the M13 forward/universal primer. The
coding region of ZmDP2, starting from the methionine codon
at position 49 and including 245 bp of 3'-untranslated region plus 20 bp of vector-derived sequence for a total of 965 bp, was similarly
amplified using the ZmDP2 cDNA as template and the
sequence-specific NdeI-tailed primer
5'-ACCATATGGGGGAGTTTGAAG. The restriction fragments obtained from
NdeI/NotI or NdeI digestion of the
ZmDP1 or ZmDP2 polymerase chain reaction
products, respectively, were then ligated into either the
NdeI/NotI sites or the dephosphorylated
NdeI site of the pET28b expression vector (Novagen) as
in-frame fusions with a vector-encoded His6 tag sequence.
After sequence verification, the pET-ZmDP1 and
pET-ZmDP2 plasmids were electroporated into BL21(DE3) cells
(Novagen). Protein expression was induced by adding 1 mM
isopropyl- Phosphatase Assay--
DPNPase activity was measured by
quantifying the inorganic phosphate released from various
phosphorylated compounds (23). Enzyme assays were conducted at 30 °C
for 30 min in 240-µl reaction mixtures containing 0.5 mM
magnesium acetate, 200 ng of the purified recombinant proteins, varying
concentrations of 3'-PAP, and 50 mM Tris-HCl, pH 8.1, for
ZmDP1 or 50 mM MES-KOH, pH 6.1, for ZmDP2. Under these
conditions, the activity of both enzymes responded linearly to both
protein amount (up to 500 ng) and reaction time (up to 1 h). The
kinetic parameters for 3'-PAP hydrolysis were determined by measuring
reaction rates at substrate concentrations ranging from 0.01 to 2 mM; nonlinear regression analysis of the data was performed
with SigmaPlot (Jandel Scientific). The activities with 2'-PAP,
3'-PAPS, ATP, 3'-AMP, D-myo-inositol
1,4-bisphosphate, L-histidinol phosphate,
O-phospho-L-serine, phosphoglycolic acid, and
NADP (all from Sigma) were measured under conditions of optimal activity for each enzyme at a fixed substrate concentration (0.1 mM).
DNA 3'-Phosphoesterase Assays--
Radioactive 32P
labeling of the 5'-OH/21-mer/3'-P (21p) and 5'-OH/21-mer/3'-OH
oligonucleotides (MWG Biotech) was carried out as described
previously (24), but with a labeling reaction time of only 30 s at
37 °C. Annealing reactions for the preparation of the 1-nucleotide
gapped p21p·23/45-mer duplex were carried out at 70 °C for 5 min
in the presence of a 2-fold molar excess of the 23-mer and 45-mer
oligonucleotides with respect to the labeled p21p oligonucleotide,
followed by cooling to room temperature over a period of 2 h. Full
oligonucleotide annealing was verified by nondenaturing polyacrylamide
gel electrophoresis. Unless otherwise specified, DNA 3'-phosphatase
assays were conducted at 37 °C for 7 min in 15-µl reaction
mixtures containing 50 mM Tris-HCl, pH 7.0, 10 mM MgCl2, 1 mM dithiothreitol,
0.035-0.15 µM oligonucleotide substrate, and 1 ng of
purified ZmDP1 or ZmDP2. Reactions were stopped by adding 7.5 µl of
denaturing loading dye solution; reaction products were fractionated on
7 M urea and 8% polyacrylamide sequencing gels, which were
subsequently subjected to phosphorimage analysis (see below). The
kinetic parameters for p21p 3'-dephosphorylation by ZmDP2 were
determined by measuring reaction rates at p21p concentrations ranging
from 1 to 160 µM in the presence of a fixed amount (4 ng)
of ZmDP2; nonlinear regression analysis of PhosphorImager data was
performed with SigmaPlot. A similar assay run under identical experimental conditions was used to analyze the 3'-phosphodiesterase activity of ZmDP2. A double-stranded DNA substrate containing a
3'-terminal phosphoglycolate group was prepared by bleomycin/iron(II) cleavage of a synthetic oligonucleotide duplex as described previously (25).
Gradient Plate DNA Repair Assays--
The repair-deficient
double mutant xth,nfo BW528 strain and the
isogenic wild-type E. coli strain BW32 (a gift of B. Weiss, Emory University, Atlanta, GA) were transformed with the empty pBluescript vector, pBluescript-ZmDP1, pBluescript-ZmDP2, or the positive control plasmid pNfo (a pBluescript derivative carrying the
E. coli endonuclease IV gene, kindly provided by D. Ramotar, Hopital Maisonneuve-Rosemont, Montreal, Canada). All the above transformants were used for DNA damage resistance assays (26), which
were conducted on LB-ampicillin-agar plates with linear concentration gradients of H2O2, tBH, or
MMS.
Other Methods--
Seed sterilization, germination, and
hydroponic culture of 15-day-old seedlings of the maize hybrid Paolo
(Dekalb, Chiarano, Italy) were carried out as described (19). Black
Mexican Sweet cells (kindly provided by S. Lobreaux, CNRS, Montpellier,
France) were maintained and propagated as described (27). E. coli XL1-Blue cells (Stratagene) were used for plasmid
propagation. DNA sequencing was performed with the dideoxy chain
termination method using the Thermo-Sequenase cycle sequencing kit
(Amersham Pharmacia Biotech). Phosphorimages of dried gels and filters
were obtained with a Personal Imager FX (Bio-Rad) and analyzed using
Multi-Analyst/PC software (Bio-Rad). Sequence similarity searches were
conducted against the NCBI Non-redundant Protein Sequence Database.
Multiple alignments were constructed with the ITERALIGN Version 1.1 program set to recognize alignment blocks including all the sequences under examination (28). Polynucleotide kinase assays were conducted at
37 °C for 30 min in 10-µl reaction mixtures containing 50 mM Tris-HCl, pH 7.0, 1 µCi of [ Isolation of Two Distinct Maize cDNAs Functionally
Complementing Bacterial and Yeast DPNPase Mutants--
A plasmid-borne
maize cDNA library was transferred into E. coli
DPNPase cysQ mutant cells, which are cysteine auxotrophs, and transformants were selected on the basis of their ability to grow
on synthetic medium containing sulfate as the sole source of sulfur.
Following the initial isolation of cysteine prototroph colonies and a
second round of selection, plasmid DNA was isolated from individual
colonies and analyzed by restriction digestion. Two different cDNA
inserts, ZmDP1 (1453 bp) and ZmDP2 (992 bp), were
identified. As shown in Fig.
1A, when reintroduced into
cysQ mutant cells, both cDNAs were capable of supporting
bacterial growth on cysteine-free synthetic medium. Sequence analysis
of the ZmDP1 cDNA revealed a coding region corresponding
to a polypeptide of 355 amino acids, very closely related throughout
its entire sequence to previously identified plant DPNPases and sharing
maximum similarity (82%) and identity (76%) with the rice RHL
phosphatase (5). Instead, the 248-amino acid-long polypeptide encoded
by the ZmDP2 cDNA did not share any sequence similarity
with known DPNPases. This was quite surprising and in apparent contrast
with the seemingly equal capacity of the ZmDP1 and
ZmDP2 cDNAs to complement the cysQ mutation.
To exclude the possibility that ZmDP2 complementation might
result from some kind of indirect, host-related effect (e.g. the stabilization or activation of a bacterial phosphatase other than
CysQ) rather than from true functional replacement, the two maize
cDNAs were inserted into a multicopy yeast expression vector and
tested for their ability to restore methionine prototrophy in an
S. cerevisiae DPNPase hal2/met22 mutant (2, 29).
The data presented in Fig. 1B show that ZmDP2 is
indistinguishable from ZmDP1 in its ability to complement
the methionine auxotrophy of the yeast hal2/met22 mutant,
thus confirming its bona fide identification as a novel
plant phosphatase functionally equivalent to, yet structurally distinct
from, previously known DPNPases.
DNA Gel Blot and Expression Analysis of ZmDP1 and
ZmDP2--
Maize genomic DNA digested with restriction
enzymes EcoRI and BamHI, which do not cut within
the ZmDP1 and ZmDP2 cDNAs, was utilized for
DNA gel blot analysis. As shown in Fig.
2A (lanes 1), a single hybridizing band was recognized by either
cDNA probe when using EcoRI-digested DNA. Similarly, a
single band pattern for ZmDP1 and one predominant
ZmDP2 hybridizing band, with an additional smaller band
probably due to an intronic cleavage site, were revealed by DNA gel
blot analysis of BamHI-digested genomic DNA (Fig.
2A, lanes 2). This indicates that
single copies of ZmDP1 and ZmDP2 are present in
the maize genome. As further shown by the RNase protection data
reported in Fig. 2B, transcripts recognized by antisense
RNAs derived from both cDNAs accumulated in the roots and shoots of
15-day-old seedlings as well as in cultured maize Black Mexican Sweet
(BMS) cells. The ZmDP1 RNA was expressed at comparable
levels in the two tissues and in BMS cells, whereas a higher
accumulation of the ZmDP2 messenger was detected in roots and in cultured cells (4- and 6-fold, respectively) compared with shoots.
No informative N-terminal sequence homology to DPNPases or other known
proteins (see below) and no significant similarity to translational
initiation consensus sequences were detected in the case of the
ZmDP2 open reading frame. A primer extension analysis was
thus conducted to map the 5'-end of the ZmDP2 mRNA. As
revealed by the data reported in Fig. 2C, a predominant
extended product, corresponding to an mRNA only 18 nucleotides
longer than the cDNA isolated by functional complementation, was
detected in maize BMS cells and tissues.
Functional Characterization of the ZmDP1 and ZmDP2
Phosphatases--
To gain insight into the enzyme activity of the two
ZmDP phosphatases, both proteins were expressed in E. coli
and comparatively analyzed for their ability to catalyze the
dephosphorylation of various phosphorylated compounds and for their
sensitivity to cation inhibition. To achieve high-level protein
expression and easy purification, the two ZmDP cDNAs
(starting from the consensus initiator methionine codon of
ZmDP1 and from the first methionine encoded by the
ZmDP2 cDNA) were inserted into the expression plasmid pET28 as in-frame fusions with a vector sequence coding for a 20-amino
acid N-terminal extension, including a metal-binding hexahistidine tag.
As shown in Fig. 3, polypeptides of the
expected molecular masses (40 and 28 kDa for histidine-tagged ZmDP1 and ZmDP2, respectively), recognized by an anti-His6 monoclonal
antibody, became detectable upon
isopropyl- ZmDP2 Is a DNA 3'-Phosphatase Acting on the 3'-Phosphorylated
Termini of DNA Single Strand Breaks--
A distinguishing feature of
the ZmDP2 phosphatase, besides its lower lithium sensitivity, was its
marked discrimination against 2'-PAP, the highest thus far reported for
any DPNPase. Such strong selectivity for 3'-phosphorylated substrates
is reminiscent of the position-specific attack of mononucleotides and
oligonucleotides by certain nucleotidases, thus suggesting a possible
relationship between ZmDP2 and enzymes acting on nucleic acids. This
hypothesis was experimentally verified by assaying the activity of
recombinant ZmDP2 on oligonucleotide substrates (schematically
represented in Fig. 4A)
previously utilized for mammalian PNKPs (24). As shown in Fig.
4B, which compares the output of dephosphorylation reactions
conducted in the presence of a fixed concentration of the 1-nucleotide
gapped p21p·23/45-mer duplex (indicated as Substrate in
Fig. 4A) and increasing amounts of either ZmDP1
(lanes 5-8) or ZmDP2 (lanes
1-4), only the latter enzyme catalyzed the nearly complete
conversion of the p21p oligonucleotide into a more slowly migrating
species, whose electrophoretic mobility was the same as that of the
5'-32P-labeled p21 3'-hydroxyl oligonucleotide indicated as
Product in Fig. 4A. This demonstrates that ZmDP2,
but not ZmDP1, acts as a DNA 3'-phosphatase that catalyzes the removal
of 3'-phosphate-blocking groups from DNA strand breaks. Moreover, the
fact that both proteins were expressed in the same bacterial strain and
purified with an identical affinity chromatography procedure rules out
the possibility that the DNA 3'-phosphatase activity exhibited by
recombinant ZmDP2 is actually due to a contaminating bacterial
enzyme.
Single-stranded and gapped double-stranded 3'-phosphate termini were
both efficiently dephosphorylated by ZmDP2 (data not shown), and the
apparent Km and Vmax values
for single-stranded p21p dephosphorylation were 12 ± 3 µM and 56 ± 4 µmol of product/h/mg of protein,
respectively. Similar to PAP hydrolysis, DNA dephosphorylation by ZmDP2
was strictly Mg2+-dependent and, as shown in
Fig. 5, was largely insensitive to Li+, Na+, and K+ inhibition.
Interestingly, however, the DNA 3'-phosphatase activity of ZmDP2 was
strongly inhibited by both Cd2+ and Cu2+ (but
not by Ni2+), with half-inhibitory concentrations of 67 and
40 µM, respectively (Fig. 5).
ZmDP2 Rescues DNA Damage Sensitivity in a DNA Repair-deficient E. coli Strain--
DNA strand breaks with 3'-blocked termini are
produced by nucleases such as DNase II as well as by various oxidative
DNA-damaging agents (30). The prototypes of 3'-phosphoesterases
involved in the repair of such lesions are E. coli
exonuclease III and endonuclease IV, which catalyze the removal of a
variety of 3'-blocking groups, including the 3'-phosphate and
3'-phosphoglycolate groups generated by the chemical oxidants hydrogen
peroxide and t-butyl hydroperoxide (26, 31, 32).
Accordingly, E. coli strains lacking both exonuclease III
(xth) and endonuclease IV (nfo) display a marked
hypersensitivity to various oxidative DNA-damaging agents. To find out
whether the newly identified plant DNA 3'-phosphatase activity may
indeed play a role in DNA repair, we tested its ability to confer DNA
damage resistance to the double mutant xth,nfo
BW528 strain (26). Besides ZmDP2, the ZmDP1
cDNA and a plasmid actively expressing bacterial endonuclease IV
(pNfo, used as positive control) were transformed into both the double
mutant BW528 strain and the isogenic wild-type BW32 strain. A gradient
plate assay, in which mutant cells grow only a short distance into a
gradient of increasing genotoxic agent concentration compared with
repair-proficient wild-type cells, was used for these experiments. As
shown in Fig. 6, ZmDP2 and endonuclease
IV (Nfo), but not ZmDP1, conferred to strain BW528 resistance against
the DNA-damaging agents H2O2 (A), tBH (B), and MMS (C). Hydrogen peroxide
sensitivity was fully rescued by ZmDP2, whereas only a partial, yet
highly significant rescue was observed in the case of tBH. Chemically
different 3'-blocked termini with either a terminal phosphate or
phosphoglycolate group are the main products of
H2O2 and tBH attack, respectively (33). Using a
partially double-stranded DNA with a 3'-terminal phosphoglycolate group
as substrate and assay conditions similar to those previously employed
for the DNA 3'-phosphatase activity, we thus directly tested the
ability of ZmDP2 to also act as a 3'-phosphodiesterase. As shown by the
results presented in Fig. 7, this appears
to be the case. In fact, increasing amounts of a lower mobility
species, comigrating with the unblocked control oligonucleotide
BL1-17, accumulated in reaction mixtures containing the
3'-phosphoglycolate-blocked substrate BL1-17PG/BL2 and increasing
amounts of ZmDP2.
Features of the Deduced ZmDP2 Protein Sequence--
When used as a
query for a similarity search, the deduced ZmDP2 protein sequence led
to the identification of partially similar amino acid sequences from
various organisms (Fig. 8). All of these sequences are from predicted proteins of unknown function, with the
sole exception of PNKP, a sequence coding for a recently identified human polynucleotide kinase 3'-phosphatase that is thought to be
involved in DNA repair (17, 18). The regions of similarity between
ZmDP2 and PNKP include two conserved amino acid blocks (underlined in Fig. 8) resembling sequence motifs recently
identified in a superfamily of phosphohydrolases as distinct domains
separated by a consensus distance of 102-191 amino acids (34).
Interestingly, a C-terminal polypeptide extension (not shown in Fig. 8)
containing a typical kinase motif is present in human PNKP (17, 18) and in all of the other non-plant sequences, but is missing in ZmDP2 as
well as in the Arabidopsis polypeptide. This suggests that at variance with PNKP, ZmDP2 may be endowed only with DNA
3'-phosphatase activity. Accordingly, no ATP-dependent
kinase activity was detected in ZmDP2-supplemented reaction mixtures
containing the 5'-OH/23-mer oligonucleotide reported in Fig.
4A (data not shown).
Two novel, structurally and functionally distinct maize
phosphatases have been identified on the basis of their ability to alleviate PAP accumulation, and thus cysteine auxotrophy, in an E. coli DPNPase cysQ mutant strain. The first of
them, ZmDP1, displays all the hallmarks of previously described,
Mg2+-dependent, Li+-sensitive
DPNPases. It shares the highest sequence similarity with the rice RHL
enzyme (5), the only other known DPNPase from monocotyledons; but its
sodium insensitivity and ability to utilize inositol 1,4-bisphosphate
as substrate (albeit with a reduced efficiency compared with PAP)
suggest that it is more related functionally to the SAL2 DPNPase from
Arabidopsis (6). As predicted by its isolation as a
cysQ-complementing activity, the other phosphatase, ZmDP2,
also efficiently dephosphorylates 3'-PAP (with a similar
Vmax and an ~4-fold higher
Km compared with ZmDP1), but strongly discriminates
against 2'-PAP and is much less sensitive to lithium inhibition. The
most remarkable feature of ZmDP2 is, however, its unique ability to
dephosphorylate 3'-phosphate-blocked single-stranded or gapped
double-stranded DNA with an apparent Km ~10-fold
lower than that determined for 3'-PAP. Despite its more favorable
Km value, the dephosphorylation of DNA 3'-phosphate
termini might, in principle, be viewed as a side activity of ZmDP2. Two
distinct lines of evidence strongly argue against this possibility,
however. The first is the complete sequence divergence between ZmDP2
and classical DPNPases, as opposed to its partial sequence similarity
to human PNKP, an enzyme likely to be involved in DNA repair (17, 18).
The second, more direct evidence is the capacity of ZmDP2, but not
ZmDP1, to restore wild-type resistance to hydrogen peroxide damage in DNA repair-deficient E. coli cells to the same extent as the
bacterial repair enzyme endonuclease IV. Hydrogen peroxide is known to
preferentially induce the formation of DNA strand breaks with
3'-phosphate termini (30, 33). This finding thus provides an important
in vivo validation of the DNA 3'-phosphatase activity of
ZmDP2, initially revealed by in vitro assays conducted on a
gapped double-stranded DNA substrate.
Besides the complete rescue of DNA damage sensitivity observed
with H2O2, ZmDP2 also conferred protection
against tBH (Fig. 6). Single strand breaks with
3'-phosphoglycolate-blocked termini are the main products of tBH attack
(33). These are not typical substrates of 3'-phosphatase action, yet
all the 3'-repair enzymes thus far identified catalyze the hydrolysis
of both 3'-phosphate and 3'-phosphoglycolate groups, often with
different specific activity ratios (25, 30). Moreover, in the case of
the Drosophila repair enzyme RRP1, mutagenesis
studies have shown that both 3'-phosphoesterase activities are
associated with the same active site (33). In keeping with the above
data, we found that ZmDP2 is also endowed with both 3'-repair
activities, albeit with a preference for 3'-phosphate-blocked termini.
Similar to human PNKP (17), ZmDP2 also conferred partial
protection against the alkylating agent MMS, whose main DNA damage products are abasic (apurinic/apyrimidinic) sites. This may reflect an
intrinsic apurinic/apyrimidinic endonuclease activity, as reported previously for other multifunctional repair enzymes such as the endonuclease/phosphoesterase encoded by the bacterial gene
nfo (30, 32, 35) and the apurinic endonuclease
redox protein ARP from
Arabidopsis (36). Alternatively, the partial protection against MMS can result from an indirect effect whereby bacterial apurinic/apyrimidinic site-processing enzymes other than
endonuclease IV and exonuclease III (e.g.
formamidopyrimidine-DNA glycosylase, endonuclease III, or endonuclease
VIII) convert MMS-generated apurinic/apyrimidinic sites into
3'-phosphate single strand breaks that are subsequently acted upon by ZmDP2.
Altogether, the present data delineate ZmDP2 as the first plant enzyme
capable of converting 3'-blocked DNA termini, generated by oxidative
damage, into priming sites competent for reparative DNA polymerization.
Based on sequence homology, a similar role in DNA repair can also be
attributed to the previously uncharacterized protein encoded by the
conceptually assembled Arabidopsis DNA sequence (37) that
scored highest in our BLAST searches.
Similar to ZmDP2, most of the eukaryotic DNA repair enzymes thus far
identified are constitutively and ubiquitously expressed even in the
absence of genotoxic agent exposure (30). This likely reflects the
involvement of such enzymes not only in the response to acute
genotoxicity, but also in the housekeeping defense against DNA damage
caused by oxygen radicals, which are normal by-products of aerobic
metabolism in all organisms. Oxygen radicals, the actual causative
agents of much oxidative DNA damage, can also be generated upon
reaction of reduced transition metals with
H2O2, a key mediator of the hypersensitive
response triggered by plant-pathogen interaction. In addition, because
of their lifestyle, plants are particularly exposed not only to oxygen
radical-generating pollutants such as ozone and redox-cycling
herbicides, but also to other compounds (e.g. heavy metals)
that, by inhibiting defense enzyme activities, may indirectly increase
oxygen radical accumulation and/or drastically impair DNA damage
repair. In the case of ZmDP2, this kind of interference is well
exemplified by the results of metal inhibition experiments, which
showed half-inhibition of DNA 3'-phosphate hydrolysis by micromolar
Cd2+ and Cu2+ concentrations, and by the
potential, competitive inhibition of ZmDP2 DNA 3'-phosphoesterase
activity by the high levels of 3'-PAP that may accumulate under
conditions of strong and persistent Li+ exposure, resulting
in the inhibition of the DPNPase activity of ZmDP1.
Given the complexity and chemical diversity of oxidative DNA damage and
of the pathways that lead to it (30), it is quite conceivable to
imagine the existence in plants of concerted interactions between
different repair enzymes belonging to the same repair machinery or to
different repair systems. In keeping with this view, direct
interactions between DNA mismatch and nucleotide excision repair
enzymes (38), as well as a synergy between base and nucleotide excision
repair systems (39, 40), have recently been documented in S. cerevisiae. A need for interaction with other protein components
can also be anticipated in the case of oxidative DNA damage repair by
ZmDP2. In fact, ZmDP2 lacks the kinase activity present in human PNKP
that may be required under certain DNA damage conditions to generate
ligation-competent 5'-termini at the other end of the break, as well as
other activities (e.g. DNA polymerase and ligase) that are
also required to complete the repair of oxidative DNA damage. In this
broader context, the maize 3'-repair phosphoesterase we have identified
should be viewed as a functional module of larger, multisubunit repair
enzyme complexes rather than as an independent functional unit. Future
experiments taking advantage of the molecular reagents reported here
will investigate the existence and significance of these interactions in the context of the repair of oxidative damage and other types of DNA
damage in plants.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3' exoribonucleases (15).
This effect is likely due to the fact that 3'-PAP mimics the monomers
of a polynucleotide chain, thus preventing phosphodiester bond attack
by RNA-processing enzymes. In keeping with this notion,
3'-phosphothymidine 5'-phosphate, the thymine analog of PAP, has been
employed previously as a 3'-phosphorylated nucleic acid analog for
nucleotidase assays measuring the removal of the 3'-phosphate group by
the bifunctional polynucleotide kinase of phage T4 (16). More recently,
the same diphosphonucleoside has been utilized as substrate to assay
the DNA phosphatase activity of human polynucleotide kinase
3'-phosphatase (PNKP) (17), an enzyme whose ability to dephosphorylate
3'-phosphate termini and to phosphorylate 5'-hydroxyl termini at DNA
single strand breaks is predictive of an important function in DNA
repair (17, 18).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside. Twenty colonies
growing in the absence of exogenously supplied cysteine were obtained from the screening of a total of ~106 transformants.
Plasmid DNA isolated from such colonies was re-transformed into MC4100
cells and again tested for its ability to confer cysteine prototrophy.
Eight clones were thus selected, which, upon restriction analysis and
sequence determination, were assigned to two distinct cDNA classes,
designated as ZmDP1 and ZmDP2.
-D-thiogalactopyranoside and allowed to
proceed for 4 h at 30 °C. After cell lysis, recombinant
proteins bearing the N-terminal hexahistidine tag were bound to a metal
affinity resin (Talon, CLONTECH) equilibrated in
10% glycerol, 300 mM NaCl, and 50 mM sodium
phosphate, pH 8.0. After washing with equilibration buffer until the
A280 of the flow-through was <0.05, bound
proteins were eluted with 100 mM imidazole in the same
buffer. Protein concentration was determined with the Coomassie
Brilliant Blue G-250 dye (Bio-Rad) using bovine serum albumin as a
standard. The composition and purity of protein fractions were assessed by gel electrophoresis on SDS-10% polyacrylamide gels (22). Monoclonal
antibodies specifically recognizing the N-terminal hexahistidine tag
(Amersham Pharmacia Biotech) were utilized for immunoblot analysis of
recombinant ZmDP1 and ZmDP2 following the manufacturer's instructions.
-32P]ATP
(Amersham Pharmacia Biotech; 5000 Ci/mmol), 10 pmol of the 5'-OH/23-mer
oligonucleotide (see above), plus either 10 ng of purified ZmDP2 or 3 units of phosphatase-free T4 polynucleotide kinase (Roche Molecular
Biochemicals) utilized as a control.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Functional complementation of microbial
DPNPase mutant strains by the ZmDP1 and
ZmDP2 cDNAs. A, bacterial
cysQ mutant cells were transformed with expression vector
constructs carrying the ZmDP1 cDNA (patches 1 and 4), the ZmDP2 cDNA (patches 2 and 5), or neither (patches 3 and 6)
and plated onto minimal M9 medium with (+Cys) or without
( Cys) cysteine. B, yeast
hal2/met22 mutant cells were transformed as
described for A (see "Experimental Procedures"
for expression vector details) and plated onto minimal synthetic
dextrose medium with (+Met) or without
(
Met) methionine.
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Fig. 2.
DNA gel blot and RNA expression analyses of
ZmDP1 and ZmDP2. A,
DNA gel blot analysis. Maize genomic DNA (20 µg/lane) digested with
EcoRI (lanes 1) or BamHI
(lanes 2) was probed with either the
ZmDP1 (1453 bp) or ZmDP2 (992 bp) cDNA. The
sizes of DNA length markers run on the same gel are indicated in
kilobase pairs (kb). B, RNase protection
analysis. Total RNA samples derived from maize roots (lane
1), maize shoots (lane 2), maize BMS
suspension culture cells (lane 3), or a control
yeast RNA (lane 4) were analyzed using antisense
ZmDP1 and ZmDP2 riboprobes as indicated on the
right. A maize glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) antisense riboprobe was included in all
hybridization reactions as an internal standard. C, primer
extension analysis of the ZmDP2 mRNA. Total RNA samples
extracted from roots (lane 1), shoots
(lane 2), or BMS cells (lane
3) were subjected to primer extension analysis using a
32P-labeled antisense oligonucleotide annealing between
positions 79 and 106 of the ZmDP2 cDNA. The
arrowhead indicates the main extension product. Shown in
lane 4 are the products of an A-track chain
termination sequencing reaction conducted in parallel with the same
antisense oligonucleotide utilized for primer extension. The position
corresponding to the first nucleotide of the ZmDP2 cDNA
is indicated with an asterisk.
-D-thiogalactopyranoside induction (A and B, cf. lanes 1 with lanes
2 and 4) and were purified to near-homogeneity by metal
affinity chromatography (A and B, lanes 3 and 5). Recombinant ZmDP phosphatases were initially
tested for their ability to release inorganic phosphate from 3'-PAP. As
shown in Table I, ZmDP1 and ZmDP2 both
catalyzed the Mg2+-dependent dephosphorylation
of this diphosphonucleoside: the apparent Km for
3'-PAP hydrolysis by ZmDP1 was ~4-fold lower than the corresponding
value measured for ZmDP2, but dephosphorylation reactions supported by
either enzyme were characterized by similar apparent
Vmax values. Using 3'-PAP as a reference
substrate, we next investigated the ability of the two maize
phosphatases to utilize other phosphorylated compounds as substrates.
As further shown in Table I, where the activity toward 3'-PAP was
arbitrarily set to 100%, ZmDP1 catalyzed the dephosphorylation of
2'-PAP and 3'-PAPS with nearly the same efficiency as that determined
for 3'-PAP and also utilized inositol 1,4-bisphosphate as substrate, albeit with an ~10-fold reduced efficiency. In contrast, 2'-PAP was
an extremely poor substrate for ZmDP2, and the only other bisphosphate
besides 3'-PAP that was efficiently dephosphorylated by this enzyme was
3'-PAPS. Furthermore, as documented by the IC50 values
reported in Table II, 3'-PAP
dephosphorylation by ZmDP2 was ~12-fold less sensitive to
Li+ inhibition than the same reaction catalyzed by ZmDP1.
Both enzymes were not inhibited by physiological Na+ and
K+ ion concentrations, whereas Ca2+ was a more
effective Mg2+-competitive inhibitor of the ZmDP2 than the
ZmDP1 enzyme.
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Fig. 3.
Expression and purification of recombinant
ZmDP1 and ZmDP2. A, Coomassie Blue-stained SDS-10%
polyacrylamide gel of total lysates derived from
isopropyl- -D-thiogalactopyranoside-induced bacterial
cells (BL21(DE3)) transformed with the empty pET28b vector (lane
1), the pET-ZmDP1 plasmid (lane 2), or the
pET-ZmDP2 plasmid (lane 4). Highly purified
fractions (0.5 µg) of histidine-tagged recombinant proteins generated
by metal affinity chromatography of either the ZmDP1 or ZmDP2 soluble
lysate are shown in lanes 3 and 5, respectively.
The migration positions of molecular mass markers (in kilodaltons) run
on the same gel are indicated on the left. B, immunoblot
analysis of the protein samples shown in A. A monoclonal
antibody specifically recognizing the N-terminal hexahistidine tag was
utilized for immunodetection. The loading order and electrophoresis
conditions are the same as described for A.
Substrate specificity and kinetic parameters of ZmDP1 and ZmDP2
Effect of different cations on the PAP phosphatase activity of ZmDP1
and ZmDP2
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Fig. 4.
DNA 3'-phosphatase activity of ZmDP2.
A, schematic representation of the oligonucleotides and
model substrates utilized for DNA 3'-phosphatase assays. The
double-stranded DNA shown at the top (Substrate) was
prepared by annealing three oligonucleotides
(5'-32P-labeled p21p, 23-mer, and 45-mer) to generate a
1-nucleotide gap with a 3'-phosphate terminus. The duplex generated by
3'-phosphate hydrolysis of the substrate DNA is shown below
(Product). p21p and p21 refer to the 21-mer oligonucleotides
with both 3'- and 5'-phosphorylated termini or only 5'-phosphorylated
termini, respectively. The termini relevant to this study are indicated
above the oligonucleotide sequences; the 3'-phosphate group undergoing
hydrolysis is circled. The 5'-phosphate of both p21p and p21
was radiolabeled. B, phosphorimage of a 7 M urea
and 8% polyacrylamide gel showing the denatured, single-stranded
substrate (p21p) and product (p21) of DNA 3'-dephosphorylation
reactions conducted with the gapped duplex reported in A (35 nM) in the presence of the indicated amounts of
either ZmDP2 (lanes 1-4) or ZmDP1
(lanes 5-8). Unreacted, 5'-labeled p21p
(lane 9) and p21 (lane 10)
were run alongside as standards; their migration positions are
indicated on the right.
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Fig. 5.
Effect of different cations on the DNA
3'-phosphatase activity of ZmDP2. The p21p oligonucleotide (0.15 µM) was incubated with recombinant ZmDP2 (1 ng) in the
presence of increasing concentrations of CdCl2 ( ),
NiSO4 (
), CuCl2 (
), LiCl (
), NaCl
(
), or KCl (
). Reaction conditions were as described under
"Experimental Procedures," except for the omission of
dithiothreitol. The results are expressed as percentages of the
activity measured in the absence of any added salt and are the average
of at least two independent experiments, performed in duplicate, that
differed by <10% of the mean.
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Fig. 6.
Resistance to DNA-damaging agents conferred
by ZmDP2 in a repair-deficient E. coli strain.
Wild-type xth+nfo+ BW32
cells harboring the empty pBluescript vector (rows 1) or
double mutant xth,nfo BW528 cells harboring the empty
pBluescript (rows 2), pBluescript-ZmDP2 (rows 3),
pBluescript-ZmDP1 (rows 4), or pNfo (rows 5)
plasmid were grown on LB-ampicillin-agar plates with the indicated
linear gradients of H2O2 (A), tBH
(B), and MMS (C).
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Fig. 7.
DNA 3'-phosphodiesterase activity of
ZmDP2. A, schematic representation of the
oligonucleotide substrate utilized for DNA 3'-phosphodiesterase assays.
The partially double-stranded 3'-phosphoglycolate substrate
BL1-17PG/BL2 was prepared using a previously described procedure (25);
the 3'-phosphoglycolate (PG) group undergoing hydrolysis is
circled. An oligonucleotide identical to BL1-17PG, but
lacking the 3'-phosphoglycolate group, referred to as BL1-17, was used
as a gel migration control of the 3'-phosphoglycolate hydrolysis
product. The 5'-phosphates of both BL1-17PG and BL1-17 were
radiolabeled. B, phosphorimage of a 7 M urea and
16% polyacrylamide gel showing the unconverted denatured BL1-17PG
substrate and the BL1-17 product that accumulated in reaction mixtures
containing a fixed concentration of BL1-17PG/BL2 (10 nM)
and the indicated amounts of ZmDP2. Unreacted, 5'-labeled BL1-17PG
(lane 1) and BL1-17 (lane 6) were run alongside
as standards; their migration positions are indicated on the
right.
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Fig. 8.
Alignment of ZmDP2 with polypeptide sequences
from other organisms. The polypeptide sequence of ZmDP2 was
aligned with the four best scoring sequences identified by the BLAST
algorithm: uncharacterized sequences from Arabidopsis
thaliana (NCBI accession number BAA97052 (37); 61%
identity and 72% similarity), Schizosaccharomyces pombe
(NCBI accession number O13911; 27% identity and 44% similarity),
Drosophila melanogaster (NCBI accession number
AAF54229; 27% identity and 41% similarity), and human PNKP (NCBI
accession number AAD50639 (17, 18); 28% identity and 43% similarity).
Amino acid residues that are conserved in at least four of the five
sequences are boxed; uppercase letters indicate
amino acid blocks that are alignable in all five sequences (28). Gaps
introduced to optimize the alignment are indicated by dots;
sequences resembling consensus phosphohydrolase motifs (34) are
underlined.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Douglas Berg, Yolande Surdin-Kerjan, and Bernard Weiss for the gift of bacterial and yeast strains; Michèle Minet and Dindial Ramotar for plasmids; Stéphane Lobreaux for BMS cells; and Michael Weinfeld for advice on DNA 3'-phosphatase assays. We are grateful to Riccardo Percudani for assistance with sequence analysis and to Alessio Peracchi for helpful discussions on enzyme activity studies. Encouragement and support from Gian Luigi Rossi are also gratefully acknowledged.
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FOOTNOTES |
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* This work was supported by grants from the National Research Council of Italy, Target Project on "Biotechnology," and the Ministry of University and Scientific and Technological Research (Rome, Italy).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF288075 and AF307152.
To whom correspondence should be addressed. Tel.: 39-521-905646;
Fax: 39-521-905151; E-mail: s.ottonello@unipr.it.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M010648200
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ABBREVIATIONS |
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The abbreviations used are: DPNPases, diphosphonucleoside phosphatases; 3'-PAP, 3'-phosphoadenosine 5'-phosphate; 2'-PAP, 2'-phosphoadenosine 5'-phosphate; 3'-PAPS, 3'-phosphoadenosine 5'-phosphosulfate; PNKP, polynucleotide kinase 3'-phosphatase; bp, base pair(s); MES, 2-(N-morpholino)ethanesulfonic acid; tBH, t-butyl hydroperoxide; MMS, methyl methanesulfonate; BMS, Black Mexican Sweet.
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