From the Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0602
Received for publication, December 11, 2000, and in revised form, January 29, 2001
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ABSTRACT |
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Polynucleotide kinase is a bifunctional enzyme
containing both DNA 3'-phosphatase and 5'-kinase activities seemingly
suited to the coupled repair of single-strand nicks in which the
phosphate has remained with the 3'-base. We show that the yeast
Saccharomyces cerevisiae is able to repair transformed
dephosphorylated linear plasmids by non-homologous end joining with
considerable efficiency independently of the end-processing polymerase
Pol4p. Homology searches and biochemical assays did not reveal a
5'-kinase that would account for this repair, however. Instead, open
reading frame YMR156C (here named TPP1) is shown to encode
only a polynucleotide kinase-type 3'-phosphatase. Tpp1p bears extensive
similarity to the ancient L-2-halo-acid dehalogenase and
DDDD phosphohydrolase superfamilies, but is specific for
double-stranded DNA. It is present at high levels in cell extracts in a
functional form and so does not represent a pseudogene. Moreover, the
phosphatase-only nature of this gene is shared by Saccharomyces
mikatae YMR156C and Arabidopsis thaliana K15M2.3.
Repair of 3'-phosphate and 5'-hydroxyl lesions is thus uncoupled in
budding yeast as compared with metazoans. Repair of transformed
dephosphorylated plasmids, and 5'-hydroxyl blocking lesions more
generally, likely proceeds by a cycle of base removal and resynthesis.
Oxidative damage to DNA can result from endogenously generated
reactive oxygen species or from exposure to exogenous agents such as
ionizing radiation or anticancer agents such as bleomycin and
neocarzinostatin (reviewed in Ref. 1). Such damage and the enzymes
involved in its repair frequently produce fragmentation of the
deoxyribose sugar backbone, resulting in DNA strand breaks bearing
abnormal structures at the 3' and 5' termini. These are termed
"blocking lesions" because they prevent the reactions necessary to
achieve final repair of the damaged strand, namely polymerization and
ligation. Since both single- and double-strand lesions can occur with
potential consequences that include replication failure and genomic
rearrangement, the resolution of blocking lesions is of major
importance in genome maintenance.
Although many chemical forms are possible, important blocking lesions
on 5' termini include hydroxyls and deoxyribose phosphates. As an
example of the redundancies in end processing, deoxyribose phosphate
moieties can be removed in short-patch base excision repair
(BER)1 by the lyase function
of DNA polymerase Polynucleotide kinase (PNK) is best known due to the utility of the T4
enzyme (14) in molecular cloning, but it was demonstrated to exist in
eukaryotes 30 years ago (15). Although not clearly indicated by their
name, the PNK proteins studied to date bear two distinct catalytic
activities, a 5'-kinase and a 3'-phosphatase (16, 17). Although the
precise biological role of eukaryotic PNK remains to be determined, it
is clearly suited to directly reverse the two reciprocal blocking
lesions that would result from a strand break with a misplaced
phosphate, i.e. a 3'-phosphate and 5'-hydroxyl. Indeed, the
preferred substrate of mammalian PNK is a DNA nick (18), and its enzyme
activities are stimulated by interaction with the XRCC1 repair protein
(19), strongly suggesting a role in BER/single-strand break repair.
As a continuation of our interest in delineating the mechanisms by
which terminal damage is resolved during DNA double-strand break
repair, we have been attempting to identify and characterize PNK from
the yeast Saccharomyces cerevisiae, whose existence we inferred from the successful repair of transformed dephosphorylated linear plasmids. The recent cloning of human PNK/3'-phosphatase (hPNKP)
(16, 17) has greatly facilitated this by allowing homology searching
against the yeast and other sequenced genomes. Surprisingly, we find
that S. cerevisiae, at least one other
Saccharomyces yeast, and species as distantly related as
Arabidopsis thaliana contain a gene with homology to only
the putative 3'-phosphatase portion of hPNKP. The S. cerevisiae protein, encoded by open reading frame (ORF) YMR156C,
here named TPP1, shares many of the biochemical properties
of the hPNKP 3'-phosphatase, but indeed is not a 5'-kinase. Despite the
observed plasmid repair, structural comparisons and enzymatic assays
failed to detect an unlinked 5'-kinase. Evolutionary models to explain
these results are discussed in the context of alternative pathways for
resolution of terminal damage during DNA repair.
Yeast Strains--
Wild-type Saccharomyces mikatae
strain 1815, obtained from Dr. Mark Johnston, is described in Ref. 20.
Schizosaccharomyces pombe strain FY254
(h Plasmid Transformation Assay--
Plasmid pES26 has been
previously described (23). Methods of plasmid preparation and
transformation were exactly as described (22), but with the following
addition. After BglII digestion, but before extraction and
precipitation, an equal volume of 2× calf intestinal alkaline
phosphatase buffer (Roche Molecular Biochemicals) was added, followed
by nothing (ligation-competent control plasmid) or 0.16 units of calf
intestinal alkaline phosphatase/µg of plasmid (dephosphorylated
plasmid) and further incubation at 37 °C for 30 min.
Multiple Sequence Alignment--
BLASTP and Psi-BLAST homology
searches were performed via the NCBI web server. Sequences
included in the overall alignment were all hPNKP BLASTP matches
from the non-redundant GenBankTM, expressed sequence tag,
and sequence tagged sites data bases with E < 0.01, in addition to Trl1p, T4 PNK, and AcNPV-2. Expressed sequence tag
and STS sequences were assembled into contigs and translated prior to
inclusion in the alignment (details are available on request).
Accession numbers for the sequences that are ostensibly complete but
uncharacterized are as follows (see Table I): Mus musculus,
AAF36487; Drosophila melanogaster (CG9601), AAF54229; At-1
(A. thaliana gene encoding a putative 3'-phosphatase),
BAA97052; At-2 (A. thaliana gene encoding a putative 5'
kinase), CAB81914; S. pombe (C23C11.04C), CAB11157;
Spodoptera exigua NPV (ORF54), AAF33584; AcNPV-1 (Ac-HisP),
AAA66663; and AcNPV-2 (A. californica PNK/ligase),
AAA66716. In the case of Caenorhabditis elegans (accession
number T21197), we extended the putative ORF F21D5.5 by appending both
amino- and carboxyl-terminal exon translations that were not originally
included. Alignments were performed with MACAW (25).
S. mikatae YMR156C Sequence--
Primers were designed that
corresponded to S. mikatae sequences (kindly provided by Dr.
Mark Johnston) homologous to S. cerevisiae ORFs YMR154C and
YMR157C (5'-TCCAGTTCAAAAGTAGGATTCC and 5'-TAGGTAAGGCCGACATCATC, respectively). These were used in a PCR with S. mikatae
genomic DNA using the HF Advantage PCR kit
(CLONTECH) according to the manufacturer's
instructions. The resulting single ~5-kilobase pair amplified
fragment was sequenced directly by the University of Michigan DNA
Sequencing Core by walking from YMR157C through YMR156C and into
YMR155W. The entire S. mikatae YMR156C coding sequence
(accession number AF326782) was read without ambiguities, including a
stop codon read clearly from two independent runs.
Extraction and Purification of Proteins from Yeast--
Yeast
strains expressing GST fusion proteins were isolated from the ORF array
described and kindly provided (via Dr. Dennis Thiele) by Dr. Eric
Phizicky and co-workers (26). Yeast cells from the YMR156C well were
streaked to single colonies, and anti-GST (Santa Cruz Biotechnology)
Western blotting was used to identify isolates expressing proteins
whose size corresponded to GST-Tpp1p and non-recombinant GST.
Purification was by glass bead lysis and salt extraction, followed by
batch chromatography on glutathione-agarose (Amersham Pharmacia
Biotech) as described (26), except that protein expression was induced
by adding 0.1 mM CuSO4 for 3 h prior to
harvest. Typical final GST-Tpp1p dialysates derived from 50 ml of yeast
culture contained 50 µg/ml fusion protein in 600 µl of 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 4 mM MgCl2, 1 mM dithiothreitol, 50 mM NaCl, and 50% (v/v) glycerol.
Crude whole-cell extracts of S. cerevisiae, S. mikatae, and S. pombe were all prepared by glass bead
disruption of cells in ~1 cell pellet volume of 50 mM
Tris-HCl (pH 7.5), 1 mM EDTA, 1 M NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 10%
glycerol, 2 µg/ml aprotinin, 1 µg/ml each leupeptin and pepstatin,
and 1 mM phenylmethylsulfonyl fluoride, followed by
centrifugation to remove cellular debris. Final extracts were diluted
to 0.5 µg/µl protein.
Enzyme Activity Assays--
Oligonucleotides with and without
3'-phosphates were purchased from Operon Technologies, Inc. (see Figs.
5 and 7 for sequences). Oligonucleotides were 5'-end-labeled with
[ Repair of 5'-Hydroxyl Blocking Lesions during Yeast NHEJ--
To
begin to determine the impact of 5'-hydroxyl blocking lesions on the
repair of double-strand breaks, plasmid pES26 was digested in
vitro with the restriction enzyme BglII and transformed in yeast cells, both with and without pretreatment with calf intestinal alkaline phosphatase. In this well established assay (22, 23), recircularization by NHEJ (7, 23, 27) is required for plasmid stability
and thus for expression of the plasmid URA3 marker gene. Because the BglII site resides in an essential region of the
ADE2 marker gene also on this plasmid, transformation to
Ade+ further requires that repair be precise (imprecise
repair yields ade2 colonies that appear red instead of
white). Dephosphorylated plasmid transformed into wild-type yeast
showed only a 2-3-fold decrease in Ura+ colony recovery as
compared with ligation-competent plasmid (Fig. 1). Moreover, there was no increase in
red colony recovery, indicating that joining remained precise.
Transformation by dephosphorylated plasmids was decreased 54-fold in
yeast deficient in the ligase required for NHEJ (dnl4-K282R)
(23), verifying that the damaged ends are not routed into another
pathway. As an additional control, it was verified that calf intestinal
alkaline phosphatase-treated plasmids could not be religated in
vitro by T4 DNA ligase unless further treated with T4 PNK, as
indicated by gel electrophoresis and a >700-fold decrease in colony
recovery following bacterial transformation (data not shown).
As shown in Fig. 1A, there are at least two ways that
NHEJ of 5'-hydroxyl lesions might be achieved. In the first, the
damaged 5'-nucleotide is nucleolytically removed and subsequently
resynthesized on the opposite side of the break. We have previously
observed that the yeast PolX family polymerase Pol4p can catalyze such base addition during NHEJ (22). pol4-D367E mutant yeast,
which expresses catalytically inactive Pol4p (22), showed the same pattern as the wild type, however. Importantly, some
polymerization-dependent NHEJ events show only a 2-fold
defect in pol4 mutants (22), so it possible that another
polymerase can substitute for Pol4p at dephosphorylated lesions as
well. Nonetheless, these observations were consistent with the
alternative model in which the 5'-hydroxyl lesion is directly reversed
by a PNK 5'-kinase. We sought to identify this in the experiments
described below.
Conservation and Modular Evolution of PNK 5'-Kinase and
3'-Phosphatase Domains--
BLAST searches against the non-redundant
GenBankTM, expressed sequence tag, and sequence tagged
sites data bases using hPNKP as the query revealed a set of 22 distinct
PNK-like genes from 20 different species and viruses (Table
I). Among these was the S. cerevisiae ORF YMR156C, but this weaker matche (E = 0.006) corresponded to only a portion of the human protein and
surprisingly lacked an apparent Walker A (i.e. P-loop) motif
for ATP binding (28), which would be expected for a 5'-kinase. Absent
as matches were three genes identified from literature searching that
are known or believed to encode 5'-kinases: TRL1 tRNA ligase
from S. cerevisiae (29, 30), bacteriophage T4 PNK (14), and
a putative PNK from AcNPV (31).
To examine the relationship between YMR156C and the other PNK-related
genes in more detail, we performed an extensive multiple sequence
alignment as shown in Figs. 2-4. Two
sequence motifs, the Walker A box and the phosphotransferase motif
DXDX(T/V) (32), were used as a means of
unambiguously identifying the 5'-kinase and 3'-phosphatase catalytic
cores, respectively, as suggested by previous alignments of smaller
numbers of bifunctional PNK sequences (16, 17). It was first apparent
that genes may contain just the 3'-phosphatase domain, just the
5'-kinase domain, or both. This was not limited to S. cerevisiae. For example, A. thaliana contains two
hypothetical genes, one encoding a 3'-phosphatase (designated for
simplicity as At-1; see "Experimental Procedures" for accession
numbers) and one encoding a 5'-kinase (At-2). These genes are on
different chromosomes, and examination of the genomic sequence
surrounding At-1 did not reveal any cryptic 5'-kinase domain exons. In
addition, in some species, there is an apparent redundancy of function.
For example, Dictyostelium discoideum has two distinct
putative 5'-kinase genes. We note, however, that these genes correspond
to two structurally evident 5'-kinase groupings (see below), so it is
possible, if not likely, that 5'-kinases perform distinct tasks in the
cell. Finally, in genes containing both domains, each of the two
possible orders are observed.
Taken together, these observations reveal a tremendous modularity in
the evolution of the PNK 3'-phosphatase and 5'-kinase catalytic
domains. As a result, it is now possible to assign clear borders to
these domains as independent units. The extensive overall conservation
can be viewed as five distinct regions of homology to the hPNKP sequence.
Regions 1 and 2 correspond to the 3'-phosphatase domain (Fig.
3), best understood by comparison with
the bacterial histidinol phosphatase domain, a homology that was
revealed by a more sensitive iterative Psi-BLAST search. This domain
catalyzes one step in histidine biosynthesis and is typified by
one portion of the bifunctional Escherichia coli HisB
(his7) protein. Most broadly, HisB-type histidinol
phosphatases and hPNKP (16, 17) are part of a large superfamily of
proteins containing the L-2-halo-acid dehalogenase fold
(33). These proteins catalyze a wide variety of hydrolytic reactions
via a covalent substrate-enzyme intermediate. More restrictively, HisB
is a member of the "DDDD" superfamily of phosphohydrolases, so
named due to the presence of four invariant aspartates (34). We
have labeled the overlapping amino acid motifs that define these
domains as DDDD motifs 1-4 in Fig. 3. Their presence in all
putative PNK-related 3'-phosphatases, including YMR156C, very strongly
indicates that these proteins form a covalent bond with their substrate
via the first aspartate of DDDD motif 1.
Two additional conserved non-DDDD motifs were evident in the putative
eukaryotic 3'-phosphatases (called motifs A and B) (Fig. 3). Motif B,
the more highly conserved, has the consensus sequence SX2DX2FAX6FXTPEX2F.
It is entirely absent in HisB, but present in YMR156C, and likely
serves in determining substrate recognition. Motifs A and B, as well as
other characteristic amino acids in the DDDD motifs (Fig. 3), thus
appear to provide a signature for identifying 3'-phosphatases. For
example, AcNPV-1 has been called Ac-HisP because the presence of HisB
motifs suggested that it was a histidinol phosphatase (see
GenBankTM accession number AAA66663). In fact, this protein
bears the more extended homology typical of 3'-phosphatases. Also, our alignment in motif 3, in contrast to that previously described (35),
contains the high-scoring 3'-phosphatase sequence
RX5MW in T4 PNK, even though this places a
threonine at the first X instead of the nearly invariant
halo-acid dehalogenase domain lysine.
Regions 3-5 correspond to the 5'-kinase domain (Fig.
4). Region 3, which includes the Walker A
motif, is strikingly conserved at non-Walker positions from
bacteriophage T4 to human, as previously described (16, 17).
This allowed T4 PNK, but not any S. cerevisiae kinases, to
be returned by the Psi-BLAST search. Region 4 is primarily responsible
for delineating what appears to be two subtypes of eukaryotic
5'-kinases. Most notably, the Dd-2 (D. discoideum gene) and
At-2 genes possess the tetrapeptide DRCN, compared with the conserved DNTN sequence in the other eukaryotic kinases.
The similarity of the former to the DRNN sequence in yeast
Trl1p might suggest that these proteins are involved in metabolism of
RNA rather than DNA. Region 5 contains a very highly conserved EGF
tripeptide specific to the eukaryotic proteins.
Subsets of PNK-related proteins showed additional homology outside of
Regions 1-5 to each other or to non-PNK proteins, further extending
the observed modularity. Of particular interest is a weak homology of
At-1 to both poly(ADP-ribose) polymerase and DNA ligase III. Both of
these proteins interact with XRCC1 and assist in BER/single-strand
break repair (36), suggesting a similar role for At-1 and this family
of proteins more generally. Indeed, hPNKP has recently been shown to
interact structurally and functionally with XRCC1 (19).
S. mikatae YMR156C Lacks a Fused 5'-Kinase Domain--
Based on
the above, we hypothesized that YMR156C would be functional as a
3'-phosphatase despite lacking the 5'-kinase domain. Nonetheless, it
was essential to exclude the alternative possibility that YMR156C is
simply a rearrangement artifact of the sequenced S. cerevisiae strain S288C or perhaps of laboratory strains more generally. We therefore sequenced the orthologous gene from the wild-type yeast species S. mikatae (strain 1815), a member
of the S. cerevisiae sensu stricto group (20). The S. mikatae locus exhibited synteny with S. cerevisiae
chromosome 13 that extended to the centromere-proximal and distal ORFs.
The encoded 245-amino acid protein was 75% identical to S. cerevisiae YMR156C and also possessed the six DDDD and
3'-phosphatase motifs described above, with the non-conservative
substitutions all clustered in the transitions between these motifs
(data not shown). Most importantly, the S. mikatae ORF ended
immediately after 3'-phosphatase motif B, so this species also clearly
lacks a fused kinase domain. It is thus highly likely that YMR156C
evolved to support a specific function in budding yeast despite lacking
the 5'-kinase domain.
YMR156C Encodes a Double-stranded DNA 3'-Phosphatase--
Although
YMR156C possesses all of the putative 3'-phosphatase motifs, it is the
most divergent among the eukaryotic species as determined by
calculation of phylogenetic distances (data not shown). As a result,
alternative phosphatase-related functions could not be ruled out based
on sequence alone. Indeed, prior to this work, the only described
function for YMR156C was that forced overexpression reduced cellular
sensitivity to ketoconazole, a non-phosphorylated antifungal agent that
acts by inhibiting ergosterol biosynthesis (37). To directly assess its
biochemical properties, YMR156C was overexpressed in yeast as a GST
fusion protein and purified. As shown in Fig.
5B (lane 2), the
purified GST-YMR156C fraction contained no other bands visible in
Coomassie Blue-stained gels as candidate 5'-kinase partners. The
protein catalyzed the efficient removal of a 3'-phosphate (Fig.
5C, lane 3) from a nicked oligonucleotide
substrate (Fig. 5A; similar to Ref. 38). Importantly, there
was no loss of the 5'-32P label, thus demonstrating the
3'-specificity of the phosphatase. As a control, no 3'-phosphatase
activity was detected when non-recombinant GST was purified in parallel
and similarly tested (Fig. 5C, lane 4).
As expected from the sequence alignment, repeated examination of the
GST-YMR156C fusion protein did not reveal an associated 5'-kinase
activity (see Fig. 7D, lane 3; and data
not shown). Thus, we conclude that YMR156C acts exclusively as a
3'-phosphatase and have designated the gene TPP1
(three prime
phosphatase-1). These results further indicate
that there is not a 5'-kinase that is tightly associated and copurifies
with Tpp1p. Given the sensitivity of the assays and amount of fusion
protein used, it is not unreasonable to expect to have
observed a copurifying activity even in the face of GST-Tpp1p
overexpression. This is supported by our observation that an
array-based two-hybrid screen (39) did not identify any
P-loop-containing Tpp1p-interacting proteins (data not shown).
Examination of the conditional dependence of GST-Tpp1p 3'-phosphatase
activity revealed the following. Activity was maximal at 100 mM NaCl, whereas salt concentrations above 200 mM were inhibitory (Fig. 5D). The enzyme was
active over a broad pH range (pH 6-9; data not shown) and displayed an
absolute requirement for a metal cofactor. Activity was optimal at 10 mM MgCl2 (Fig. 5E), but the enzyme
was similarly active in buffer containing 1 mM
MnCl2 (Fig. 5F). Other divalent cations,
including Ca2+, Ni2+, Co2+, and
Zn2+, could not support enzyme activity (data not shown).
Utilization of different DNA substrates revealed that GST-Tpp1p removed
3'-phosphates from nicks and single-nucleotide gaps with equal
efficiency (Fig. 6). Although active at
blunt ends, the enzyme was inactive on single-stranded
oligonucleotides, further demonstrating that it is a structure-specific
DNA 3'-phosphatase. Thus, it is highly likely that Tpp1p acts in the
repair of damaged DNA. At present, it is unclear how this pattern of
biochemical activity might explain the observed suppression of
ketoconazole resistance by TPP1.
Removal of 3'-Phosphates and 3'-Phosphoryl-terminated Nucleotides
by Yeast Cell-free Extracts--
Crude whole cell extracts were next
tested to assess the constitutive levels of Tpp1p-dependent
and -independent 3'-phosphatase activities in yeast cells. It has been
previously demonstrated that Apn1p, the major apurinic-apyrimidinic
endonuclease/3'-diesterase in S. cerevisiae, can remove
3'-phosphates (13). We therefore anticipated that Apn1p activity would
substantially compete with Tpp1p in this experiment and so tested
extracts prepared from wild-type as well as isogenic tpp1,
apn1, and tpp1 apn1 yeast cells. Wild-type
extract resulted in the formation of the expected 3'-dephosphorylated
22-mer oligonucleotide product as well as an unexpected 21-mer product
corresponding to removal of the entire 3'-nucleotide at the nick (Fig.
7A, lane 3).
Again unexpectedly, nucleotide removal was not observed with either of
the extracts lacking Apn1p (i.e. apn1 and
tpp1 apn1) (lanes 5 and 6,
respectively). These data suggest that Apn1p itself is able to remove
both a 3'-phosphate and a nucleotide at a nick, a hypothesis that has been validated in a separate
study.2 Of primary
interest here, 3'-phosphate removal, evidenced by disappearance of the
substrate, proceeded efficiently in both the tpp1 and
apn1 mutant extracts (lanes 4 and 5,
respectively), but was completely absent in the tpp1 apn1
mutant extract (lane 6). Comparison of the apn1
and tpp1 apn1 extracts (lanes 5 and 6,
respectively) specifically demonstrated that TPP1
contributes an abundant 3'-phosphatase activity.
Uncoupling of 3'-Phosphatase and 5'-Kinase Activities in
Saccharomyces as Compared with Schizosaccharomyces
Extracts--
Finally, crude extracts of S. cerevisiae,
S. mikatae, and S. pombe were prepared and
assayed using nicked oligonucleotide substrates to compare the relative
levels of 5'-kinase and 3'-phosphatase activities. As predicted from
the above results, all extracts possessed a 3'-phosphatase activity,
although its level was considerably lower in the S. pombe
extract (Fig. 7B). In contrast, but again predicted by the
homology searches, only the S. pombe extract showed a
detectable 5'-kinase (Fig. 7D, lanes 4-7) in an
assay that depends on conversion of the nick to a ligatable form by 5'-phosphorylation (Fig. 7C) (similar to Ref. 38).
Importantly, the appearance of the 47-mer product was not the result of
displacement polymerization because it was dependent on addition of T4
DNA ligase (data not shown). In addition to the S. pombe
5'-kinase, these assay conditions also detected T4 PNK activity when it
was added to the Saccharomyces extracts (Fig. 7D,
lanes 9-12). We conclude that, in contrast to their
abundant 3'-phosphatase, Saccharomyces yeast cells do not
constitutively express a detectable DNA 5'-kinase and likely lack one
entirely, despite the initial observations with the plasmid
transformation NHEJ assay.
Given the potentially disastrous consequences of persistently
blocked DNA termini, it is not surprising that multiple mechanisms have
evolved to deal with these lesions. Blocking lesions can be resolved by
strand degradation mechanisms such that nucleotides must be
resynthesized in excess of those that have been directly damaged.
Alternatively, direct reversal can occur by removal of only the
fragmented nucleotide to leave the next available 3'-hydroxyl or
5'-phosphate or by simple rephosphorylation of a 5'-hydroxyl. The
recently described bifunctional hPNKP appears to have evolved to
directly reverse both of the reciprocal lesions created by damage that
results in a misplaced phosphate (16, 17). The results presented here
reinforce that this is not the only mechanism of dealing with such
lesions, however, and that coupling is not obligatory.
Ultimately, it must be assumed that the PNK domains maintained through
evolution reflect the biology of lesion repair in a given organism
(Figs. 2-4 and Table I). Both 5'-kinase and 3'-phosphatase domains are
present in metazoans and fission yeast in a linked fashion, indeed
suggesting the potential for efficient reversal of coupled 3'-phosphate
and 5'-hydroxyl lesions. In at least some plants, this gene linkage was
lost or never established. Importantly, it is as yet uncertain whether
all putative 5'-kinase genes listed in Table I will function in DNA
metabolism (note especially At-2, the only 5'-kinase gene in A. thaliana), and so these plants may represent 3'-phosphatase-only
species from the standpoint of DNA repair.
Budding yeast stands out as the clearest example of a eukaryotic branch
that possesses only a 3'-phosphatase. Caution is indicated, however,
given that this conclusion is based on essentially negative results,
namely that no PNK 5'-kinase homolog is revealed by comparison searching; no 5'-kinase is detected in the same crude extracts that
display abundant 3'-phosphatase activity (Fig. 7); and no apparent
5'-kinase has been identified as interacting with Tpp1p in
copurification (Figs. 5 and 7) and two-hybrid approaches (data not
shown). It is possible that another gene provides a cryptic unlinked
5'-kinase activity. For example, Trl1p possesses coupled 5'-kinase and
ligase activities whose biological role is in tRNA processing (29, 40).
The inviability of trl1 mutants prevents a simple testing of
the hypothesis that there is a functional overlap between tRNA
processing and DNA repair, but this seems unlikely, especially since
Trl1p-dependent DNA 5'-kinase activity was not detected in
the crude extracts. An alternative search approach based on pattern
matching identified two ORFs that contain the PNK-like P-loop consensus
sequence (FYWVLIMC)GXP(GAS)XGKS(TSHY)(FYWVLIMC) (Fig. 4), but that have no clear function based on literature or
homology searching (YFR007W and YOR262W). These ORFs have no similarity
to hPNKP beyond the P-loop, however; and it is difficult to reconcile
why evolution would have resulted in a clearly conserved 3'-phosphatase
and yet a highly divergent 5'-kinase. Even if these or another
ORF is ultimately shown to provide cryptic DNA 5'-kinase activity, our
results nonetheless demonstrate that there has been both a biochemical
and structural uncoupling of 5'-kinase and 3'-phosphatase functions in
Saccharomyces yeast.
It must be emphasized that no eubacterial or archaebacterial
proteins were uncovered in sequence comparisons or literature searches
as containing either 3'-phosphatase or 5'-kinase domains, despite the
large number of complete genomic sequences that are available. It is
clear that a common DDDD-type phosphatase domain precursor was
transmitted to both eukaryotes and bacteria, which, in bacteria, became
histidinol phosphatase (34). In many bacteria, this was fused to create
a bifunctional enzyme containing the additional activity
imidazole-glycerophosphate dehydratase (e.g. E. coli HisB). Interestingly, the yeast histidinol phosphatase His2p
evolved from a structurally distinct phosphatase precursor of the
"PHP" type that is also found in DNA polymerases in all branches of
life (41). A tremendous modularity is thus apparent in the evolution of
phosphatase domains, with the ancient precursor types being variably
adapted to fulfill critical cellular functions and, in some cases,
fused to novel domains. In contrast, no clear bacterial counterpart to
the 5'-kinase domain was made evident in the Psi-BLAST search, beyond
the ubiquitous Walker A motif. This pattern strongly suggests that the
PNK 3'-phosphatase domain is evolutionarily older, whereas the DNA
5'-kinase arose independently and was fused only after the divergence
of budding yeasts (Fig. 8). An
alternative hypothesis would be that the 5'-kinase and 3'-phosphatase
domains were only uncommonly split and/or lost in some eukaryotic
lineages from a common coupled PNK progenitor. Redundant mechanisms for
5'-blocking lesion resolution might have simply allowed the 5'-kinase
domain to be lost without detriment as a result of a rare deletion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(2, 3) or certain glycosylases (4, 5) or in
long-patch BER by flap excision and resynthesis (6). The extensive
5'-resection that occurs in the first steps of recombination also
likely removes blocking lesions at double-strand breaks (7, 8). Common
blocking lesions on 3' termini include phosphates,
,
-unsaturated
aldehydes resulting from
-elimination reactions (9), and
phosphoglycolate moieties that are the primary product of bleomycin
action (10). The most potent 3'-processing enzymes are the
apurinic-apyrimidinic endonucleases, which, in addition to cleaving
strands at abasic sites, possess 3'-diesterase activities capable of
removing most nucleotide fragments (11-13). It is again possible that
3'-lesions might be resolved by a more extensive degradation during
recombination, e.g. by RecBCD (8).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, leu1-32, ura4-048,
ade6, can1-1) was obtained from Dr. Dennis Thiele. The S. cerevisiae strains used in the plasmid assay
were the wild-type strain YW389 (MAT
,
ade2
0, his3
200,
leu2, lys2-801, trp1
63,
ura3
0) and its isogenic derivatives YW513
(dnl4-K282R) and YW514 (pol4-D367E). The
chromosomal point mutations in these strains were constructed by the
de novo mutation strategy described by Erdeniz et
al. (21). Strains were verified by diagnostic PCR, allele
sequencing, and documenting their deficiencies in previously described
DNL4- and POL4-dependent
non-homologous end joining (NHEJ) assays (22, 23). The S. cerevisiae strains used for biochemical assays were the wild-type
strain YW465 and its isogenic derivatives YW573
(tpp1
::MET15), YW605
(apn1
::HIS3), and YW619
(tpp1
::MET15,
apn1
::HIS3). The deletions in these strains were constructed by PCR-mediated gene replacement and verified
by PCR as described (24). Single mutants were constructed directly in
YW465 and YW619 by disruption of APN1 in YW573.
-32P]ATP using 3'-phosphatase-free polynucleotide
kinase (Roche Molecular Biochemicals). Final substrates were prepared
by annealing labeled oligonucleotides to a 2-fold molar excess of the
required unlabeled strands by heating to 90 °C, followed by slow
cooling. Standard assays of 3'-phosphatase activity contained 50 fmol
of DNA substrate and 10 fmol of GST-Tpp1p or 1 µg of crude cellular
protein in a reaction volume of 10 µl such that the final buffer was
50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, and
50 µg/ml bovine serum albumin. Kinase assays were similar, except
they used 1 pmol of GST-Tpp1p and 25 mM NaCl and also
included 100 units of T4 DNA ligase (New England Biolabs Inc.), 1 mM ATP, and, where indicated, 5 units of T4 PNK (New
England Biolabs Inc.) as an internal control. After incubation at
30 °C for 10 min, formamide/EDTA loading buffer was added, and
samples were electrophoresed on 7 M urea and 12%
polyacrylamide gels, followed by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Efficient POL4-independent
NHEJ of transformed plasmids bearing dephosphorylated
BglII ends. A, the schematic shows
potential pathways for processing of dephosphorylated BglII
ends during NHEJ. Direct reversal of the 5'-hydroxyl by PNK or
nucleotide removal and resynthesis through the combined action of a
nuclease and polymerase (nuc + pol) might each occur either
before or after end annealing. B, after normalization to
parallel transformations with uncut plasmid to correct for small
differences in plasmid uptake, ligation-competent and dephosphorylated
plasmid transformation efficiencies from wild-type (wt),
dnl4-K282R (dnl4), and pol4-D367E
(pol4) yeast strains were plotted relative to the wild
type/non-calf intestinal alkaline phosphatase (CIP)-treated
combination. White circles represent white (ADE2)
colonies and therefore simple religation of the BglII ends,
whereas black circles represent red (ade2)
colonies. Results are from four independent transformation experiments
performed on three different days (mean ± S.D.).
Species distribution of 3'-phosphatase and 5'-kinase domains identified
by homology and literature searches
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Fig. 2.
PNK multiple sequence alignment. This
schematic depicts the alignment of a representative subset of PNK
sequences. Tall and short boxes represent aligned
and unaligned ("unlinked") regions, respectively; blank
regions represent gaps; and diagonal lines indicate
that sequences were deleted from the figure to conserve space.
Gene/species designations are abbreviated as in Table I, with a
hyphenated number corresponding to the gene number. Sequence Dd-1 is
shown twice because the expressed sequence tags for this single
cDNA could not be assembled into an unbroken contig. AcNPV-2 and T4
PNK are shown twice because the order of the 3'-phosphatase (3'
Pase) and 5'-kinase domains are inverted relative to the other
genes, and so a simple linear alignment is not possible. Homologies to
non-PNK sequences are indicated above the relevant regions.
PARP, poly(ADP-ribose) polymerase; LIG3, DNA
ligase III; YOR258W, yeast hypothetical protein.
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Fig. 3.
Structural motifs of the PNK 3'-phosphatase
domain. Selected sequence regions from the complete
alignment are shown that correspond to the conserved motifs of the
3'-phosphatase (3' Pase) domain. Positions identical in all
species from human to bacteriophage T4 are highlighted as
yellow-on-red. Positions identical or similar to a consensus
of 50% of the sequences are highlighted as yellow-on-blue
and white-on-green, respectively. Two PNK phosphatase
subtypes evident in the alignment are separated by a horizontal
line. Motifs discussed under "Results" are indicated,
with the corresponding E. coli HisB sequence shown above the
alignment where relevant. Asterisks mark amino acid
positions in motifs 1-4 that, in addition to motifs A and B, provide
strong discrimination between the histidinol and DNA 3'-phosphatase
families. Gene/species designations are abbreviated as in Table I, with
a hyphenated number corresponding to the gene number.
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Fig. 4.
Structural motifs of the PNK 5'-kinase
domain. Selected sequence regions from the complete alignment are
shown that correspond to the conserved motifs of the 5'-kinase domain.
The depiction is the same as in Fig. 3. Additionally, the Walker A box
is indicated along with the PNK-type P-loop consensus sequence
(h, hydrophobic; x, any amino acid).
Asterisks mark amino acid positions that delineate the three
apparent 5'-kinase subtypes, which are separated by horizontal
lines. Gene/species designations are abbreviated as in Table I,
with a hyphenated number corresponding to the gene number.
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Fig. 5.
S. cerevisiae ORF YMR156C
(TPP1) encodes a DNA 3'-phosphatase.
A, a nicked oligonucleotide substrate was used to detect
3'-phosphatase activity. Removal of the 3'-phosphate from the
32P-labeled 22-mer (indicated by an asterisk)
leads to decreased mobility in a sequencing gel. nt,
nucleotides. B, GST-Tpp1p (i.e. GST-YMR156C)
(lane 2) and non-recombinant GST (lane
3) proteins were purified from yeast, run on a 10%
SDS-polyacrylamide gel, and stained with Coomassie Blue. Relative
molecular masses are indicated by standards (M,
lane 1). C, the 5'-end-labeled
substrate (50 fmol) was incubated with 10 fmol of GST-Tpp1p (lane
3) or GST protein (lane 4) for 10 min at 30 °C and
analyzed on a sequencing gel, followed by autoradiography. Lanes
1 and 2 contain the corresponding 22-mer
oligonucleotide synthesized without and with a 3'-phosphate,
respectively. These marker lanes are repeated in D and Fig.
7A. D, salt dependence of GST-Tpp1p activity was
assayed as described for C, except the NaCl concentration
was varied as shown. E and F, divalent cation
dependence of GST-Tpp1p activity was assayed as described for
C, except the MgCl2 and MnCl2
concentrations were varied as shown. In F, the
triangles indicate decreasing amounts of GST-Tpp1p (from
left to right: 10, 4, 2, 1.3, and 1 fmol).
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Fig. 6.
Tpp1p is active on double-stranded DNA, but
not single-stranded DNA. Oligonucleotides containing 3'-phosphates
in different configurations were tested for their ability to act as
substrates for varying amounts of GST-Tpp1p, as in the left
panel of Fig. 5F. Substrates are schematized above the
relevant lanes and represent 3'-phosphates at a nick, single-nucleotide
gap, single-stranded DNA end, extended 5'-overhang, and double-stranded
DNA blunt end (from left to right and from top to bottom). All were
approximately equally efficient substrates, except single-stranded DNA,
which was not dephosphorylated by even the highest amount of
GST-Tpp1p.
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Fig. 7.
Uncoupling of 3'-phosphatase and 5'-kinase
activities in cell-free extracts of Saccharomyces
yeast. A, glass bead extracts from wild-type
(wt), tpp1, apn1, and tpp1
apn1 strains (lanes 3-6, respectively) were tested for
3'-phosphatase activity as described in the legend to Fig. 5. The
extracts resulted variably in removal of the 3'-phosphate or
3'-terminal nucleotide, yielding 22- and 21-mer oligonucleotide
products, respectively. B, GST-Tpp1p and glass bead extracts
from S. cerevisiae (Sc), S. mikatae
(Sm), and S. pombe (Sp) were used in
3'-phosphatase assays as described for A to provide an
activity comparison with D. C, a nicked
oligonucleotide substrate was used to detect 5'-kinase activity.
Following phosphorylation of the 5' terminus at the nick, ligation by
exogenously added T4 DNA ligase led to an increase in size of the
labeled strand (indicated by an asterisk) from 22 to 47 nucleotides (nt). D, T4 PNK (5 units) and the
same protein samples used in B were incubated with the
5'-kinase substrate (50 fmol) in the presence of 100 units of T4 DNA
ligase at 30 °C for 10 min and analyzed on a sequencing gel,
followed by autoradiography. S. cerevisiae apn1
mutant extracts were used to rule out inhibition by the Apn1p nuclease.
Also, T4 PNK was included in a duplicate of all reactions to
demonstrate that the extracts did not prevent detection of 5'-kinase
activity (note that nicked DNA is a poor substrate for T4 PNK).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (12K):
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Fig. 8.
Modular evolution of 3'-phosphatase and
histidinol phosphatase domains. The diagram illustrates the
idealized relationships between the PHP- and DDDD-type
phosphotransferase superfamilies and eukaryotic and prokaryotic DNA
3'-phosphatases (3' Pase) and histidinol phosphatases
(H'ol Pase). See "Discussion" for details.
Dashed lines indicate less favored possibilities. Many other
putative relationships are not illustrated, notably the derivation of
the 5'-kinase domain from its precursors as well as its presumed
duplication and subsequent fusion with still other domains in the
derivation of tRNA ligase, etc. IGP-deHase,
imidazole-glycerophosphate dehydratase.
As sequences are generated from still more budding yeasts as a result
of comparative genomic efforts (42), it should be possible to determine
whether the 5'-kinase domain is indeed absent from this lineage. At
present, we favor this hypothesis in part because it is less cumbersome
than a cycle of fusion and loss. Moreover, the alternative hypothesis
presupposes that coupling of 5'-kinase and 3'-phosphatase functions is
inherently advantageous in most species. In fact, the fragmentation of
bases generates a great diversity of blocking lesions, of which coupled
3'-phosphates and 5'-hydroxyls are only a subset. For example,
3'-phosphate generation by Fpg entails sequential - and
-elimination reactions, leaving 3'- and 5'-phosphates at a single
nucleotide gap (5). Also, cleavage of topoisomerase I-DNA covalent
complexes by tyrosyl-DNA phosphodiesterase (Tdp1p in S. cerevisiae) is thought to occur after replication fork collapse
caused by collision-induced double-strand breaks, and so the resulting
3'-phosphate and 5'-hydroxyl would no longer be physically linked in a
single-strand nick (43). We suggest that the overall pattern of PNK
conservation reflects this lack of coupling. The greatest question is
whether domain linkage in fission yeast and metazoans underlies a true
increase in nick repair efficiency, fulfills a biological role specific to cells that divide by fission, or was more simply a way of ensuring co-inheritance once the domains each occupied a unique niche in nick repair.
In the case of 5'-blocking lesions, higher eukaryotes depend on DNA
polymerase lyase activity to remove 5'-deoxyribose phosphate lesions in the rate-limiting step in BER (2, 3). In contrast, the only
yeast
-like polymerase, Pol4p, is not required for BER (44, 45).
Instead, polymerization function is provided primarily by DNA
polymerase
(45, 46), presumably in conjunction with more extensive
5'-base loss in a long-patch repair. Similarly, budding yeast cells
process double-strand breaks almost exclusively by recombination
mechanisms that entail highly efficient 5'-degradation, whereas
end-preserving NHEJ plays a far greater role in higher eukaryotes (7,
27). We argue that this emphasis on more extensive 5'-degradation also
applies to 5'-hydroxyl lesions in S. cerevisiae. As a
result, the need for a 5'-kinase, just as for the
5'-deoxyribose-phosphate lyase, does not exist. The observed repair of
dephosphorylated plasmids would thus occur by base removal and
resynthesis, which, based on our results, must occur by a
Pol4p-independent pathway of end processing in NHEJ. Because NHEJ
entailing simple nucleotide gaps shows a minimal pol4
effect, this is perhaps not surprising (compare Fig. 1 and Ref.
22).
In the case of 3'-blocking lesions, further studies will be required to
determine the specific role(s) that TPP1 has been maintained
to fulfill. The observed specificity of this enzyme for 3'-phosphates
present in double-stranded DNA strongly suggests a role in genome
repair (Fig. 6). More specifically, the competitiveness of Tpp1p and
the promiscuous 3'-diesterase Apn1p observed using cell-free extracts
seems to foreshadow a complex interplay between multiple pathways of
3'-processing (Fig. 7). Results to be presented elsewhere indicate that
several DNA repair pathways display functional overlap between Tpp1p
and Apn1p.2 In contrast, bacteria apparently require only
the universally conserved 3'-diesterases (47). Why the seemingly
redundant Tpp1p/Apn1p enzyme combination was established and maintained
in budding yeast (and indeed, all eukaryotes characterized to date)
while the PNK 5'-kinase was not will be important issues to resolve.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Elissa Karathanasis for technical assistance on Fig. 1 and Peter Uetz (of the laboratory of Stan Fields) for performing the array-based two-hybrid analysis with YMR156C.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Pew Scholars Program in the Biomedical Sciences of the Pew Charitable Trusts (to T. E. W.) and by National Institutes of Health Postdoctoral Training Grant 5T32HL07157 (to J. R. V.).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.
To whom correspondence should be addressed: Dept. of Pathology,
University of Michigan Medical School, 1301 Catherine Rd., M4214
Medical Science I, P. O. Box 0602, Ann Arbor, MI 48109-0602. Tel.:
734-936-1887; Fax: 734-763-6476; E-mail: wilsonte@umich.edu.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M011075200
2 J. R. Vance and T. E. Wilson, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: BER, base excision repair; PNK, polynucleotide kinase; hPNKP, human polynucleotide kinase 3'-phosphatase; ORF, open reading frame; PCR, polymerase chain reaction; NHEJ, non-homologous end joining; AcNPV, A. californica nucleopolyhedrovirus; contig, group of overlapping clones; GST, glutathione S-transferase.
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