Characterization of SpPol4, a unique X-family DNA
polymerase in Schizosaccharomyces pombe
Sergio Gonza ´lez-Barrera, Arancha Sa ´nchez, Jose ´ F. Ruiz, Raquel Jua ´rez,
Angel J. Picher, Gloria Terrados, Paula Andrade and Luis Blanco*
Centro de Biologı ´a Molecular Severo Ochoa (CSIC-UAM), Universidad Auto ´noma, Madrid, Spain
Received May 25, 2005; Revised and Accepted August 3, 2005
protein coded by Schizosaccharomyces pombe
SPAC2F7.06c is a DNA polymerase (SpPol4) whose
biochemical properties resemble those of other
X family (PolX) members. Thus, this new PolX is
template-dependent, polymerizes in a distributive
and its preferred substrates are small gaps with a 50-
phosphate group. Similarly to Polm, SpPol4 can
incorporate a ribonucleotide (rNTP) into a primer
DNA. However, it is not responsible for the 1–2
rNTPs proposed to be present at the mating-type
Unlike Polm, SpPol4 lacks terminal deoxynucleotidyl-
transferase activity and realigns the primer terminus
to alternative template bases only under certain
sequence contexts and, therefore, it is less error-
prone than Polm. Nonetheless, the biochemical prop-
erties of this gap-filling DNA polymerase are suitable
for a possible role of SpPol4 in non-homologous
end-joining. Unexpectedly based on sequence ana-
lysis, SpPol4 hasdeoxyribose phosphatelyase activ-
ity like Polb and Poll, and unlike Polm, suggesting
also a role of this enzyme in base excision repair.
Therefore, SpPol4 is a unique enzyme whose enzym-
atic properties are hybrid of those described for
mammalian Polb, Poll and Polm.
Efficient DNA repair is essential to maintain genome stability
and cell viability (1,2). In spite of a variety of DNA repair
mechanisms, one common step is DNA synthesis, carried out
by specialized DNA polymerases. DNA polymerases are clas-
sified into four different groups according to their biochemical
properties and to the biological processes in which they are
involved. Among them, only family X DNA polymerases
(PolX) are devoted to DNA repair, being evolutionarily
conserved in prokaryotes, eukaryotes and archaea (3–5).
However, their number ranges from five members inmammals
[Polb, Poll, Polm, terminal deoxynucleotidyltransferase (TdT)
and Pols] to one member in yeasts, plants, and some bacteria
Arabidopsis thaliana (AthPolX), Bacillus subtilis (BsPolX)
and African swine fever virus (ASFVPolX). Interestingly,
twomodelorganisms, Caenorhabditis elegans and Drosophila
melanogaster, whose genomes
sequenced, have no putative PolX (3).
PolX enzymes most probably share a common modular
organization (Polb core) consisting of an 8 kDa domain and
a 31 kDa polymerization domain comprising ‘fingers’, ‘palm’
and ‘thumb’ subdomains. Such a structural organization has
been demonstrated for Polb (6,7), TdT (8), Poll (9,10) and
ASFVPolX (11,12). Unlike Polb, ASFVPolX, bacterial and
archaea PolX members, other family enzymes (Poll, Polm,
TdT and ScPol4) have an additional domain, the Brca1
C-terminal, named BRCT, which has been suggested to
take part in protein–protein and protein–DNA interactions
(13). Besides this BRCT domain, Poll AthPolX and ScPol4
have a proline/serine-rich region in their central part with a yet
unknown function (3).
Regarding their biochemical properties, all DNA poly-
merases from this family are single-subunit enzymes, lacking
the 30!50exonuclease activity and displaying very low pro-
cessivity during primer extension reactions [reviewed in (14)].
Polb, the paradigm of the PolX family, inserts nucleotides in
a template-dependent manner and is moderately accurate
(15,16). Its preference for small gaps with a 50-phosphate
group (17) and its deoxyribose phosphate (dRP) lyase activity
that relies on the 8 kDa domain (18) are properties consistent
with a role in base excision repair (BER), a major pathway
*To whom correspondence should be addressed. Tel: +34 91 497 8493; Fax: +34 91 497 4799; Email: firstname.lastname@example.org
Jose ´ F. Ruiz, Departamento de Gene ´tica, Facultad de Biologı ´a, Universidad de Sevilla, Spain
? The Author 2005. Published by Oxford University Press. All rights reserved.
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Nucleic Acids Research, 2005, Vol. 33, No. 15
involved in the repair of damaged nucleotides (19,20). This
multistep process is initiated with the removal of the modified
base by a specific DNA N-glycosylase yielding apurinic/
apyrimidinic (AP) sites. AP sites are recognized and incised
by an AP endonuclease leaving single strand breaks (SSBs)
the 50-dRPase activity of Polb (short-patch BER) or by the
combined action of a DNA polymerase (Polb, and Pole or
Pold) and the 50-flap endonuclease FEN1 (long-patch BER)
Poll has 32% amino acid identity to Polb and contains an
intrinsic dRP lyase activity that can substitute for Polb in BER
in vivo and in vitro (23,24). However, the high affinity of Poll
for deoxynucleotides (dNTPs) (37-fold over Polb) is consis-
tent with its possible involvement in DNA transactions occur-
ring under low cellular levels of dNTPs, i.e. in non-replicating
phases of the cell cycle (25). Similar to Polb, Poll inserts
dNTPs in a DNA template-dependent manner and is proces-
sive in small gaps containing a 50-phosphate group (25). In
addition, immunodepletion of nuclear extracts of HeLa cells
(26) and recent studies in which Poll associates with a Ku-
XRCC4–DNA ligase IV–DNA complex (27–29) suggest a
possible role for Poll in non-homologous end-joining (NHEJ).
Polm has 41% identity to TdT, a template-independent DNA
Pol X responsible for the N-addition during V(D)J recomb-
ination of the immunoglobulin genes and T-cellreceptor genes
(30,31). Polm-deficient mice are impaired in V(D)J recomb-
ination of the immunoglobulin k light chain (32), which is
initiated by an induced double strand break (DSB) that is
repaired by an NHEJ mechanism, similar to that employed
by other tissues to repair DSBs. However, unlike TdT, whose
expression pattern is restricted to lymphoid tissues, Polm is
expressed in additional tissues (33), suggesting a more general
role of Polm in DNA repair (34).
Polm behaves as an error-prone DNA polymerase, since it is
able to induce/accept dislocations of the template strand (35).
Unlike Polb and Poll, Polm is able to insert ribonucleotides
(rNTPs) to a DNA chain (36,37) and lacks dRP lyase activity
(24). Based on these properties and on the physical and func-
tional interactions with the Ku-XRCC4–DNA ligase IV–DNA
complex (38), it has been proposed that Polm functions in
NHEJ and V(D)J recombination by promoting microhomol-
ogy search and pairing activities (27,29). Polm is not a strictly
template-dependent DNA polymerase, since it has an intrinsic
terminal transferase activity (33) that probably plays a role
in microhomology-mediated NHEJ reactions (R. Jua ´rez,
J. F. Ruiz, S. A. Nick McElhinny, D. A. Ramsden and
L. Blanco, manuscript submitted).
In contrast to mammals, budding and fission yeasts have
only one DNA PolX enzyme (3). Whereas ScPol4 is
closely related to Poll (25% amino acid identity to the
Poll core), the putative DNA PolX from the fission yeast
Schizosaccharomyces pombe (SPAC2F7.06c) is more closely
related to Polm than to Poll (27% versus 24% identical core
residues, respectively). ScPol4 was the first DNA PolX shown
to play a role in NHEJ (39). In agreement with that, it has been
shown to have a direct interaction of the BRCT domain of
ScPol4 with the Lig4/Lif1 complex (40), and a physical
and functional interaction of Rad27 with both ScPol4 and
Dnl4/Lif1 (41). No functional data have been reported for
the putative DNA PolX (SPAC2F7.06c) from the fission
yeast S.pombe, a unicellular eukaryotic organism whose
properties closely resemble those of higher eukaryotic organ-
isms. For this reason, S.pombe is a good model system for the
analysis of gene products involved in DNA repair. Here, we
report the cloning, expression and biochemical characteriza-
tion of the SPAC2F7.06c gene product from S.pombe.
DNA polymerization properties and the presence of a dRP
lyase activity support a role of this DNA polymerase in
both NHEJ and BER reactions. In spite of the closer similarity
to Polm, this enzyme combines Polb, Polm and Poll properties,
and therefore, it should be more unambiguously referred to
MATERIALS AND METHODS
Strains and growth conditions
Cells were grown at 30?C in rich medium (YES; 0.5% yeast
extract, 3% glucose and supplemented with 200 mg/l of
leucine and uracil) or in minimal medium (EMM). Appropri-
ate amino acids and thiamine were added to EMM when
required to a final concentration of 200 mg/l and 25 mM,
respectively. Geneticin selection was performed using YES
medium containing100 mg/l
pol4D::KanMX strains, sp8 and sp10, were created from the
wild-type strains, sp7 (h- leu1-32 ura4D18) and sp968 (h90),
respectively, using the PCR-based method and the primers,
nucleotide sequences in boldface overlap to the KanMX
cassette of plasmid pFA6a-kanMX4 (42). The deletion was
confirmed by PCR using primers pol4.C (50-AGATCTGTT-
CAAAATGAAGATT-CTTGC-30) and pol4.D (50-CTGCA-
GAGTAATGTGGCGATCTTA-AGG-30) and by Southern
blot (data not shown).
G418 (Sigma). The
Nucleotides and proteins
[a-32P]dCTP and [a-32P]ddATP (3000 Ci/mmol) were pur-
chased from Amersham Biosciences. T4 polynucleotide
kinase, UDG and T4 DNA ligase were purchased from
New England Biolabs; TdT was obtained from Promega;
Fidelity were obtained from Roche; hAPE was a gift from
Dr S. H. Wilson (NIEHS, Research Triangle Park, NC).
Purified human Poll and Polm were obtained as described
unlabeleddNTPs and rNTPs,[g-32P]ATP,
Oligonucleotides, templates and substrates for
Invitrogen [P15, 50-TCTGTGCAGGTTCTT-30; P15 (C),
50-TCTGTGCAGGTTCTC-30; SP1C, 50-GATCACAGTGA-
GTAC-30; P6, 50-CTGCAGCTGATGCGCUGTACGGATC-
CCCGG-GTAC-30; T32 (A), 50-TGAAGTCCCTCTCGAC-
AAAGAACCTGCACAGA-30; T32 (C), 50-TGAAGTCCCT-
CTCGACCAAGAACCTGCACAGA-30; T32 (G), 50-TGAA-
G-TCCCTCTCGACGAAGAACCTGCACAGA-30; T32 (T),
DNA oligonucleotides wereobtainedfrom
Nucleic Acids Research, 2005, Vol. 33, No. 154763
TC-30; T18 (T), 50-ACTGGCCGTCGTTCTATTGTACT-
CACTGTGATC-30; T4, 50-GTACCCGGGGATCCGTACG-
TCT-30; DG5, 50-AACGACGGCCAGT-30; D16, 50-GTCGA-
GAGGGACTTCA-30; D15, 50-TCGAGAGGGACTTCA-30].
All the oligonucleotides were purified by 8 M urea–20%
PAGE. Oligonucleotides SP1C, P15 (C), P15, P6 and oli-
go(dT)15were 50-labeled with [g-32P]ATP and T4 polynu-
cleotide kinase. FordRP
oligonucleotide P6 was 30-labeled with [a-32P]ddATP and
TdT. Polymerase activity was evaluated by using synthetic
double-stranded oligonucleotides as substrates. These sub-
strates were prepared by annealing a 50-32P-end-labeled primer
to different oligonucleotides to generate open (P15/T32 or
SP1C/T18(T) and 1 or 2 nt gapped (P15/T32/D16; SP1C/
T13(C)/DG1 or P15/T32/D15) template/primer substrates in
the presence of 0.2 M NaCl and 60 mM Tris–HCl, pH 7.5. The
polymerization reactions were done in 12.5 ml of incubation
mixture containing 50 mM Tris–HCl, pH 7.5, 2 mM MgCl2
or 1 mM MnCl2, 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA,
different concentrations of the indicated dNTPs or rNTPs,
4 nM of 50-labeled substrate and the indicated concentrations
of purified hPolm, hPoll, SpPol4 or calf thymus TdT. After
incubation for 15 min at 30?C, reactions were stopped by
adding gel loading buffer [95% (v/v) formamide, 10 mM
EDTA, pH 8, 0.1% (w/v) xylene cyanol and 0.1% (w/v)
bromophenol blue]. Products were resolved and analyzed
by denaturing 8 M urea–20% PAGE and autoradiography.
Quantification was done in a Fujix BAS1000.
Cloning and purification of S.pombe DNA polymerase X
Cloning of the S.pombe SpPol4 gene was started from the
(SPAC2F7.06c), in the public database S.pombe/GeneDB
(http://www.genedb.org/) that codifies for a putative DNA
polymerase from the X family. Specific primers with restric-
tion sites (in boldface) in their 50ends SpPol4.50BgX
CTTT) were designed to amplify yeast genomic DNA. PCR
was performed with Taq Expand High Fidelity (Roche) as
follows: 35 cycles at 95?C for 30s, 50?C for 30s and 68?C
for 120s. The 1551 bp SpPol4 PCR product was cloned in
pGEM-T Easy (Promega) to generate plasmid pGEM-T
Easy::SpPol4, verified by sequencing, digested with BglII
and subcloned in the BamHI site of the expression vector
pDS473a, which allows the expression of recombinant pro-
teins as fusions with a glutathione S-transferase (GST)-tag, to
generate the yeast expression plasmid pDS473-SpPol4.
Expression of SpPol4 was carried out in the S.pombe wild-
type strain sp7 transformed with plasmid pDS473-SpPol4. A
10 liters culture was grown at 30?C for 18–20 h in EMM
supplemented with leucine (final OD600¼ 1). Subsequently,
the cultured cells were harvested at 4?C and washed with stop
buffer (150 mM NaCl, 50 mM NaF, 10 mM EDTA and 1 mM
NaN3, pH 8). The pelleted cells were weighted (10 g) and
frozen (?70?C). Just before purification, which was carried
out at 4?C, frozen cells were thawed on ice in the presence of
50 ml ice-cold lysis buffer [phosphate-buffered saline
(PBS), 50 mM NaF, 2 mM EDTA, pH 8, 1% NP-40, 1.3 mM
p-NH2-benzamidine, 1 mM phenylmethylsulfonyl fluoride
(PMSF) and 1 tablet of protease inhibitor cocktail; Roche]
and broken with a French Press (twice at 20000 psi). KCl
was added to the lysate upto a final concentration of 0.2 M.
Cell debris were separated from the soluble lysates by ultra-
centrifugation (50000 g for 1 h at 4?C in a Beckman JA-25.50
rotor). The protein from the soluble fraction was subjected to
affinity chromatography. Gluthathione–Sepharose 4B (1 ml)
of lysis buffer. Soluble lysate was then loaded at 3 ml/h flow
rate into the column. Afterwards, the column was extensively
washed with buffer IPP150 (PBS and 0.1% NP-40) and equi-
librated with native binding buffer (100 mM Tris–HCl, pH 8
and 100 mM NaCl). After several washing steps with native
binding buffer containing 20 mM gluthathione, polymerase-
containing fractions estimated by Coomassie blue staining
were collected, 2-fold diluted with buffer A (50 mM
Tris–HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA and 1 mM
DTT), and bound to a phosphocellulose column (1 ml) and
eluted with buffer A containing 500 mM NaCl. This fraction
contains highly purified GST-tagged SpPol4. Protein concen-
tration was estimated by densitometry of Coomassie blue-
stained 10% SDS–PAGE gels, using standards of known
concentration. Under these conditions, the yield was 26 mg
of purified GST-tagged SpPol4/g of S.pombe cells. This
purified final fraction, adjusted to 50% (v/v) glycerol and
supplemented with 0.1 mg/ml BSA, was stored at ?70?C.
Construction and purification of a
polymerization-deficient form of SpPol4
Site-directed mutations were introduced into pGEM-T
Easy::SpPol4 plasmid by using a PCR-based method
(QuickChange? Site-Directed Mutagenesis kit; Stratagene)
with the oligonucleotide 50-GCCTGTTGGAGCGGCCGTT-
GCTATGGTGTTGAGTCC-30and its reverse complementary
GCTCCAACAGGC-30for the double mutation D355A/
D357A. The plasmid pGEM-T Easy::SpPol4D355A/D357Agen-
erated was sequenced and a BglII fragment containing
sequence was subcloned in the
BamHI site of pDS473a. SpPol4D355A/D357Aprotein, which
has two of the three catalytic aspartates mutated to alanines,
was purified tohomogeneity as the wild-type SpPol4described
DNA polymerization on activated DNA
The incubation mixture contained, in 25 ml, 50 mM Tris–HCl,
pH 7.5, 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA, 13.2 nM
[a-32P]dCTP, 1 mM (dATP, dCTP, dGTP, dTTP), 1 mM
calf thymus DNA and 250 nM of the purified GST-tagged
SpPol4 or SpPol4D355A/D357A. After incubation for 30 min
at 37?C, the reactions were stopped by adding 10 mM TE/
0.1% SDS and the samples were filtered through Sephadex
G-50 spin columns in 10 mM TE/0.1% SDS. The excluded
volume, corresponding to the labeled DNA, was counted
(LiquidScintillationCounter;Pharmacia) and the
4764Nucleic Acids Research, 2005, Vol. 33, No. 15
polymerization activity of SpPol4 was calculated as the
amount of incorporated dCMP.
The incubation mixture, in 12.5 ml, contained 50 mM
Tris–HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT, 4% glycerol,
0.1 mg/ml BSA, 50 nM SpPol4 and 1.5 nM single-stranded
labeled P15 or P15/T32(C) hybrid. Reactions were incubated
at 30?C for 15 min and were stopped by adding denaturing
loading buffer. 30!50exonucleolysis, expected to produce a
degradation ladder of the labeled P15 primer, was analyzed by
8 M urea–20% PAGE and autoradiography.
Electrophoretic mobility shift assay (EMSA)
EMSA was performed using 1 [SP1C/T13(C)/DG1] and 5 nt
[SP1C/T18(T)/DG5] gapped molecules to analyze the inter-
action of SpPol4 and DNA. Gel mobility shift assays were
performed in a final volume of 12.5 ml containing 50 mM
Tris–HCl, pH 7.5, 0.1 mg/ml BSA, 1 mM DTT, 4% glycerol,
4 nM labeled DNA and different concentrations of SpPol4
(250, 600 and 1200 nM). Samples were incubated for
10 min at 30?C to allow the formation of enzyme–DNA
complexes. For competition analysis, 100 nM SpPol4 was
incubated with labeled 1 nt gapped 50-phosphate molecules
(4 nM) for 15 min at 30?C in a final volume of 25 ml. After the
complexes were formed, unlabeled 1 nt gapped 50-phosphate
molecules were added to the reaction mixture and incubated
for another 10 min at 30?C. After incubation, samples were
mixed with 3 ml of 30% glycerol and resolved by native gel
electrophoresis on a 4% polyacrylamide gel (80:1 monomer/
bis). After autoradiography, DNA polymerase–DNA com-
plexes were detected as mobility retardation in the migration
position of the labeled free DNA. Quantification of the com-
petition experiments was done in a Fujix BAS1000. The
amount of the labeled GAP1-P forming SpPol4::GAP1-P
complexes was calculated by normalizing the radioactive
signal in the shifted band to the total radioactivity.
dRP lyase activity assay
As a substrate, the 30end 34mer-labeled P6 oligonucleotide
was annealed to the 34mer T4 oligonucleotide. This labeled
double-stranded substrate (500 nM) was treated with UDG
(100 nM) for 20 min at 37?C in buffer containing 50 mM
HEPES, pH 7.5, 20 mM KCl and 2 mM DTT to remove
the uracil. After incubation, the mixture was supplemented
with 10 mM MgCl2and 40 nM hAPE for 10 min at 37?C;
thus, generating the substrate for dRP lyase activity. Reaction
mixtures (25 ml) containing 50 mM HEPES, pH 7.5, 10 mM
MgCl2, 20 mM KCl, 2 mM DTT, 70 nM concentration of the
treated substrate and different amounts of either SpPol4 (20,
60 and 120 nM), SpPol4D355A/D357A(20, 60 and 120 nM),
hPolm (70 nM) or hPoll (60 nM) were incubated at 37?C
for 20 min. After incubation, NaBH4was added to a final
concentration of 340 mM, and the reactions were kept for
20 min on ice. Stabilized (reduced) DNA products were
ethanol precipitated in the presence of 0.1 g/ml of tRNA,
resuspended in water and analyzed with 8 M urea–20%
PAGE and visualized by autoradiography.
In vitro reconstitution of BER
A 34mer double-stranded DNA substrate that contained a G
opposite uracil at position 16 was used. This molecule was
treated as described above for the dRP lyase activity assay to
generate the dRP-containing substrate. Reactions (25 ml) con-
taining 70 nM substrate, 50 mM HEPES, pH 7.5, 10 mM
MgCl2, 20 mM KCl, 2 mM DTT, 0.3 mM [a-32P]dCTP and
either SpPol4 (125 nM) or human Poll (245 nM) were incu-
bated for 20 min at 37?C. Later, each reaction was divided into
two halves. One was incubated with 1 mM ATP and 40 U T4
DNA ligase for 10 min at 37?C and the other was mock-
treated. Reactions were terminated by the addition of dena-
turing loading buffer, analyzed by 8 M urea–20% PAGE and
visualized by autoradiography.
Genomic DNA preparation and imprint analysis
Yeast chromosomal DNA was purified from a logarithmically
growing culture (10 ml; OD600?0.5–1). Cells were harvested
and resuspended in 200 ml breaking buffer (2% Triton X-100,
1% SDS, 100 mM NaCl, 10 mM Tris–HCl, pH 8 and 1 mM
EDTA), ?200 ml glass beads and 200 ml phenol/chloroform/
isoamyl alcohol (25:24:1). After 15 min vortexing at high
speed, 200 ml TE was added and the mixture was centrifuged
for 5 min at 11000g. The aqueous layer was transferred to
a clean tube and DNA was precipitated with ethanol. The
pellet was resuspended in TE. HindIII-digested DNA
(50 mg) was separated by agarose gel electrophoresis and
analyzed by Southern hybridization using a 1 kb32P-labeled
mat1-P PCR probe. The oligonucleotides sequences used for
the PCR were mat1-50, 50-AGAAGAGAGAGTAGTTGAAG-
30; and mat1P-30, 50-CCAATTCCTTCT-TGTATATGTTA-
TAC-30. The mat2 (6.3 kb) and mat3 (4.2 kb) bands result
from hybridization to the mat1 probe as they share cassette
homology. The imprint could be converted into a DSB during
standard methods of DNA purification (43,44) and visualized
by autoradiography. To determine the efficiency of mating-
type switching, a standard iodine staining assay was carried
out. Individual colonies were replicated onto EMM supple-
mented with the appropriate amino acids and then were grown
for 3 days at 22?C before being exposed to iodine vapors.
SpPol4 a unique X-family DNA polymerase in S.pombe
The S.pombe ORF SPAC2F7.06c coding for a putative 736
amino acids DNA polymerase from the X family was over-
produced in fission yeast wild-type cells and purified to near
homogeneity as described in Materials and Methods. The pro-
tein, expressed as a fusion protein containing a GST-tag at its
N-terminus, was purified by gluthathione–Sepharose affinity
and phosphocellulose chromatography. After the purification
steps a unique polypeptide was observed in the final fraction,
identified by Coomassie blue staining after SDS–PAGE ana-
lysis, migrating at the expected position for GST-tagged
SPAC2F7.06c (?84 kDa) (data not shown). The purified
fraction was assayed for DNA polymerase activity on an acti-
vated DNA. As expected, the purified fraction was able to
catalyze dNTP incorporation in the presence of either
Nucleic Acids Research, 2005, Vol. 33, No. 154765
Mg2+(2 · 10?5pmol/minng)orMn2+(9 · 10?6pmol/minng)
as activating divalent metal ions. As a control of specificity,
we carried out a parallel purification of a catalytically inactive
mutant (see Materials and Methods). In this case, no DNA
polymerization activity was detectable in the final fraction
(data not shown). Therefore, SPAC2F7.06c codifies for a
DNA polymerase that we refer to as SpPol4.
SpPol4 is a distributive polymerase that lacks
30! 50exonuclease activity
Processivity is a common feature of DNA polymerases
involved in extensive DNA synthesis (i.e. replicative poly-
merases), and relies on a tight DNA binding and an efficient
nucleotide insertion. Conversely, DNA repair enzymes fre-
quently display weaker DNA interactions and incorporate
nucleotides more slowly and consequently synthesize DNA
in a distributive mode. Distributive polymerization is a com-
mon feature of all DNA polymerases from the X family
(16,25,35,36,45,46). We assessed SpPol4 processivity on a
DNA template/primer substrate by analyzing the chain length
in Supplementary Figure 1, the length of the elongated primer
decreased with the enzyme/DNA substrate ratio in agreement
with a fully distributive polymerization pattern. This distribu-
tive behavior of SpPol4 is also maintained usingMn2+as metal
activator (data not shown). Therefore, we conclude that
SpPol4 is a distributive polymerase suited for short-stretch
Another distinctive feature of replicative DNA polymerases
is its proofreading 30!50exonuclease activity. Three con-
served amino acid motifs, named Exo I, Exo II and Exo III,
are responsible for the 30!50exonuclease active site of all
proofreading DNA polymerases (47). However, these motifs
are absent in the DNA polymerases from the X family includ-
ing SpPol4, suggesting that, as other PolX enzymes, SpPol4
has no proofreading activity. We tested this prediction using
either a single-stranded oligonucleotide or a template/primer
as substrates for 30!50exonucleolysis. Purified SpPol4 failed
to display any nucleolytic activity on both substrates after
15 min at 30?C (data not shown). This result demonstrates
that SpPol4 does not possess 30!50proofreading activity.
SpPol4 prefers small gaps with a 50-phosphate group
To further characterize the DNA polymerization activity pre-
sent in the purified SpPol4 fraction, we tested different in vitro
assay conditions using defined templated-DNA molecules in
the presence of Mg2+as a cofactor. The purified protein was
able to catalyze dNTP incorporation very efficiently either in a
template/primer (data not shown) or in the 1 nt gapped sub-
However, a significant increase (10-fold as an average) in the
polymerization capacity was observed when a phosphate
group was present at the 50-side of the gap compared with
the same gapped DNA molecule having a hydroxyl group at
the 50end of the gap (Figure 1B). Therefore, the DNA sub-
strate preference of SpPol4, small gaps with a 50-phosphate
group, is compatible with a role in DNA repair.
In Polb and Poll a 50-phosphate-dependent increase in pro-
of the N-terminal 8 kDa domain (5,7,9,48). Since SpPol4 also
contains an N-terminal 8 kDa domain, and is stimulated by the
presence of a 50-phosphate group in the 1 nt gapped substrate,
we tested if this stimulation was primarily due to differences
in the DNA-binding capacity, a step preceding dNTP binding
Figure 1. Gap-filling synthesis and substrate preferences of SpPol4. (A)
Scheme of the two types of DNA molecules used: 1 nt gap (GAP1-OH) and
1 nt gap with a 50-phosphate (GAP1-P). Labeled primers (asterisk) and the 50
in Materials and Methods using 125 nM SpPol4 and the indicated concentra-
tions of dGTP. Primer extension was analyzed by 8 M urea–PAGE and auto-
radiography. Mobility of the unextended primer (P) and the 1 nt (+1) extended
primers are indicated at the autoradiograph. (C) DNA-binding capacity of
SpPol4 to the 1 nt gapped molecules. EMSA was performed as described in
Materials and Methods using none (lanes 1 and 5), 250 nM (lanes 2 and 6),
600 nM (lanes 3 and 7) and 1.2 mM (lanes 4 and 8) SpPol4. Formation of
SpPol4:DNA complexes was resolved by native gel electrophoresis on a 4%
polyacrylamidegel (80:1; monomer/bis). Mobility ofthe free DNA (F) and the
SpPol4/DNA complexes (C) are indicated at the autoradiograph. (D) Competi-
tion analysis of SpPol4 bound to the GAP1-P. EMSA was performed as de-
gapped 50-phosphate (G1-P) or 50-hydroxyl (G1-OH) as unlabeled competitor
DNA. The plotted values represent the percentage of labeled GAP1-P that
remains bound to SpPol4 after competition, and are the mean of four indepen-
4766 Nucleic Acids Research, 2005, Vol. 33, No. 15
and catalysis. The formation of stable SpPol4/DNA com-
plexes, assessed by EMSA, required a lower enzyme concen-
tration when the 1 nt gapped DNA had a 50-phosphate
group (Figure 1C). Even more, the affinity of SpPol4 for
the 50-phosphate group is so strong that when the primer strand
is removed, SpPol4 still binds this molecule almost with the
same efficiency (data not shown).
To further analyze the stabilizing effect of the 50-phosphate
on DNA binding by SpPol4, competition analysis were carried
out as indicated in Materials and Methods. As expected, the
amount of SpPol4::GAP1-P (labeled) complexes formed in a
previous step progressively decreased when increasing
amounts of unlabeled competitor DNA (either having a
50-phosphate or not) were added, being greater the competition
with unlabeled GAP1-P (Figure 1D). However, even at 150-
fold molar excess of the 1 nt gapped 50-phosphate competitor,
the amount of SpPol4::GAP1-P labeled complexes was
reduced only by 5% in comparison with that in the absence
of the competitor (Figure 1D). Taken together, these results
clearly demonstrate that SpPol4 binds stably and preferentially
to a 50-phosphate containing DNA gap.
SpPol4 is a template-instructed polymerase with
preference for purines
We evaluated the ability of SpPol4 to discriminate among the
four dNTPs in order to catalyze template-directed DNA syn-
thesis. We used a set of 1 nt gapped template-primer substrates
with each of the four (X ¼ A, C, G or T) bases as template and
having a 50-phosphate flanking the gap (Figure 2A). For each
substrate, the four dNTPs, one complementary to the template
and the other three non-complementary, were assayed indi-
viduallyatdifferent concentrations.As showninFigure2B, on
the four 1 nt gapped substrates, SpPol4 preferentially incor-
porated the nucleotide complementary to the first template
base, even when non-complementary nucleotides were pro-
vided at a 100-fold higher concentration. Therefore, these
results suggest that SpPol4 is template-instructed, i.e. it per-
forms DNA synthesis following the Watson–Crick base pair-
ing rules. Interestingly, quantification of the efficiency of
incorporation of each complementary dNTP demonstrated a
strong imbalance in correct dNTP incorporation with prefer-
ence for purines: dG>>dA>dT>dC (Figure 2C).
Template dislocation and primer realignment
capacities of SpPol4
Human Polm behaves as an error-prone DNA polymerase,
since it is able to induce/accept dislocations of the template
strand (35), which is likely crucial for its NHEJ function. To
examine whether SpPol4 is similarly error-prone, we analyzed
DNA synthesis in some template sequence contexts that are
appropriate for evaluating: (i) slippage-mediated dislocation
(Figure 3A); (ii) dNTP selection-mediated dislocation
(Figure 3B); and (iii) primer realignment versus direct mis-
match extension (Figure 3C). Each dNTP was provided indi-
vidually to identify the opted mechanism for each DNA
polymerase. In the substrate with the dA-track repeat, normal
DNA synthesis would lead to dT incorporation, whereas slip-
page of the primer terminus (dT) to the next template base
(dA) would result in dG incorporation and a ?1 frameshift
DNA synthesis (see schematic representation in Figure 3A).
Unlike Polm, which clearly preferred to insert dG by a
slippage-mediated dislocation mechanism, SpPol4 and Poll
predominantly incorporated dT (Figure 3A). However, both
polymerases also incorporated dG and therefore, they can
misalign the template-primer to some extent. As shown in
Figure 3B, changing the third dA of the track for a dG reduces
the possibility of template slippage; therefore, DNA synthesis
is more restricted to the canonical incorporation of dC, like
SpPol4 and Poll do. Besides this normal DNA incorporation
event, only Polm was also able to insert the complementary
base (dG) to the position +2 in the template (dC). As reported
stabilized by the incoming dNTP (49).
To examine the mismatch extension capacity of SpPol4, we
performed a primer extension assay starting from a dA:dC
base pair mismatch(see
Figure 3C). Because SpPol4 and other PolX enzymes cannot
remove mismatched nucleotides at the primer 30end, only two
outcomes are possible: (i) direct mismatch extension inserting
dC; and (ii) primer terminus realignment inserting dG. As can
be seen in Figure 3C, Poll uses both alternatives almost
equally well. In agreement with its extreme error-proneness,
Polm is able to extend the mismatch with any of the four
dNTPs. Interestingly, SpPol4 has a more restricted behavior,
as it is only able to insert dG, indicating a significant primer
realignment capacity that enables this enzyme for a role
Figure 2. SpPol4 preferentially incorporates complementary nucleotides. (A)
(P) used in this assay, only differing in the templating base (X). The primer
strand was 50end labeled (asterisk). The oligonucleotides used to obtain these
assays using any of the four 1 nt gapped DNA substrates, and the four dNTPs
(dA, dC, dG and dT) individually provided. Reactions were carried out as
described in Materials and Methods using 125 nM SpPol4. Extension of the
(100 mM) dNTP was analyzed by 8 M urea–20% PAGE and autoradiography.
(C) Quantification of the complementary dNMP incorporation for the four 1 nt
gapped molecules at different dNTP concentrations. The values plotted repre-
mean of four independent experiments.
Nucleic Acids Research, 2005, Vol. 33, No. 154767
SpPol4 inserts both rNTPs and dNTPs with
the same efficiency
Polm and TdT have the striking ability to incorporate both
rNTPs and dNTPs to nucleic acid chains (36,37,45). This
unusual capacity mainly relies on a single glycine residue
that opens the ‘steric gate’, which is frequently closed by a
conserved aromatic residue present in Polb, Poll and in other
members of the PolX family of DNA-dependent DNA poly-
merases (Figure 4A) (36). Since SpPol4 also has a glycine
residue at this position (Gly434), it seemed very likely that
SpPol4 could incorporate rNTPs. By using the same four
1 nt gapped template–primer substrates as in Figure 2B, it
was shown that SpPol4 efficiently incorporates rNTPs on a
DNA primer strand (Figure 4B) with almost equal efficiency
as dNTPs and displaying the same preference pattern for
purines (compare Figures 4B and 2C).
Using a competition assay in which both sugars (ribose and
deoxyribose) are simultaneously provided, the sugar selectiv-
ity factor of a given DNA polymerase can be calculated
(36,45). Since the rNTP and the dNTP have different molecu-
lar weights, the +1extended primerscan beeasily separated by
gel electrophoresis and quantified. The sugar selectivity factor
is given as the ratio between the amounts of primer extended
with rNTP versus dNTP. Irrespective of the nature of the base,
the sugar selectivity factors obtained for SpPol4 were very
similar (0.74–0.91) and proximal to one, indicating a lack
of discrimination between rNTPs and dNTPs (Figure 4C).
These values are similar to those obtained for TdT (43) and
Polm (36) in untemplated and templated reactions, respec-
tively, being several orders of magnitude smaller than the
ones reported for other DNA polymerases (50–52).
SpPol4 is not required for imprinting at the mat1 locus
It is known for many years that mating-type switching in
S.pombe depends on a strand-specific imprint at the mat1
locus (53,54). The imprint was characterized either as an
alkali-labile modification or as a nick that could be converted
into a DSB during standard methods of DNA purification
(43,44). More recently, this imprint has been characterized
as an RNase-sensitive modification that consists of one or
two RNA residues incorporated into the mat1 locus (55).
Based on these results, it was tempting to speculate with
the possibility that SpPol4 might be responsible at the incorp-
oration of these one or two RNA residues. S.pombe genomic
DNA was prepared by a standard yeast extraction protocol
(see Materials and Methods), digested with HindIII and
analyzed by Southern hybridization using a 1 kb mat1-P as
a probe. The h90wild-type strain yielded the typical bands
of uncleaved (10.4 kb; mat1) and cleaved (5.4 kb; mat1*)
products, together with two other bands representing cross-
hybridization of the mat1-P probe with the mat2 (6.3 kb) and
mat3 (4.2 kb) loci (Supplementary Figure 2A and B, lane 2).
As expected, the smt-0 mutant strain (56), containing a dele-
tion of the cis-acting elements SAS1 and SAS2 and thus
preserving the integrity of the cleavage site sequence while
abolishing mating-type switching, did not produce the mat1*
band (57) (Supplementary Figure 2B, lane 1). However, the
the level obtained in the h90wild-type strain (Supplementary
Figure 2B, compare lanes 2 and 3). Moreover, direct measure-
ment of the mating-type switching efficiency by the iodine
staining assay (see Materials and Methods) was carried out.
The starch reaction with iodine vapors stains spore-containing
colonies black, whereas slow-switching mutants exhibit
streaky iodine staining patterns and colonies unable to switch
Figure 3. Characterization of SpPol4 template dislocation and primer realign-
ment capacities at gapped DNA intermediates. Schemes representing template
sequence contexts that are appropriate to evaluate slippage-mediated disloca-
tion (A), dNTP selection-mediated dislocation (B), and primer realignment
versus direct mismatch extension (C) are shown (see text for details). Labeled
primer (asterisk) and 50end phosphate group (P) are indicated. Transiently
Polymerization assays were carried out as described in Materials and Methods
individualdNTPeither at10 mM in all cases (C), orat a different concentration
for each DNA polymerase: Polm (500 nM), Poll (100 nM) and SpPol4 (5 mM)
(A and B). Primer extension was analyzed by 8 M urea–20% PAGE and auto-
(+2) extended primers are indicated at the autoradiographs.
4768Nucleic Acids Research, 2005, Vol. 33, No. 15
the mating type appear yellowish. As expected, iodine vapors
stained the smt-0 mutant colonies yellowish, whereas the h90
wild-type and the pol4D colonies were stained black (data not
shown). Therefore, the imprint at mat1 remains unaffected in
the absence of SpPol4 and if there were one or two RNA
residues in the DNA, the incorporation would be SpPol4-
SpPol4 has no TdT activity
Polm, as TdT, displays an intrinsic deoxynucleotidyltrans-
ferase activity, which is stronger in the presence of Mn2+as
cofactor (33). This enzymatic activity requires a region of the
palm subdomain called loop1, which is absent in SpPol4, Polb
and Poll (Supplementary Figure 3A). It has been demon-
strated that the deletion of this loop abolishes the TdT-like
activity of human Polm (29) (R. Jua ´rez, J. F. Ruiz, S. A. Nick
McElhinny, D. A. Ramsden and L. Blanco, manuscript sub-
mitted). TdT activity can only be unambiguously determined
by using single-stranded homopolymeric DNA as primer and
any of the three dNTPs not included in the primer sequence.
Thus, using a32P-labeled 15mer dT oligonucleotide, in the
presence of either Mg2+or Mn2+, SpPol4 displayed no TdT
activity (Supplementary Figure 3B).
SpPol4 has an intrinsic dRP lyase activity most
probably involved in BER
The amino acid residues that are critical for dRPase activity
are conserved in the 8 kDa domain of Polb and Poll (18,24)
and are indicated with dots in Figure 5A. Among them, a
specific lysine (Lys72in Polb and Lys312in Poll) is the cat-
alytic residue acting as a Schiff-base during b-elimination of
the dRP moiety. As shown in Figure 5A, Polm and SpPol4 lack
the catalytic lysine residue, and as it has been demonstrated for
Polm(24),it was probable thatSpPol4was devoid ofdRP lyase
activity. However, by using standard BER assays (18,24), here
we show that SpPol4 is able to remove a dRP group and
Figure 4. Lack of sugar discrimination by SpPol4. (A) Multiple amino acid alignmentof the amino acid region (connecting subdomains palm and thumb)probably
involved in sugar discrimination in the Pol X family. Numbers between slashes indicate the amino acid position relative to the N-terminus of each polymerase.
Invariant (in white letters over a black background) and conservative substitutions referred to SpPol4 residues are boxed in dark gray. The two amino acid residues
are: Hom.sa. (Homo sapiens), Sch.po. (S.pombe), and Sac.ce. (S.cerevisiae). (B) SpPol4 inserts rNTPs efficiently. The assay, essentially as described in Figure 2,
evaluatescomplementary rNMP incorporationinto the four1 nt gapped molecules,at different rNTP concentrations. The plottedvaluesrepresent the ratiobetween
the amount of extended versus total primers, and are the mean of four independent experiments. (C) Lack of sugar discrimination by SpPol4. The four 1 nt gapped
DNA substrates described in Figure 2, differing in the templating base (X) (a scheme is shown), were used. In this competition assay, each complementary duo of
nucleotides (rNTP + dNTP) was simultaneously provided at 100 nM. Reactions were carried out as described in Materials and Methods using 125 nM SpPol4.
is given as the ratio between the amount of rNTP- versus dNTP-extended primers.
Nucleic Acids Research, 2005, Vol. 33, No. 154769
promote single-patch BER in vitro. By adding UDG and hAPE
to a uracil-containing substrate, a nicked strand with a
3-hydroxyl and a 50-dRP is produced. The strand containing
the 50-dRP moiety, which is 30end labeled, migrates at the
expected position of an 18mer + dRP (Figure 5B). By using
increasing amounts of SpPol4, this product was converted to a
shorter product (18mer), indicating that SpPol4 as Poll, and
unlike Polm, has an intrinsic dRP lyase activity (Figure 5B).
Similarly, the SpPol4D355A/D357Apolymerization-deficient
mutant was proficient in dRP lyase activity similar to
the wild-type SpPol4 (Figure 5B). Unexpectedly from the
alignment shown in Figure 5A, SpPol4 has dRP lyase
activity though it lacks the lysine residue conserved in Polb
(Lys72) and Poll (Lys312) responsible for their dRP lyase
Removal of a dRP residue is an essential step for the com-
pletion of single nucleotide BER. Polb and Poll are able to
efficiently promote in vitro BER of a uracil-containing duplex
Figure 5. Characterization of SpPol4 dRP lyase activity and reconstitution of BER in vitro. (A) Multiple amino acid alignmentof the 8 kDa domainof SpPol4 with
other family X DNA polymerases members. Numbers between slashes indicate the amino acid position relative to the N-terminus of each polymerase. Residues
Lys312) or may be involvedin (ScPol4 Lys248) dRP lyase activity are in bold type. The position of SpPol4 Lys240that might substitute for HsPolb Lys72is indicated
used are: Hom.sa. (H.sapiens), Sch.po. (S.pombe), and Sac.ce. (S.cerevisiae). (B) In vitro analysis of the dRP lyase reaction. The scheme shows a 34mer double-
stranded oligonucleotide containing an uracil residue (at position 16) in the strand which is 30end labeled (asterisk). After treatment with UDG and hAPE, a dRP-
nM) was included as a negative control, lacking dRP lyase activity. (C) In vitro reconstitution of a BER reaction with SpPol4. A 34mer double-stranded
oligonucleotide containing an uracil residue at position 16 in one strand is treated with UDG (100 nM) and hAPE (40 nM) to release a dRP-containing nicked
electrophoresis and autoradiography: (i) a 16mer product generated by a single nucleotide insertion at the 30-hydroxyl end of the 50-incised AP site; (ii) a 34mer
product that corresponds to the complete repair of the DNA strand upon T4 DNA ligase action.
4770 Nucleic Acids Research, 2005, Vol. 33, No. 15
DNA in the presence of hUDG, hAPE and DNA ligase I
(24,59,60). As shown in Figure 5C, two main products
were observed in a human Poll-based reconstituted BER reac-
tion: a 16mer product generated by a single nucleotide inser-
tion (dCTP labeled) at the 30-hydroxyl end of the 50-incised AP
site, and a 34mer product that corresponds to the complete
repairofthe DNA strand upon DNA ligase action. Asshownin
Figure5C, bothSpPol4and hPollare able toproduce the same
16 and 34mer labeled products. Thus, SpPol4 is able to coord-
inate both the gap-filling and dRP excision steps of repair
preceding DNA ligase action. These data are consistent
with a role for SpPol4 in BER and predict that the dRP
lyase-containing enzyme SpPol4 could participate in BER
In mammals, there are five members belonging to the X family
of DNA polymerases: Polb, Poll, Polm, Pols and TdT. On the
contrary, yeasts, plants, and some bacteria and viruses have
only onePolX enzyme (3).
SPAC2F7.06c (GeneDB http://www.genedb.org/) predicted
a putative DNA PolX as inferred by sequence comparison
analysis. Based on the results presented here, it can be con-
cluded that SPAC2F7.06c does codify for a novel DNA poly-
merase belonging to the PolX family that would be adequately
designated as SpPol4.
The structural organization of SpPol4 as an N-terminal
BRCT domain followed by a C-terminal 39 kDa Polb-like
core domain resembles members of the family X DNA poly-
merases, such as Polm, TdT and Poll. Excluding the more
variable N-terminal BRCT domain (61), SpPol4 is more clo-
sely related to Polm (27% identical core residues), followed by
Poll (24% identity) and Polb (20% identity). It is worth noting
that budding yeast has also one DNA PolX (ScPol4) that,
unlike SpPol4, resembles Poll in its core domain (25% iden-
tity) and in its structural organization (62). Therefore, based
only on their coding sequences, it was speculated that SpPol4
is a yeast orthologue of Polm, whereas ScPol4 would be an
orthologue of Poll (3).
As summarized in Table 1, our biochemical analysis
demonstrated that SpPol4 is capable of carrying out DNA
synthesis in a template-dependent manner and exhibits low
processivity during primer extension. Such properties are
shared by all members of the eukaryotic X family, except
TdT [reviewed in (63)]. EMSAs showed that SpPol4 binds
to 50-phosphate gapped substrates better than to those with
S.pombe, the entry
a 50-hydroxyl, and this should imply an improvement in
polymerization on the former substrates. As indicated in
Table 1, the improved polymerization activity dependent
on a 50-phosphate group described here for SpPol4 is also
an attribute of Polb and Poll (23), and Polm (R. Jua ´rez,
P. Andrade and L. Blanco, unpublished data), but not of
Most residues involved in dRP lyase activity are conserved
between Polb and Poll (24). Among them, the nucleophile
residue at position Lys72(Polb) or Lys312(Poll), responsible
for 90% of the activity (18,24), is conserved in ScPol4
(Lys248), but not in Polm and TdT (which lack dRP lyase
activity), and is also absent in SpPol4. Unexpectedly,
SpPol4 was shown to have dRP lyase activity although it
lacks this conserved residue. Nonetheless, other residues pro-
posed in Polbto facilitate removal of the dRP group are indeed
present in SpPol4, and an alternative lysine (Lys240) could be
acting as the attacking nucleophile (for details see Figure 5). In
any case, and based on our in vitro assays, we propose that
SpPol4 could play a role in BER, as Polb and Poll. Based on
the demonstration of an intrinsic dRP lyase activity, a similar
role for ScPol4 has been proposed recently (64).
As shown in this paper, the relative nucleotide usage
of SpPol4 is different from that observed for other DNA-
dependent DNA polymerases of the X family (Polb, Poll
and Polm). In particular, SpPol4 preferentially inserts purine
Hydrolysis, alkylation, oxidation and deamination are the
major forms of DNA damage in all living cells, which are
mainly repaired by BER. It is worth noting that, at least
in mammalian cells, purines are lost 20-fold more frequently
than pyrimidines (?10000/cell/day versus ?500/cell/day,
respectively). Additionally, purines are the most frequently
alkylated bases and guanine is the base more prone to
oxidation, resulting in 8-oxoG (100–1000/cell/day) and
along with adenine in a ring-opened form called formami-
dopyrimidine (FaPyG and FaPyA). Only deamination, another
prevalent form ofDNA damage, occurs predominately at cyto-
sine, turning it into uracil (100–500/cell/hour) (1,21). There-
fore, it is tempting to speculate that the preference of SpPol4
for purine nucleotides has been adapted to cope with a more
intensive role of repairing purine bases.
In addition to its preference for small gaps, the unusual
capacity of SpPol4 to accept misaligned template–primer
molecules as a substrate and to realign 30-terminal mismatches
would be very convenient for microhomology-mediated
NHEJ. Moreover, some BER intermediates, as staggered
nicks made by an AP endonuclease in opposite strands, origi-
nate DSBs that would trigger the NHEJ pathway. Under these
circumstances, a DNA repair polymerase endowed with dRP
lyase activity would be very convenient to process the
damaged DNA ends and eliminate the dRP residues.
Physical and functional interactions with factors of NHEJ
(27,38,39,41), occurring through the BRCT domain of these
proteins. The presence of a BRCT domain at the N-terminus of
SpPol4 would support similar interactions with NHEJ factors
operating in S.pombe.
Most DNA polymerases have an exquisite sugar selectivity
and prefer toincorporate dNTPs over rNTPsby afactor of104-
to 106-fold (50). Sugar discrimination has been shown to
Table 1. Comparison of SpPol4 properties to other template-dependent
members of the DNA PolX family
aTaken from (64).
Nucleic Acids Research, 2005, Vol. 33, No. 154771
depend on a steric barrier for the 20-hydroxyl of an incoming
rNTP (4,50,65). Accordingly, Polb and Poll are unable to
incorporate rNTP since they have bulky residues close to
the 20position of the ribose of the incoming nucleotide
and TdT, which efficiently insert rNTPs (36,37,45), have a
was shown to be responsible for rNTP insertion (34). As
shown here, SpPol4 resembles Polm and TdT, as it also incor-
porates rNTP very efficiently. This property was expected
because the two residues equivalent to Polm (Gly433-Trp434)
are strictly conserved in SpPol4 (Gly434-Trp435). Strikingly, it
has been recently reported that ScPol4, although having two
aromatic residue at these positions (His517-Tyr518) also incor-
porates rNTPs with a high efficiency (64).
It has been demonstrated that NHEJ is a predominant repair
pathway in G1 phase and probably in non-cycling cells
(66–68). In contrast to dNTPs, abundant during S phase,
rNTPs are available at high levels in all phases of the cell
cycle (69,70). Therefore, as suggested for human Polm (38)
and ScPol4 (64), the extraordinary ability of SpPol4 to incorp-
orate rNTPs would be very convenient for a role in NHEJ.
Moreover, insertion of rNTPs might also be useful in BER to
repair modified or damaged bases into DNA throughout the
cell cycle that could be removed by the sequential action of
RNaseH35/RNaseH type II and Rad27/FEN-1 (71). Further
work should be carried out to ascertain this specific pathway
It has been reported that the imprinting step during mating-
type switching in S.pombe is an RNase-sensitive modification
that consists of one or two RNA residues incorporated into the
mat1 locus (55), which becomes a fragile chromosome site.
Taking into account the capacity of SpPol4 to incorporate a
few rNTPs in the DNA, it was tempting to speculate with a
probable involvement of SpPol4 in mating-type switching in
S.pombe. As shown here, the lack of SpPol4 (h90pol4D strain)
did not affect either the level of DSBs in the mat1 locus or the
mating-type efficiency with respect to the h90wild-type strain;
therefore, we conclude that the imprint should remain unaf-
fected. Thus, if there were some RNA residues in the mat1
locus, the incorporation would be SpPol4-independent.
Alternatively, the imprint could imply a strand-specific nick
with no flanking RNA residues (72).
In conclusion, the results presented here demonstrate that
SpPol4 shares biochemical properties with different members
of the PolX super-family; thus, it must be considered to be a
unique enzyme (see Table 1 for a comparison). Mammalian
PolXs became specialized to play a role in BER (Polb), in
NHEJ coupled with BER (Poll), microhomology-mediated
NHEJ (Polm) or V(D)J recombination (TdT). However, the
fact that both fission and budding yeasts had only one DNA
Pol X suggests that they are evolutionarily closer to the stem
ancestor of the family, which is also consistent with a less
specialized and multipotential role in different forms of DNA
repair, enabled by a combination of the biochemical properties
of their mammalian homologues.
Supplementary Material is available at NAR Online.
We thank Juan Jime ´nez for S.pombe strains and plasmids and
Aurelia Lahoz for her inestimable advice and help with
S.pombe handling and techniques. This work was supported
by Ministerio de Ciencia y Tecnologı ´a Grant BMC 2003-
00186, and by an institutional grant to Centro de Biologı ´a
Molecular ‘Severo Ochoa’ from Fundacio ´n Ramo ´n Areces.
A.S., R.J., A.J.P. and G.T. were recipients of a fellowship
from the Ministerio de Educacio ´n y Ciencia. Funding to pay
the Open Access publication charges for this article was
provided by the Spanish Ministry of Science and Technology.
Conflict of interest statement. None declared.
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