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J Biol Chem, Vol. 274, Issue 34, 24176-24186, August 20, 1999
From the Mammalian polynucleotide kinases catalyze the
5'-phosphorylation of nucleic acids and can have associated
3'-phosphatase activity, predictive of an important function in DNA
repair following ionizing radiation or oxidative damage. The sequences
of three tryptic peptides from a bovine 60-kDa polypeptide that
correlated with 5'-DNA kinase and 3'-phosphatase activities identified
human and murine dbEST clones. The 57.1-kDa conceptual translation
product of this gene, polynucleotide kinase 3'-phosphatase
(PNKP), contained a putative ATP binding site and a
potential 3'-phosphatase domain with similarity to
L-2-haloacid dehalogenases. BLAST searches identified
possible homologs in Caenorhabditis elegans,
Schizosaccharomyces pombe, and Drosophila
melanogaster. The gene was localized to chromosome 19q13.3-13.4.
Northern analysis indicated a 2-kilobase mRNA in eight human
tissues. A glutathione S-transferase-PNKP fusion protein
displayed 5'-DNA kinase and 3'-phosphatase activities. PNKP
is the first gene for a DNA-specific kinase from any organism. PNKP expression partially rescued the sensitivity to
oxidative damaging agents of the Escherichia coli DNA
repair-deficient xth nfo double mutant. PNKP
gene function restored termini suitable for DNA polymerase, consistent
with in vivo removal of 3'-phosphate groups, facilitating
DNA repair.
Polynucleotide kinases catalyze phosphorylation of 5'-OH termini
of nucleic acids. In a number of biochemical experiments over several
decades, evidence for a mammalian polynucleotide kinase
(PNK)1 activities with an
acidic pH optimum has mounted (reviewed in Refs. 1-8). We and others
have purified such a PNK to near-homogeneity from bovine tissue, which
lacks significant 5'-phosphorylation activity when assayed with RNA
substrates (5, 6, 9). This activity, denoted SNQI-PNK, corresponded to
a polypeptide of approximately 60 kDa in our experiments (6). Highly
purified SNQI-PNK fractions contain a 3'-phosphatase activity (6),
originally discovered in the PNK from bacteriophage T4 (10, 11) and
also observed in PNKs from rat liver nuclei (2-5, 12). Furthermore, there are reports of mammalian PNK activities with a greater substrate specificity for RNA than DNA (8, 13,
14)2 and of conservation of
yeastlike tRNA ligation (with its requirement for a PNK activity) as a
minor pathway in HeLa cells (15).
Because of its widespread presence in mammalian cells, the acidic pH
optimum PNK is likely to be a key enzyme in DNA metabolism, and its
biochemical functions immediately suggest a role in the critical
process of DNA repair. One of its enzymatic activities, DNA
3'-phosphatase, implies an ability to repair strand breaks terminated
by 3'-phosphate, a type of DNA damage seen in cells treated with
ionizing radiation or hydrogen peroxide (16). Removal of this 3'-end
blocking lesion allows synthesis by DNA polymerase and joining of nicks
by DNA ligase. DNA purified from irradiated thymocytes and irradiated
thymus, but not DNA irradiated in vitro, contains strand
breaks with 5'-OH termini (17, 18). The 5'-phosphorylation activity of
the SNQI-PNK enzyme suggests a possible model in which 5'-OH termini
are repaired prior to ligation. 5'-OH termini in DNA also occur in
ischemia in rat brain (19), after cleavage by nucleases with the
appropriate specificity such as DNase II (20), and as intermediates
during topoisomerase cleavage (21, 22). The highest concentration of
5'-DNA termini occurs during DNA replication, and Pohjanpelto and
Hölttä (23) proposed that a small fraction of Okazaki
fragments contain 5'-OH termini; this fraction decreases upon
incubation of extracts with ATP at pH 6.0, which was inferred to
reflect 5'-phosphorylation by a cellular PNK.
Despite extensive biochemical studies, to date there are no molecular
reagents such as antibodies or cDNAs available for mammalian PNKs,
hampering further investigation. We present here the molecular cloning
of the PNKP gene, the first gene for a mammalian PNK and the
first gene for a DNA-specific kinase from any organism. Concomitantly, the PNKP gene also represents the second gene for a
mammalian DNA 3'-phosphatase; the previously described human APE/HAP1
AP endonuclease has a weak 3'-phosphatase activity (24, 25). Using
Escherichia coli as a model biological system, we report the
first evidence for participation in DNA repair in vivo by the PNKP gene product.
Protein Purification--
In studies of bovine thymus, we
observed multiple activities that 5'-phosphorylate
oligo(dT)25 (8). The SNQI-PNK fraction was named on the
basis of fractionating into the supernatant after a Polymin P
precipitation and eluting first among the activities from the
supernatant on a Q-Sepharose column (8). The SNQI-PNK polypeptide of
approximately 60 kDa (SNQI-PNK) that correlated with 5'-DNA kinase and
3'-phosphatase activities assayed as described (6, 8) was purified to
near-homogeneity from the thymus glands of 6-month-old calves (6).
Briefly, the purification scheme involved preparation of a crude cell
extract; precipitation of nucleic acids and some acidic proteins with
Polymin P (Sigma Aldrich, Oakville, Ontario, Canada; Ref. 26); then
sequential chromatography on Q-Sepharose (Amersham Pharmacia Biotech,
Baie d'Urfe, Quebec, Canada), SP Sepharose, Blue Sepharose (all
obtained from Amersham Pharmacia Biotech), DNA cellulose (Sigma), and
Superose 12 (Amersham Pharmacia Biotech) columns (method 1). If the
order of the DNA cellulose and Superose 12 columns was reversed, three polypeptides were observed, one of which was similar in estimated molecular mass to the active enzyme as observed in renaturation gel
activity experiments (method 2).
Amino Acid Sequence Analysis of the Purified
Protein--
Protein concentration was measured using the method of
Bradford (27) with a commercially obtained reagent (Pierce). Protein (2 µg) from a method 2 purification was electrophoresed through a 10%
SDS-polyacrylamide gel (28). The gel was briefly stained with Coomassie
Blue R-250, and the band at around 60 kDa excised for tryptic
digestion. Tryptic peptides were purified by HPLC, and the sequences
were analyzed by mass spectrometry using a Finnigan LCQ ion trap mass
spectrometer at the Harvard Microchemistry Facility (Cambridge, MA). In
addition, near-homogeneous protein (0.2 µg) from a method 1 preparation was also further purified by SDS-polyacrylamide gel
electrophoresis and HPLC-purified tryptic peptides were subjected to
sequencing both by Edman degradation and mass spectrometry. The
isobaric pairs of amino acid residues I/L, Mr = 113, and Q/K, Mr = 128, could not be
unambiguously differentiated in mass spectrometric sequencing.
Identification and Isolation of cDNAs--
Peptide sequences
of suitable length were used to search the dbEST section of GenBank
using the BLAST algorithm (29). Further overlapping EST clones were
identified in subsequent searches. I.M.A.G.E. consortium clones were
ordered from ATCC (Rockville, MD) or GenomeSystems (St. Louis, MO). DNA
was prepared using Wizard Minipreps (Fisher, Laval, Quebec, Canada) or
Mini or Midi Preps (Qiagen, Mississauga, Ontario, Canada), and the
identity was confirmed by DNA sequencing at Sheldon Biotechnology
(McGill University, Montreal, Quebec, Canada). Clone 32798 (human) was
fully sequenced on both strands using an overlapping primer walk
strategy. Additional human cDNA clones were obtained independently
by screening of a cDNA library from a lymphoblastoid cell line
cloned into pREP4 (Ref. 30; kindly provided by Dr. Manuel Buchwald,
University of Toronto, Toronto, Ontario, Canada) using a probe that was
prepared by PCR from the 5' end of the 32798 clone. The PCR primers
(U221, 5'-GCGTATGCGGAAGTCAAACC-3'; and L400,
5'-TCGGAGCTTACGGGGAATCT-3') also generated a signal of
about 220 base pairs in the cDNA library, confirming the presence
of the cDNA. Positive colonies were detected by colony
hybridization using the 220-base pair fragment labeled with the
RediPrime kit (Amersham Pharmacia Biotech) and
[ Southern Blot Analysis--
A Southern blot containing 4 µg of
EcoRI-digested DNA from nine eukaryotic species was obtained
from CLONTECH (Palo Alto, CA). The blot was
prehybridized for 5 h at 65 °C in ExpressHyb (CLONTECH) according to the directions of the
manufacturer. A KpnI/HindIII fragment from a
pcDNA 3.1/His C construct containing the full-length
PNKP cDNA3 was
used as a probe. Hybridization was for 16 h at 65 °C. The blot
was washed quickly four times with 2× SSC, 0.05% SDS, three times for
10 min at room temperature with 2× SSC, 0.05% SDS, and twice for 20 min each at 65 °C with 0.1× SSC.
Chromosomal Localization--
A bacterial artificial chromosome
clone was identified by hybridization screening of the RPCI-11 human
genomic library (33) with the HindIII/NotI insert
from I.M.A.G.E. clone 32798. This genomic clone (429D20) was then used
for mapping by fluorescence in situ hybridization (FISH;
Refs. 34 and 35) at the MRC Genome Resource Facility.
Northern Blot Analysis--
A Northern blot of 2 µg of
poly(A)+ RNA extracted from eight different human tissues
(Human Multiple Tissue Northern blot II, CLONTECH)
was hybridized to a cDNA probe from the middle portion of the
coding sequence (HindIII/PstI fragment of 32798)
and to a probe including the 3' portion of the PNKP cDNA
(32798 digested with HindIII and NotI). A
GAPDH cDNA probe was used as a loading control. Probes
were labeled as described above, and prehybridization and hybridization
were carried out with ExpressHyb (CLONTECH). The
blot was prehybridized for 2 h at 68 °C and hybridized for 3 h at 68 °C. Washing protocols were as described in Sambrook et al. (32). Between hybridizations, the previous probe was stripped by heating at 95-100 °C for 10 min in 0.1× SSC, 0.5% SDS. The filter was then exposed to x-ray film for at least 96 h
to confirm the absence of prior signals.
Expression of I.M.A.G.E. Consortium Clone 32798 as a Fusion
Protein in E. coli (GST PNKP) and Assay of Enzymatic Activity--
DNA
from clone 32798, a 1.45-kb insert with an open reading frame of 452 amino acids comprising residues 69-521 of the full-length PNKP conceptual translation cloned into the Lafmid BA
vector, was cleaved with HindIII. The 5' terminus of the
gene fragment was treated with Klenow polymerase and dNTPs to generate
a blunt end, after which the DNA was cleaved with NotI and
purified from an agarose. DNA from the vector pGEX-4T-3 (Amersham
Pharmacia Biotech) was cleaved with SmaI and
NotI. The insert and vector were incubated with T4 DNA
ligase (Canadian Life Technologies, Burlington, Ontario, Canada) and
the ligation mixture was used to transform DH5 Preparation of Antiserum--
A 14-mer peptide, EPRLGRLYCQFSEG,
comprising the C terminus of the conceptual translation product of the
PNKP gene was synthesized and conjugated to keyhole limpet
hemocyanin. The conjugated peptide was then used to produce a rabbit
polyclonal antiserum, termed AC-IV, using a standard inoculation
protocol. This work was carried out at Research Genetics (Huntsville, AL).
Immunoblot Analysis--
Protein samples were
electrophoresed through 10% SDS-polyacrylamide gels (28) and
transferred to a nitrocellulose membrane using a Bio-Rad MiniTransblot
apparatus as recommended by the manufacturer. Immunoblots were carried
out using the ECL kit (Amersham Pharmacia Biotech) according to the
directions of the manufacturer, with an anti-rabbit horseradish
peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech).
In some experiments, alkaline phosphatase activity was used for
detection (36). Antibodies against glutathione S-transferase
were purchased from Amersham Pharmacia Biotech. Secondary antibodies
conjugated to alkaline phosphatase were from Jackson Laboratories (West
Grove, PA).
Gradient Plate Assay--
Cells were grown overnight in 1 ml of
Luria broth in the presence of 50 µg/ml ampicillin. Cells were
replicated onto various Luria broth agar drug gradient plates, which
were prepared as described in detail elsewhere (37, 38).
Preparation of Chromosomal DNA and Measurement of
[methyl-3H]dTMP Incorporation--
Exponentially growing
cells in 5 ml of Luria broth were either untreated or treated with 25 mM H2O2 for 1 h at 37 °C.
Cells were harvested, washed twice with M9 buffer, and the cell pellet stored frozen for 1 h at Identification of dbEST Clones Containing Peptide Sequence from the
Bovine DNA Kinase SNQI-PNK--
Two bovine peptide sequences were
found from analysis of method 2 purified material,
I/LVI/LFTN[Q/K]MGI/LGR (peptide 1), and I/LI/LYI/LEI/L(PR) (peptide
2) where I/L or Q/K symbolizes an isobaric amino acid, the brackets
indicate an ambiguous residue assigned according to the sequence found
in a dbEST hit, and the parentheses indicate residues assigned with
uncertainty. The peptide 1 sequence identified murine and human
cDNAs in the dbEST data base in searches using the BLAST algorithm
(29). A human cDNA, clone 32798, contained the peptide 2 sequence
in its longest open reading frame upon conceptual translation. This
cDNA clone, with an insert of 1.45 kb, was fully sequenced. In the
analysis of method 1 purified material, a peptide of sequence
GPI/LI/LTQ/KVTDR (peptide 3) was determined by mass spectrometry. A
murine cDNA, clone 598211, contained peptide 3 in its longest ORF
in EST sequence from the 5' end (GenBank accession no. AA162545),
indicating that peptide 3 probably mapped close to the N terminus of
the protein.
Assembly of the Composite Full-length Human cDNA
Sequence--
Another cDNA clone (clone 27) was obtained by
screening a human lymphoblastoid cell line cDNA library in pREP4
(30) using a colony hybridization protocol. Other clones were generated
by anchored PCR (Fig. 1A). The
combined DNA sequence information from all of the available cDNA
clones allowed the inference of the sequence of the full-length
cDNA (Fig. 1B). The gene was given the name
PNKP, for polynucleotide
kinase 3'-phosphatase.
Features of the PNKP Protein--
The PNKP gene encodes
a polypeptide of 521 amino acids, with a predicted molecular mass of
57,148 daltons. Salient features of the protein include a consensus
nucleotide binding site, GFPGAGKS, at residues 372-379 (40). Such a
nucleotide binding site is also found in the T4 polynucleotide kinase
peptide sequence (41), although in T4 PNK it maps near the N terminus
at residues 9-17. Several motifs found in the L-2-haloacid
dehalogenase superfamily (42, 43), also observed in the T4
polynucleotide kinase sequence (41), are present in the PNKP
translation product (Fig. 2,
double underline). Motif 1 includes an aspartate
residue (171) and a threonine residue (175), motif 2 includes threonine
217, and motif 3 comprises aspartates 283 and 289. These motifs are
likely to be involved in the 3'-phosphatase activity of the PNKP
protein. Biochemical studies of the rat liver enzyme indicated a
nuclear localization (1, 44), although we used whole cell extracts for
our purification. There is no obvious candidate for a nuclear localization sequence, but there is a grouping of four basic residues, RKKK at 301-304, that might be involved in nuclear localization (45).
All three of the bovine peptides were observed in the conceptual
translation product; only one difference (a glycine in peptide 1 instead of a serine in the human conceptual translation at residue 221)
in sequence was apparent, although this analysis cannot take into
account the possibility of differences at positions of isobaric amino
acids.
Conservation during Evolution as Detected by BLAST Searches of the
GenBank Data Base and Southern Analysis--
There is a high degree of
homology between the murine and human PNKP genes as seen in
dbEST, at least 70% identical amino acids. GenBank data base searches
using the BLAST algorithm (29) were conducted with the PNKP conceptual
translation product in order to identify similar genes.
Caenorhabditis elegans (F21D5) and
Schizosaccharomyces pombe (c23c11) clones with scores of 159 and 111 and E values of 2e-66 and 3e-43, respectively,
representing possible homologs, were retrieved from the NR data base
(Fig. 3). The C. elegans
cosmid F21D5 gene contained two regions of similarity with 46% and
47% identical residues. These regions spanned amino acids 160-504 of
the human PNKP protein and contained both the putative phosphatase and
nucleotide binding site motifs. As well, the S. pombe
chromosome 1 gene had two segments with scores over 80, from 147 to 267 in hPNKP (46% identical or conserved amino acids) and from 268 to 464 in hPNKP (50% identical or conserved amino acids) and spanned both
putative domains. A Drosophila dbEST (LPO5621) clone with a
score of 140 and expected value of 5e-32 was identified, with 45%
identical residues. A ClustalW alignment of human PNKP polypeptide with
the three related polypeptides, indicating the identical or conserved
residues, is shown in Fig. 3. In Saccharomyces cerevisiae,
BLAST searches revealed a protein (YMR156c) with a lower score (43, E value 0.014). T4 PNK is retrieved by a PSI-BLAST search
using the PNKP peptide sequence within 4 iterations, with an expected
value of 1e-38. There are two regions of similarity, one with 20%
identity and 34% conserved or identical residues, and one with 16%
identity and 32% conserved or identical residues (data not shown). A
eukaryotic viral gene with homology to T4 PNK and T4 RNA ligase is
encoded by the ORF86 gene product of Aplysia californica
nucleopolyhedrovirus (46). In PSI-BLAST searches, this ORF had a region
of similarity from residue 361 to residue 484 of PNKP, with
22% identical and 35% conserved or identical residues. The protein
has not yet been demonstrated to have any enzymatic activity (47).
The availability of cDNA for the PNKP gene prompted us
to see if this gene is conserved across the various eukaryotic species. Southern analysis of EcoRI-digested genomic DNA from
multiple species probed with the full-length cDNA for
PNKP (Fig. 4) indicated a
strong hybridization signal in lanes containing mammalian DNA, and a
weak signal in DNA from chicken. Reproducible bands were observed in
S. cerevisiae DNA. These data suggest that the
PNKP gene is conserved among mammals and as far as avian
species (chicken). Taken together, the results from the BLAST searches
and the Southern analysis provide ample evidence that the exons of the
PNKP gene have been highly conserved during evolution, implying an
important function in many species.
Localization of the PNKP Gene in the Human Genome--
It was
important to investigate the chromosomal localization of the
PNKP gene to ascertain if it corresponded to the position of
any potential disease gene and to initiate a study of the genomic organization of the PNKP locus. Genomic clone 429D20 was
isolated by hybridization screening and used for fluorescence in
situ hybridization (Fig. 5).
Panel A indicates the fluorescence results, and
panel B the overall chromosomal staining. The
gene maps to chromosome 19q13.3-13.4 as indicated in the ideogram
(Fig. 5, panel C). The results of the screening
for genomic clones (data not shown) and the chromosomal localization
(Fig. 5A) indicate that there are no other closely related
loci in the human genome.
Expression of the PNKP Gene in Human Tissues--
Since
biochemical studies had indicated presence of acid pH optimum DNA
kinase activity in many tissues (reviewed in Ref. 1), we were
interested to investigate tissue-specific gene expression, which was
studied in eight human tissues using a commercially obtained Northern
blot. A message of 2 kb was observed in all of the tissues (Fig.
6A). The size of this mRNA
would be sufficient to encode the 60-kDa polypeptide found in
preparations of SNQI-PNK. The strongest intensity of this signal was
observed in spleen and in testis (Fig. 6A, lanes
1 and 4, respectively), and the weakest in small
intestine (Fig. 6A, lane 6). In order
to confirm whether each lane of the blot contained an equal amount of
RNA, the blot was stripped and probed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. The result is shown in Fig.
6B; lane 6 (small intestine) was
observed to be underloaded. Normalization to GAPDH levels
indicated about 3-fold higher expression in spleen and testis (data not
shown). Despite the highly stringent washing conditions, a second
signal of about 7.5 kb with highest intensity in spleen was also
observed, either with an internal probe (Fig. 6A) or a probe
containing the 3' region of the cDNA (data not shown). The
intensity of this signal relative to the 2-kb signal varied somewhat
depending on the tissue source. The genomic library screen and the
chromosomal localization suggest that this signal also arises from the
PNKP locus, since there were no data indicating genomic
clones of a highly related gene that cross-hybridized with
PNKP clones. Currently, it is not known whether this signal may represent an alternatively spliced transcript; we have at present
characterized no cDNA clones that indicated that an alternative transcript might be present. Another possibility is that this larger
transcript represents retained intron sequences. There is also a weak
band appearing between 2.4 and 4.4 kb.
Immunoblot Analysis of Bovine PNKP--
The AC-IV antiserum
against the C terminus of the conceptual translation product of human
PNKP produced a signal of slightly below 60 kDa in partially
purified (SP Sepharose step; Ref. 6) bovine SNQI-PNK preparations (Fig.
7A, lane
1), while no signal was apparent with the same dilution of
preimmune serum (Fig. 7A, lane 2).
This figure demonstrates that an antiserum produced against a
conceptual translation product with features expected in a
polynucleotide kinase identifies a polypeptide of a similar size to the
purified protein. Importantly, in samples from the same stage of the
SNQI-PNK purification, renaturation gel activity studies demonstrated
an active polypeptide of about 60 kDa (6). These results also show that
the cDNA sequence obtained probably accounts for the size of the
purified mammalian protein. Also evident is the conservation of the
epitope(s) recognized by the antiserum between the bovine and human
proteins.
Expression of the PNKP Gene Product as a 5'-DNA Kinase and
3'-Phosphatase in E. coli--
A plasmid, pGST-PNKP, was constructed
by fusing in-frame the clone 32798 next to the C-terminal end of GST in
the vector pGEX-4T-3. The expression construct consisted of amino acids
69-521 of the PNKP conceptual translation. When the plasmid was
introduced into strain E. coli BL21, it produced a
polypeptide that migrated with an apparent molecular mass of 80 kDa,
which is consistent with the predicted mass of the fusion protein.
Protein from cells expressing pGST-PNKP and from control cells
expressing the empty vector pGEX-4T-3 was purified on
glutathione-Sepharose 4B and analyzed by SDS-polyacrylamide gel
electrophoresis and immunoblotting. As expected, the AC-IV antibodies
directed against the C terminus of the conceptual translation product
of the PNKP gene detected the GST-PNKP fusion protein (Fig.
7B, lane 4). A signal of about 80 kDa
was detected, reflecting the anticipated size of the fusion protein.
Control GST protein, detected with anti-GST antibodies, migrated at
about 29 kDa (Fig. 7B, lane 1).
The bacterially expressed GST-PNKP was tested for DNA kinase function
using oligo(dT)25 as a substrate. Protein from crude extracts of E. coli expressing pGEX-4T-3 contained no
detectable DNA kinase activity, while 5'-phosphorylation of the
oligonucleotide substrate was observed in protein from crude extracts
expressing pGST-PNKP (data not shown). This finding led us to test
purified GST-PNKP and corresponding amounts of control GST protein for DNA kinase activity. In Fig.
8A, there was again no
detectable activity in control samples (lanes
1-3), but the DNA kinase activity of GST-PNKP protein was
clearly detectable (lanes 4-6). The specific activity was increased compared with the bovine SNQI-PNK preparations at the final step of purification (data not shown). The 3'-phosphatase activity of GST-PNKP was investigated using a TLC assay monitoring conversion of 5' [32P]TP to 5'
[32P]TOH (6, 10). The region of the
autoradiogram showing 5' [32P]TOH is
displayed in Fig. 8B. As seen with T4 polynucleotide kinase
run as a positive control, the GST-PNKP (lanes
5-7) functioned as a 3'-phosphatase. No detectable
3'-phosphatase activity was observed in reactions containing control
GST protein (lanes 2-4). The GST-PNKP had a
comparable specific activity to the bovine SNQI-PNK preparation at the
final step of the purification (data not shown).
Human PNKP Confers Resistance to Some DNA Damaging Agents in E. coli Lacking 3'-Phosphodiesterase--
Several oxidants are known to
engender genetic instability by inducing DNA single strand breaks (ssb)
with blocked 3'-termini that prevent DNA repair synthesis (25, 48). For
example, H2O2 and the antitumor drug bleomycin
produce ssb bearing 3'-phosphate and 3'-phosphoglycolate, respectively.
These blocked 3'-termini are typically removed by enzymes with
3'-phosphodiesterase activity, thereby permitting DNA repair synthesis
to proceed. In E. coli, exonuclease III and endonuclease IV
constitute the major 3'-phosphodiesterases that process the 3'-blocking
groups in damaged DNA. In addition, these enzymes each possesses an
apurinic/apyrimidinic (AP) endonuclease activity that hydrolyzes AP
sites produced indirectly by alkylating agents, such as methyl methane
sulfonate (MMS). Thus, E. coli mutants lacking both
exonuclease III and endonuclease IV display marked hypersensitivity to
both oxidative agents and MMS (Fig. 9,
A, C, and D; Ref. 39). We predicted
that if the apparent 3'-phosphatase activity of PNKP indeed plays a
role in DNA repair, then this enzyme should restore some oxidant
resistance to E. coli strain BW528 (xth nfo) that
is deficient in both exonuclease III (xth) and endonuclease
IV (nfo). Plasmid pGST-PNKP and its control empty vector
pGEX-4T-3 were introduced into BW528 cells. Purification of the fusion
protein from a glutathione affinity column, and subsequent confirmation
of its 3'-phosphatase activity, revealed that GST-PNKP was actively
expressed in strain BW528 as an 80-kDa polypeptide with a similar
specific activity as obtained from BL21 cells (data not shown). When
GST alone was purified from the affinity column, no 3'-phosphatase
activity was detected, again similar to the data from BL21 cells (data
not shown).
We next examined whether the expressed GST-PNKP is capable of restoring
resistance to DNA damaging agents to strain BW528 by using a gradient
plate assay. In this assay, cells deficient in DNA repair grow only a
short distance into the gradient of increasing chemical concentration,
as compared with cells that are proficient in DNA repair (39). The
plasmid pGST-PNKP restored to strain BW528 partial resistance to the
chemical oxidants H2O2 and
tert-butylhydroperoxide (tBH, Fig. 9, panel
A, bar 7 and panel C, bar 5, respectively). No drug
resistance was conferred to strain BW528 either by the empty vector
pGEX-4T-3 (Fig. 9, panel A, bar 6; panels C and D,
bar 4), or by a plasmid carrying the human LIG1 gene fused to GST (Fig. 9, panel
A, bar 8). These latter observations
preclude the possibility that the GST domain itself is contributing to
the enhanced drug resistance of strain BW528 harboring plasmid
pGST-PNKP. In control experiments, and as previously reported (39), the
plasmid pNfo, which actively expresses bacterial endonuclease IV, also
restored drug resistance to strain BW528 (Fig. 9, panels
A and C, bar 5 and
bar 3, respectively). It was important to
determine that overproduction of GST-PNKP did not confer additional
resistance to wild type AB1157; this was not the case (Fig. 9,
panels A and B, bar
1 (AB1157/pGEX-4-T-3) compared with bar
2 (AB1157/pGST-PNKP). We further tested whether the partial restoration of drug resistance to strain BW528 conferred by pGST-PNKP is specific for oxidative agents. A gradient plate assay was performed on cells treated with the alkylating agent MMS. Surprisingly, pGST-PNKP
also conferred MMS resistance to strain BW528, but not to the same
extent as pNfo (Fig. 9D; bar 5 versus bar 3). One possible
interpretation of this finding is that the endogenous AP lyases, such
as endonuclease III, formamidopyrimidine-DNA glycosylase, and/or
endonuclease VIII, may cleave the AP sites to generate 3'-blocked
termini, which are then further processed by the 3'-phosphatase activity of GST-PNKP (see "Discussion"). It should be noted that strain BW528 is no more sensitive than the parental strain AB1157 to
the DNA damaging agent formaldehyde (Fig. 9B), which
produces DNA lesions other than strand breaks with blocked 3'-termini
and AP sites. Moreover, the expressed GST-PNKP conferred no additional formaldehyde resistance to strain BW528 (Fig. 9B). From
these data, it would appear that the drug resistance conferred by
pGST-PNKP to strain BW528 may be due to the enzyme's ability to
process DNA lesions.
Human PNKP Acts in Vivo to Process
H2O2-induced 3'-Blocking DNA Lesions--
To
directly test whether PNKP is acting in vivo to remove
3'-blocking DNA lesions at ssb, we examined if chromosomal DNA isolated from H2O2-treated cells could sustain in
vitro DNA repair synthesis by E. coli DNA polymerase I
(39). Three exponentially growing strains BW528/pNfo, BW528/pGEX-4T-3,
and BW528/pGST-PNKP were either untreated or treated with 25 mM H2O2 for 1 h, the
chromosomal DNA was immediately isolated from each strain, and examined
for the extent of [methyl-3H]dTMP
incorporation by DNA polymerase I. Chromosomal DNA isolated from any of
the untreated cells showed virtually no incorporation of
[methyl-3H]dTMP by DNA polymerase I (Fig.
10, A-C, open
circles). In contrast, H2O2-damaged
chromosomal DNA isolated from strain BW528/pNfo showed a substantial
level, at least 30-fold increase, of
[methyl-3H]dTMP incorporation (Fig.
10A, closed circles). The
incorporation of [methyl-3H]dTMP was directly
dependent on the in vivo processing of the damaged DNA by
the 3'-phosphodiesterase activity of endonuclease IV, as no
incorporation was observed into H2O2-damaged
DNA derived from strain BW528 carrying only the vector pGEX-4T-3 (Fig.
10B, closed circles). However, if the
damaged DNA from strain BW528/pGEX-4T-3 was pretreated with purified
endonuclease IV, the extent of [methyl-3H]dTMP
incorporation was greatly enhanced, and reached the same level as
damaged DNA derived from strain BW528/pNfo (Fig. 10, A and
B, closed squares). The most striking
observation was the incorporation of
[methyl-3H]dTMP into the
H2O2-damaged DNA derived from strain BW528
harboring the pGST-PNKP (Fig. 10C, closed
circles). This finding can only be explained if the
3'-phosphatase activity of PNKP acts in vivo to process
H2O2-induced DNA lesions. It is noteworthy,
however, that the extent of [methyl-3H]dTMP
incorporation into the damaged DNA derived from BW528/pGST-PNKP was
only 65% of the level incorporated with
H2O2-damaged DNA obtained from strain
BW528/pNfo (Fig. 10, A and C). Endonuclease IV
pretreatment of the H2O2-damaged DNA derived
from strain BW528/pGST-PNK permitted an additional 30% of
[methyl-3H]dTMP incorporation by DNA
polymerase I (Fig. 10C). This latter finding suggests that
the level of GST-PNKP in strain BW528 may be insufficient to process
all the H2O2-induced DNA lesions. Nonetheless, the level of label incorporation into the
H2O2-damaged DNA was unchanged if the
endogenous level of GST-PNKP was increased from the
isopropyl-1-thio- The identification of a cDNA encoding a human PNKP
represents the first mammalian polynucleotide kinase and the second
mammalian 3'-phosphatase to be cloned. Importantly, this gene is the
first DNA-specific kinase from any organism to be characterized at the molecular level. This discovery has allowed significant progress toward
an understanding of its role in DNA metabolism, particularly in DNA
repair of damage by oxidative damaging agents.
We conclude that we have obtained a gene encoding a polynucleotide
kinase 3'-phosphatase due to the 5'-DNA kinase and 3'-phosphatase activities of the GST-PNKP construct expressed in E. coli.
The observation of 3'-phosphatase activity of the PNKP gene
expressed in E. coli BW528, which lacks significant
endogenous 3'-phosphatase activity that might have copurified with
GST-PNKP, is incontrovertible evidence that the two activities are
encoded by the same gene. Sequence similarity to T4 PNK in certain key
motifs also supports our conclusion that a PNK gene has been cloned.
Furthermore, antibodies raised against the PNKP peptide sequence
recognize in immunoblots a bovine polypeptide of the size of
the active DNA kinase detected in renaturation gel activity
assays (6).
The PNKP gene product contains a motif found in adenine
nucleotide-binding proteins (40), GFPGAGKS, at residues 372-379. There
is a corresponding conserved motif, GCPGSGKS, at positions 9-16 in the
T4 polynucleotide kinase peptide sequence (41). The importance of this
conserved region for the polynucleotide kinase activity of the T4
enzyme correlates with mapping of the kinase domain to the N terminus
(49). We identified in the T4 PNK peptide sequence (6) a series of
three motifs that have been found to be important in numerous studies
of proteins in the L-2-haloacid dehalogenase superfamily
(42, 43). In the PNKP gene product, a particularly important residue in
catalysis is predicted to be aspartate 171, which is the conserved
residue found to be critical for function of L-2-haloacid
dehalogenase (50) and observed to be phosphorylated when human
phosphomannomutase, also a member of the superfamily, is incubated with
substrate (51). Interestingly, in the T4 sequence these motifs map to the C-terminal half of the protein (6), but in the mammalian PNKP
protein sequence the motifs are centrally located between residues 171 and 290. The C. elegans and S. pombe genes that
scored high in BLAST searches contained the putative 3'-phosphatase and nucleotide binding domains in the same order as in the PNKP protein. Thus, the organization of the putative active domains of the T4 PNK and
PNKP proteins seems to differ considerably, with the relative locations
of the putative PNK and 3'-phosphatase motifs switched. The observation
that a GST fusion construct containing amino acids 69-521 of the
conceptual translation product was active in E. coli was not
entirely unexpected, since this construct contained 87% of the coding
sequence, including the putative phosphatase motifs at residues
171-289 and the putative nucleotide binding site at residues 372-379
that would be anticipated to be critical for the 3'-phosphatase and
5'-kinase activities, respectively.
The PNKP gene maps to chromosome 19q13.3-13.4, a region of
the genome rich in well-characterized genes, including POLD1
at 19q13.3-13.4 (52), and LIG1 at 19q13.2-q13.3 (53, 54)
among those involved in DNA metabolism. DNA repair genes that localize proximally to PNKP include ERCC1 at 19q13.2-q13.3
(54), ERCC2/XPD at 19q13.2-q13.3 (55), and XRCC1
at 19q13.2 (54, 56). A search of the OMIM data base (57) revealed no
obvious candidate human disease mapping to this area of the genome;
however, since many human genetic diseases have not been mapped, a
contribution of PNKP to human disease burden cannot be ruled
out. This part of chromosome 19 is involved in certain translocations
found in malignancies (57). In addition, loss of heterozygosity of this portion of chromosome 19 has been reported in some neoplasms
(58-62).
Preliminary gene expression studies revealed two major signals upon
Northern analysis of samples of human poly(A)+ RNA,
corresponding to sizes of 2 and 7.5 kb. The cDNA sequences obtained
correspond well to the size of the smaller of the two messages. In the
tissues that we examined, testis and spleen displayed greater amounts
of the 2-kb message when the results were normalized to expression of
the GAPDH gene. The larger message is of unknown biological
significance; it was detectable with a probe containing the 3' end of
the cDNA and a probe from the interior of the cDNA. The genomic
DNA library screening results and FISH mapping results are strongly
indicative of one gene. The larger message may be an alternatively
spliced transcript, although dbEST clones analyzed so far do not
support this notion, or perhaps may represent retained intron sequences.
A potential physiological role in DNA repair has been suggested
repeatedly from biochemical studies of mammalian DNA kinase. Findings
from in vitro reconstitution experiments with synthetic substrates containing 3'-phosphate and/or 5'-OH termini support the
notion that polynucleotide kinase 3'-phosphatases can function in DNA
repair (4, 63). Another correlation with DNA repair is that the first
human DNA 3'-phosphatase to be cloned is the APE/HAP1 gene,
which has a firmly established DNA repair function supported by many
biochemical and genetic studies. The APE/HAP1 gene is a member of the
exonuclease III family, which together with the endonuclease IV family
includes enzymes detected in E. coli and eukaryotes that are
able to repair 3'-phosphate damage at DNA strand breaks (25, 64). These
proteins have multiple functions, including Type II AP endonuclease
activity, 3'-phosphodiesterase activity, and sometimes an exonuclease
activity. Biochemical studies of APE/HAP1 function (25) indicate that
it is unlikely to be responsible for repair of all 3'-blocking residues
arising from radiation or oxidative damage to DNA. Moreover,
fractionation of mammalian cells suggests the presence of multiple
3'-phosphodiesterase activities (24, 65).
To investigate the role of PNKP in DNA repair in
vivo, we performed heterologous complementation experiments.
Rescue of a mutant phenotype in another species can often provide
compelling evidence for a physiological role. We took advantage of the
availability of E. coli with vastly reduced
3'-phosphodiesterase activity, xth nfo mutants deficient in
AP endonuclease activity and 3'-phosphodiesterase activity (37). We
performed experiments testing sensitivity to the DNA damaging agents
hydrogen peroxide and tBH. As expected, the BW528 (xth nfo)
cells were highly sensitive, and overexpression of the bacterial
nfo gene provided resistance. Our discovery that overexpression of pGST-PNKP, but not an empty vector or pGST-hLIG1, partially overcame the sensitivity to these oxidative DNA damaging agents but not to formaldehyde used in control experiments, supports a
role for PNKP in DNA repair in living cells. Importantly,
isolated DNA from H2O2-treated cells expressing
pGST-PNKP, but not cells expressing the empty vector, pGEX-4-T-3, was a
better substrate for DNA polymerase. This reflects in vivo
DNA repair and shows that the partial mutant rescue was correlated with
events at the DNA level rather than some other effect on cellular
response to H2O2.
The ability of PNKP to confer partial resistance to the
alkylating agent MMS to strain BW528 can be explained if endogenous AP
lyases, i.e. formamidopyrimidine-DNA glycosylase,
endonuclease III, or endonuclease VIII, cleave the MMS-induced AP sites
to produce blocked 3'-termini, such as 3'-phosphate. Alternatively, the
PNKP enzyme might directly cleave the AP sites. This latter possibility
is supported by the finding that GST-PNKP purified from AP
endonuclease-deficient strain BW528 weakly cleaves a substrate with a
centrally located AP site (66). Experiments are in progress to
determine if the enzyme acts as an AP lyase or as a hydrolytic AP
endonuclease that directly produces 3'-hydroxyl termini, which are
compatible with DNA repair synthesis. The inability of PNKP to fully
restore drug resistance to strain BW528 is not entirely surprising,
since the enzyme is not in its natural environment. In fact, expression
of the yeast homologue of endonuclease IV, Apn1, in strain BW528 also
only partially substitutes for endonuclease IV (39). We cannot
completely exclude the possibilities that the N-terminal 13% of the
polypeptide may be required for full complementation or that the GST
domain may interfere with the enzyme's ability to process DNA lesions.
Furthermore, in mammalian cells, PNKP may require accessory factors,
which are lacking in E. coli, to efficiently repair damaged DNA.
This work provides support for a crucial function of the 3'-phosphatase
activity of the PNKP gene product in repair of oxidative DNA
damage in mammalian cells. Importantly, this type of damage arises
endogenously during normal cellular metabolism, and its repair is
essential for cellular survival. The role of the 5'-DNA kinase activity
of the gene product may also reside in DNA repair, restoring 5'-OH
termini arising during the life of the cell to 5'-P termini suitable
for ligation. The PNKP gene product may also participate in
DNA replication. The molecular reagents reported here, together with
the appropriate genetic model systems that we have now identified, will
allow further detailed analysis of the biological implications of
juxtaposition of 5'-DNA kinase and 3'-phosphatase activities in the
same enzyme.
We thank Dr. William S. Lane and the staff of
the Harvard Microchemistry Facility for peptide sequence analysis and
Nancy Qiang of Sheldon Biotechnology for DNA sequencing. We thank
Suzana Anjos, Didier Look, and Dr. Fortunato Manganaro for technical assistance, and Nadine Beaulieu and Dr. Hung-The Hyunh for help with
colony hybridization. We thank Dr. Manuel Buchwald for providing a
cDNA library. We are grateful to Drs. Lenore Beitel, Marco
DiFruscio, Rongtuan Lin, Stéphane Richard, and Rima Rozen for
helpful suggestions.
Karimi-Busheri et al. (67)
describe the cloning of human polydeoxyribonucleotide kinase, a gene
identical to PNKP, in an accompanying article.
*
This work was supported in part by the National Cancer
Institute of Canada, with grants from the Canadian Cancer Society (to D. D. L. and D. R.) and the Medical Research Council of
Canada (to S. W. S.).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) AF126486.
¶¶
Scholar of the Medical Research Council of Canada.
||
To whom correspondence should be addressed:
Rm. 502, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Côte-Ste-Catherine Rd.,
Montreal, Quebec H3T 1E2, Canada. Tel.: 514-340-8260 (ext. 3454); Fax:
514-340-7576; E-mail: dlasko@ldi.jgh.mcgill.ca.
2
C. Ong and A. Jilani, unpublished observations.
3
A. Jilani, unpublished observations.
The abbreviations used are:
PNK, polynucleotide
kinase;
FISH, fluorescence in situ hybridization;
ssb, single strand breaks;
tBH, tert-butylhydroperoxide;
HPLC, high pressure liquid chromatography;
EST, expressed sequence tag;
PCR, polymerase chain reaction;
kb, kilobase(s);
GST, glutathione
S-transferase;
AP, apurinic/apyrimidinic;
MMS, methyl
methane sulfonate.
Molecular Cloning of the Human Gene, PNKP, Encoding a
Polynucleotide Kinase 3'-Phosphatase and Evidence for Its Role in
Repair of DNA Strand Breaks Caused by Oxidative Damage*
§,
,**,§
,§¶¶||
Molecular Oncology Group,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP (ICN, Mississauga, Ontario, Canada) with a
modification (31) of the procedure in Sambrook et al. (32).
The cDNA sequence of the 5' end of the gene was obtained by PCR
from the aforementioned pREP4 cDNA library, using the pREP4
sequencing primer (30) as an anchored primer together with the L400 primer.
-competent cells (Life
Technologies, Inc.). Plasmid DNA was prepared (Promega Wizard kit,
Fisher, Town of Mount Royal, Quebec, Canada), and the DNA sequence of
the pGST-PNKP construct was verified by sequencing. Expression of the
fusion protein in exponentially growing BL21 cells was induced with 1 mM isopropyl-1-thio-
-D-galactopyranoside. A
freeze-thaw method with buffer consisting of 50 mM
Tris-HCl, pH 7.5, 30 mM NaCl, 0.5 mM
dithiothreitol, 0.5 mM EDTA, and a mixture of protease
inhibitors (aprotinin, leupeptin, chymostatin, N
-p-tosyl-L-lysine
chloromethyl ketone, and phenylmethylsulfonyl fluoride) was used to
lyse the cells. The GST protein or the GST PNKP protein was purified on
glutathione-Sepharose 4B (Amersham Pharmacia Biotech) according to the
instructions of the manufacturer. Purified protein (130 ng) from the
cell extracts expressing GST or GST PNKP 69-521 was assayed for PNK
activity with oligo(dT)25 at pH 5.5 (8) and for
3'-phosphatase activity using the procedure of Cameron and Uhlenbeck
(6, 10). The same procedure was followed to purify GST or GST PNKP
expressed in BW528, with slightly lower yields of GST PNKP.
80 °C. Extraction of the chromosomal DNA was performed as described previously (39), with a typical yield of
120 µg of DNA from a 5-ml culture. To measure the incorporation of
[methyl-3H]dTMP, 150 µM
untreated or H2O2-treated chromosomal DNA in 25 µl of HE buffer (10 mM Hepes-KOH, pH 7.0, 1 mM EDTA) was added to 225 µl of an ice-cold reaction
mixture. The reaction mixture consisted of 25 mM Hepes-KOH,
pH 7.6, 25 mM KCl, 10 mM MgCl2, 50 µg/ml bovine serum albumin, 100 µM dATP, 100 µM dCTP, 100 µM dGTP, 30 µM
dTTP, and 3 units/ml E. coli DNA polymerase. The labeled [methyl-3H]dTTP (NET221X from NEN Life Science
Products; 37.0 MBq) was added to the reaction mixture to a specific
activity of 1260 cpm/pmol. The reaction was started when the samples
were immersed into a 37 °C water bath. At the indicated time,
40-µl samples were withdrawn and added to tubes containing 200 µl
of 0.1 M sodium pyrophosphate and 1 mg/ml bovine serum
albumin, followed by the addition of 200 µl of 0.8 M
trichloroacetic acid, mixed, and placed on ice for 10 min. The samples
were processed on a 12-hole filtration apparatus (Millipore, Bedford,
MA) using GF/C circle filters (Whatman). The trapped DNA was washed
three times with 3 ml of 0.1 M sodium pyrophosphate,
briefly rinsed with ethanol, air-dried, and counted with 5 ml of
scintillation fluid (BCS, Amersham Pharmacia Biotech). In the case of
chromosomal DNA pretreated with purified endonuclease IV, 10 ng of the
enzyme was incubated with the DNA for 20 min at 37 °C in 10 µl of
endonuclease reaction buffer (25 mM Hepes-KOH, pH 7.6, 50 mM KCl, 1 mg/ml bovine serum albumin). Endonuclease IV was
heat-inactivated at 70 °C for 3 min.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (51K):
[in a new window]
Fig. 1.
A, cloning strategy and cDNAs
available. As described under "Experimental Procedures," a human
dbEST clone from an infant brain cDNA library (32798, 1.45-kilobase
pair insert) was obtained by BLAST searches using the sequence of a
13-mer tryptic peptide, and sequenced using an overlapping primer
walking strategy. An additional clone (pREP4 clone 27) of about the
same size was identified in a human lymphoblastoid cell line cDNA
library in the vector pREP4. The latter library was the source for 5'
clones generated by anchored PCR (TA13, TA21, TA26). These clones were
sequenced in their entirety using the M13 forward and M13 reverse
primers. B, sequence of composite cDNA. The DNA sequence
and several features are shown. Underlined are the presumed
ATG start codon, the first in-frame stop codon, and the putative
poly(A) binding site.
View larger version (42K):
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Fig. 2.
Conceptual translation of composite cDNA
sequence. The 521-amino acid PNKP gene product contains regions
A-E (single underline) and residues
corresponding to motifs 1, 2, and 3 of the haloacid dehalogenase fold
(double underline, see "Results").
A, bovine tryptic peptide 3 (GPI/LTQ/KVTDR); B,
bovine tryptic peptide 2 (I/LI/LYI/LEI/L(PR)); C, bovine
tryptic peptide 1 used to identify I.M.A.G.E. clone 32798 (I/LVI/LFTN[Q/K]MGI/LGR); D, Walker consensus nucleotide
binding site identified in a PROSITE search; E, 14-mer
peptide used to generate antiserum AC-IV. I/L and
Q/K indicate isobaric amino acids could not be distinguished
with the peptide sequencing methods used but were assigned based on
dbEST clones.
View larger version (54K):
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Fig. 3.
Alignment of human PNKP with high scoring
C. elegans, S. pombe, and D. melanogaster
genes. The conceptual translation product of the human
PNKP gene was used to search the NR and dbEST sections of
GenBank. An alignment of three polypeptides scoring over 80 with the
BLAST algorithm was compiled using ClustalW. F21D5: C. elegans cDNA predicted from genomic sequence of cosmid F21D5
from chromosome IV, denoted protein F21D5.5, accession no. Z54271.
c23C11, an open reading frame identified in the DNA sequence
of S. pombe chromosome 1, accession no. Z98559.
LP05621, a D. melanogaster larval-early pupal EST
cDNA clone, accession no. AI257304. On the consensus line
underneath each section of the alignment, asterisk indicates
identical or conserved residues in all sequences in the alignment,
colon indicates conserved substitutions, and dot
denotes semiconserved substitutions.
View larger version (62K):
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Fig. 4.
Conservation of exons of the PNKP
gene analyzed by Southern blotting. A Southern blot
containing 4 µg of DNA from nine species digested with
EcoRI was probed with the full-length PNKP cDNA. All
genomic DNAs were isolated from kidney tissue, except human DNA was
isolated from placental tissue, while chicken DNA was isolated from
liver tissue.
View larger version (31K):
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Fig. 5.
Chromosomal localization. A,
FISH results from the bacterial artificial chromosome (BAC)
clone 429D20 containing the PNKP gene are shown.
B, staining of the chromosomes is indicated. C,
the ideogram shows the results from analysis of the hybridization. The
gene was mapped to chromosome 19q13.3-13.4.
View larger version (65K):
[in a new window]
Fig. 6.
Northern blot. A commercially obtained
blot containing 2 µg of poly(A)+ RNA was probed with an
internal portion of the PNKP cDNA (panel A)
or, following a stripping procedure as described under "Experimental
Procedures," a GAPDH cDNA probe (panel B).
RNA was isolated from the following human tissues: lane
1, spleen; lane 2, thymus;
lane 3, prostate; lane 4,
testis; lane 5, ovary; lane
6, small intestine; lane 7, colon
(mucosal lining); lane 8, peripheral blood
leukocytes. The blot was washed under high stringency conditions (0.1×
SSC, 65 °C) and exposed to x-ray film.
View larger version (39K):
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Fig. 7.
Immunoblot analysis of bovine protein and
GST-PNKP. Protein samples were electrophoresed through 10%
SDS-polyacrylamide gels and transferred to nitrocellulose membrane.
Immunoblots were carried out using the ECL kit (panel
A) or with alkaline phosphatase detection (panel
B) as described under "Experimental Procedures."
Panel A, lane 1,
SP-Sepharose step bovine preparation (2 µg) probed with AC-IV
antiserum (1:1000 dilution); lane 2, SP-Sepharose
preparation (2 µg) probed with preimmune serum (1:1000 dilution).
Panel B, lane 1, purified
GST protein (0.4 µg) from BL21 cells expressing pGEX-4T-3 detected
with anti-GST antibodies (1:2000 dilution); lane
2, purified GST-PNKP (0.4 µg) probed with preimmune serum
from rabbit AC-IV (1:2000 dilution); lane 3,
purified GST protein probed with AC-IV serum (1:2000 dilution);
lane 4, purified GST-PNKP probed with AC-IV serum
(1:2000 dilution).
View larger version (43K):
[in a new window]
Fig. 8.
GST-PNKP functions as a 5'-DNA kinase and a
3'-phosphatase. A, 5'-DNA kinase activity. Purified
control GST protein (130 ng, lanes 1-3) or
purified GST-PNKP (130 ng, lanes 4-6) was
incubated in a standard DNA kinase assay with oligo(dT)25
substrate. The autoradiogram was exposed for 3 h, and the bands
were excised for quantitation of DNA kinase activity. B,
3'-phosphatase activity. A 3'-phosphatase assay employed 5'
[32P]Tp as substrate. Reactions were
monitored by TLC and autoradiography. The portion of the autoradiogram
corresponding to the 5' [32P]TOH product is
shown. For quantitation, the corresponding area of the TLC plate was
excised and counted by liquid scintillation. Lane
1, T4 polynucleotide kinase (positive control);
lanes 2-4, purified control GST protein (130 ng); lanes 5-7, purified GST-PNKP (130 ng).
View larger version (101K):
[in a new window]
Fig. 9.
Resistance to DNA damaging agents conferred
by human PNK in the DNA repair-deficient E. coli
strain BW528 (xth nfo). In panels A
and B the numbers represent the following
strains: 1-3, AB1157
(Xth+Nfo+) harboring the
vector pGEXT-4T-3, the plasmids pNfo, and pGST-PNKP, respectively;
4-8, BW528 (xth nfo) harboring the vector
pBluescript S/K, pNfo, pGEX-4T-3, pGST-PNKP, and pGST-LIG1,
respectively. For panels C and D, the strains are
numbered as follows: 1, AB1157; 2-5, BW528
harboring the plasmids pBluescript S/K, pNfo, pGEX-4T-3, and pGST-PNKP,
respectively. The initial amount of drug in the bottom layer of the
gradient was 75 µmol of H2O2
(panel A), 0.01% formaldehyde (HCHO,
panel B); 3.9 µmol of tBH (panel
C), and 0.4 mmol of MMS (panel D). The
concentration increases in a gradient from left to right. Photographs
were taken after cells were incubated overnight at 37 °C.
Complementation was scored as complete restoration to growth to that
observed in the wild type strain; partial complementation was scored as
increased growth compared with the mutant strain.
-D-galactopyranoside-inducible
lac promoter of the pGEX-4T-3 vector (data not shown). It is
certainly possible that PNKP may be unable to access and or repair all
the H2O2-induced DNA lesions in vivo
(see "Discussion"), thus accounting for the partial drug-resistance
conferred by GST-PNKP to strain BW528.
View larger version (17K):
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Fig. 10.
In vitro incorporation
of [methyl3H]dTMP by DNA polymerase I
into untreated (open circles) and
H2O2-treated (closed
circles) chromosomal DNA isolated from E. coli strains. Where indicated, DNA was pretreated with
10 ng of purified endonuclease IV for 20 min before monitoring
[methyl-3H]dTMP incorporation (open
squares, untreated cells; closed
squares, treated cells).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
Note Added in Proof
FOOTNOTES
Research Scientist of the National Cancer Institute of Canada.
Supported by a Clark Fellowship in Cancer Research, Jewish
General Hospital, and by a studentship from the Faculty of Medicine, McGill University.
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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