Molecular modeling and functional characterization
of the monomeric primase–polymerase domain from
the Sulfolobus solfataricus plasmid pIT3
Santina Prato
1
, Rosa Maria Vitale
2
, Patrizia Contursi
1
, Georg Lipps
3
, Michele Saviano
4
, Mose
´
Rossi
1,5
and Simonetta Bartolucci
1
1 Dipartimento di Biologia Strutturale e Funzionale, Universita
`
degli Studi di Napoli Federico II, Naples, Italy
2 Istituto di Chimica Biomolecolare, CNR, Pozzuoli, Naples, Italy
3 Institute of Biochemistry, University of Bayreuth, Germany
4 Istituto di Biostrutture e Bioimmagini, CNR, Naples, Italy
5 Istituto di Biochimica delle Proteine, CNR, Naples, Italy
In all cell types, chromosomal DNA replication is a
complex process entailing three enzymatic activities:
helicase activity for double-helix unzipping and prim-
ase and DNA polymerase for RNA primer de novo
synthesizing and elongation respectively [1,2].

plasmid pIT3 from Sulfolobus solfataricus strain IT3 was identified using a
structural–functional approach. The N-terminal domain of the pIT3 repli-
cation protein encompassing residues 31–245 (i.e. Rep245) was modeled
onto the crystallographic structure of the bifunctional primase–polymerase
domain of the archaeal plasmid pRN1 and refined by molecular dynamics
in solution. The Rep245 protein was purified following overexpression in
Escherichia coli and its nucleic acid synthesis activity was characterized.
The biochemical properties of the polymerase activity such as pH, tempera-
ture optima and divalent cation metal dependence were described. Rep245
was capable of utilizing both ribonucleotides and deoxyribonucleotides for
de novo primer synthesis and it synthesized DNA products up to several kb
in length in a template-dependent manner. Interestingly, the Rep245 prim-
ase–polymerase domain harbors also a terminal nucleotidyl transferase
activity, being able to elongate the 3¢-end of synthetic oligonucleotides in a
non-templated manner. Comparative sequence–structural analysis of the
modeled Rep245 domain with other archaeal primase–polymerases revealed
some distinctive features that could account for the multifaceted activities
exhibited by this domain. To the best of our knowledge, Rep245 typifies
the shortest functional domain from a crenarchaeal plasmid endowed with
DNA and RNA synthesis and terminal transferase activity.
Abbreviations
AEP, archaeo-eukaryotic replicative primases; dNTP, deoxyribonucleotide; MD, molecular dynamics; prim–pol, primase–polymerase; TdT,
terminal deoxyribonucleotidyl transferase; TP, template ⁄ primer.
FEBS Journal 275 (2008) 4389–4402 ª 2008 The Authors Journal compilation ª 2008 FEBS 4389
-independent activities that these enzymes perform in
addition to primer synthesis. For example, Sulfolobus
DNA primase has the additional catalytic property of
performing 3¢-terminal nucleotidyl transferase activity
[7,8], and archaeal replicative primases can use deoxy-
ribonucleotides (dNTPs) as a substrate for synthesizing

polymerases [11]. Furthermore, the conservation of
catalytic aspartate residues and their 3D arrangement
suggest that the catalysis mode is probably comparable
with the two-metal-ion mechanism of both RNA and
DNA synthesis [17].
In a previous study, we reported the findings of an
analysis of the complete sequence of the cryptic plas-
mid pIT3 isolated from the crenarchaeon S. solfatari-
cus strain IT3 [18]. The fully sequenced plasmid
contains six ORFs, the largest of which (ORF915)
spans over half the plasmid genome and encodes a
putative 100 kDa replication protein designated as
RepA [18]. Bioinformatic analyses of the predicted
amino acid sequence showed that the C-terminal half
of the RepA of the pIT3 plasmid is sequence-similar to
the helicases of the phage-encoded superfamily III pro-
teins. The N-terminal half of the pIT3 protein RepA
shows little sequence similarity to both the related
RepA of crenarchaeal plasmids and the ORF904 pro-
tein of the plasmid pRN1, which is the only enzyme
biochemically characterized to date in Sulfolobales
plasmids. Despite low sequence identity, multisequence
alignment highlighted major similarities in short
sequence motifs, e.g. two conserved aspartates in a
local group of hydrophobic amino acid residues which
are known to serve as ligands for divalent cations and
as tags revealing the presence of DNA polymerases in
the active site [18–20].
In this study, we report on the structural and func-
tional characterization of the shortest tri-functional

Despite low sequence identity (29% for the N-termi-
nal 32–103 region, but  17% for the modeled
sequence as a whole), the pairwise alignment in the
modeling procedure (Fig. 1A) shows no gaps and ⁄ or
insertions of more than two residues, highly conserved
residues (highlighted in yellow) are evenly distributed
among archaeal plasmids prim–pol domains, and both
Analysis of the pIT3 prim–pol domain S. Prato et al.
4390 FEBS Journal 275 (2008) 4389–4402 ª 2008 The Authors Journal compilation ª 2008 FEBS
the acidic residues D101, D103 and D166 and the
adjacent H138 are present in the active site. Moreover,
the construction of a reasonable model for the Rep245
prim–pol domain (as we designate it from now on)
from the pRN1 prim–pol structure was supported by
both the reliable FUGUE server score value (12.45,
with a recommended cut-off of 6) and the secondary
structure profile (data not shown), both of which point
to considerable fold similarity. To build the Rep245
model, we performed 16 pairwise and multiple
alignments of template and target sequences and used
deleted versions of the template structure. In overall
terms, the final model selected by reference to quality
score indices (Modeller objective function, Procheck
and 3D profile) was in agreement with the template.
Its rmsd value was 0.391 A
˚
and had been derived from
backbone superimposition at the Ca atom level in the
following regions: 31–60, 61–123, 128–130, 136–141,
150–159, 164–184, 199–230 and 233–244 of the Rep245

reported above the alignment and colored
according to the ribbon representation (cyan
cylinders for a helices, light-cyan cylinders
for 310 helices and light-blue arrows for
b strands). Highly conserved residues within
prim–pol domain sequences from archaeal
plasmids are highlighted in yellow, the three
acidic residues with the histidine of the
active site in red, the loop region in
magenta and the corresponding 1RNI
Zn-stem in gray. Cysteine residues are high-
lighted in green with the disulfide bonds
drawn as green lines. Sequence alignment
of the conserved motif between Pfu-prim-
ase and Rep245 is also reported in the
brown boxed region. (B) Ribbon representa-
tion of Rep245 homology model with a -
helices colored in cyan and b strands in
light-blue. The three acidic residues and the
adjacent histidine are shown as stick bonds
and colored in violet.
S. Prato et al. Analysis of the pIT3 prim–pol domain
FEBS Journal 275 (2008) 4389–4402 ª 2008 The Authors Journal compilation ª 2008 FEBS 4391
Zn-binding motif and the two disulfide bonds respec-
tively connecting the a4-helix to the b4 strand and the
b9 strand to the b10 strand at the bottom of the
Zn-stem loop in the pRN1 prim–pol structure. How-
ever, because the Zn-stem loop is a fairly self-standing
structure protruding from the interface between the
DNA binding and the active site subdomains, we man-

and 133-TGKGYH-138 in Rep245; Fig. 1A), although
not in the prim–pol domain of pRN1. The sequence
similarity observed reflects a comparable spatial
arrangement, because this motif is part of a b-strand-
loop situated close to the active site in either protein.
Again, a strong parallelism was observed for the latter
pair of residues: in the Pfu-primase structure, the
K300 residue is located in a loop left on the active site
and because of its poorly defined electronic density
other authors have suggested that it was likely to
change conformation upon DNA binding [14]; simi-
larly, as in Rep245, the R186 residue lies in the loop
(corresponding to the 1RN1 zinc knuckle motif) posi-
tioned left of the active site, we assumed that it could
plausibly be involved in sequence recognition and
DNA binding.
In sum, sequence–structure analysis highlighted that
the Rep245 domain of the pIT3 plasmid replication
protein shares structural features with other replicative
archaeal and eukaryotic enzymes and suggested simi-
larity at the functional level as well.
Expression and protein purification
Initially, we checked if the orf915 of the pIT3 plasmid
from the archaeal S. solfataricus strain IT3 actually
encoded a DNA polymerase. When the corresponding
protein was produced in E. coli, we found that it could
synthesize DNA products in a template ⁄ primer (TP)-
dependent polymerase reaction.
We designed a truncated variant of the full-length
pIT3 replication protein comprising the N-terminal

polymerase activity
Based on the results of structure–sequence analysis, we
characterized the functions of the Rep245 protein and
tried to determine optimal DNA polymerase activity
conditions.
Analysis of the pIT3 prim–pol domain S. Prato et al.
4392 FEBS Journal 275 (2008) 4389–4402 ª 2008 The Authors Journal compilation ª 2008 FEBS
The pH dependence of DNA polymerase activity
was investigated in the 5.0–10.0 range using the hetero-
polymeric 40 ⁄ 20-mer TP (Table 1). As shown in
Fig. 3A and Fig. S1, Rep245 was found to be active
over a broad pH range with maximal DNA template
elongation at pH 8.0.
Because all polymerases require divalent cations for
catalysis, we tested the effect of metal ions on enzyme
activity. The influence of Mg
2+
,Mn
2+
and Zn
2+
ions
on the synthesis function of Rep245 was assessed on
TP heteropolymeric DNA as a template (Fig. 3B).
First, because the protein was unable to perform DNA
synthesis without a metal ion activator (Fig. 3B) we
concluded that Rep245 polymerase activity was strictly
dependent on divalent cations. Second, because DNA
synthesis started promptly after the addition of 1 mm
MgCl

Thus, to verify if this unexpectedly low thermophi-
licity level was correlated to structural protein unfold-
ing, far-UV CD spectroscopy was used to assess the
structural stability of the Rep245 mutant. Following
30 min incubation at 60, 70 and 80 °C, we recorded
the CD spectra of the incubated Rep245 samples at
these temperatures. The absence of thermal unfolding
transitions provided evidence that temperature
increases did not result in detectable changes in the
secondary structure of the Rep245 protein (data not
shown). Based on this finding, we could rule out that
the loss of DNA polymerase activity sparked off by
temperature increases in the tested range was to be
traced to thermal enzyme inactivation.
30 prim-pol 245
915
1
Walker A motif
RepA
A
B
C
Rep245
1
245
6His
prim-pol
Rep516
6His
1

protein extracts at various stages of the purification of Rep245.
Lane M, molecular mass markers; lane 1, crude extract from unin-
duced Escherichia coli control culture; lane 2, crude extract from
induced E. coli (pET-Rep245) cells; lane 3, heat-treated sample; lane
4, eluate from the nickel affinity chromatography; lane 5, eluate
from the Resource-Q cation-exchange column. (C) Purified trun-
cated proteins. SDS ⁄ PAGE of purified Rep245 and Rep516 pro-
teins. Lane M, molecular mass markers; lane 1 and 2, purified
C-His
6
-tagged Rep516 (59 kDa) and Rep245 (29 kDa), respectively.
Table 1. DNA substrates used in this study. The position of the
radioactive label is marked with an asterisk.
Template-primer used for polymerase assay
TP 40 ⁄ 20-mer
40-mer 3¢-GCGCCTCTAACGAAGATAGGATCCGTGTGTCTTAGCTTCC-5¢
20-mer *5¢-CGCGGAGATTGCTTCTATCC-3¢
Oligonucleotides used for TdT assay
TEMP
20-mer *5¢-CGAACCCGTTCTCGGAGCAC-3¢
oligo(dT)
28
S. Prato et al. Analysis of the pIT3 prim–pol domain
FEBS Journal 275 (2008) 4389–4402 ª 2008 The Authors Journal compilation ª 2008 FEBS 4393
Eventually, heat resistance tests conducted by assay-
ing residual polymerase activity after 15 min incuba-
tion at temperatures between 50 and 80 °C showed
that Rep245 was fairly stable even after incubation at
80 °C, when its residual activity was found to be 60%
of the corresponding level of non-preincubated samples

DNA primers into longer products (Fig. 4C). Temper-
ature increases were seen to influence the size of DNA
products: small amounts of DNA primers between 16
and 20 nucleotides in size were synthesized at 30 °C; in
the temperature range between 40 and 65 °C, DNA
primer formation was both more clearly observable
and accompanied by the appearance of longer DNA
products. Because no product was observed when
the protein was not included in the reaction mixture,
this reaction was clearly template dependent and
specific.
The fact that the Rep245 variant retained the capabil-
ity of the RepA full-length protein of synthesizing and
elongating DNA products, although with a reduced
80
100
120
20
40
60
Relative acitivity (%)
Temperature (°C)
0
40 50 60 70 80 90
60
80
100
Relative activity (%)
20
40

Fig. 3. Effects of pH, divalent cations and temperature on Rep245 polymerase activity. Polymerase activity was assayed on TP heteropoly-
meric 40 ⁄ 20-mer DNA as the substrate. Reaction products were separated on a 20% polyacrylamide ⁄ urea gel and quantified by PhosphoIm-
ager. (A) Graphical representation of the pH dependence. Buffer systems (25 m
M final concentration and pH measured at 65 °C) were as
follows: Na-acetate (pH 5.0, 5.4 and 5.8), Tris ⁄ HCl (pH 6.5, 7.0, 7.5 and 8.0) and glycine ⁄ NaOH (pH 8.6, 9.0 and 9.6). (B) Dependence of
Rep245 polymerase activity on metal ions. The results are the means of three independent experiments. (C) The dependence of polymerase
activity on the temperature was determined by assaying the enzyme in the standard reaction mixture at the indicated temperatures. (D)
Thermal stability of Rep245 was tested by pre-incubating the enzyme for 20 min at the indicated temperatures (NP, not pre-incubated);
enzyme residual activity was then assayed on TP heteropolymeric 40 ⁄ 20-mer DNA, as described in Experimental procedures.
Analysis of the pIT3 prim–pol domain S. Prato et al.
4394 FEBS Journal 275 (2008) 4389–4402 ª 2008 The Authors Journal compilation ª 2008 FEBS
specific activity value (0.607 nmol dNTPsÆmin
)1
Æmg
)1
protein i.e.  20% of the corresponding level of the
RepA full-length protein’s polymerase activity measured
by the DE-81 filter binding assay) was evidence that our
structural homolog model included an active DNA
polymerase and primase domain within the N-terminal
1–245 amino acids of the pIT3 replication protein.
Furthermore, the progressive accumulation of smal-
ler length products observed for Rep245 might point
to high-frequency enzyme–DNA dissociation during
catalysis as a result of the higher temperatures. When
Rep516 was tested under identical assay conditions we
observed a more pronounced increase in RNA ⁄ DNA
synthesis. As shown in Fig. 4C, Rep516 mainly synthe-
sized larger molecular size DNA products that had not
entered the polyacrylamide gel; a negligible accumula-

incorporate dATP and dGTP used for the test at the
3¢-end of the 28-mer homo-oligomer (oligodT) and
20-mer heteropolymeric (TEMP) substrates, respec-
tively (for sequence details see Table 1), albeit at
different levels of efficiency (Fig. 5A,C). Interestingly,
template
KOH
A
C
B
Rep245
+
–
+
–
+–
+
+
++–
–
1234
16 nt
20 nt
28 nt
28 nt
20 nt
35 n
t
ATP
ATP

elongated RNA primers (lane 1) that can be extended to longer
products by further 30 min incubation in the presence of 0.2 m
M
dNTPs (lane 3) or 0.2 mM dNTPs and 0.5 U Taq DNA polymerase
(lane 4). Neither primer nor extension products were seen when
Rep516 was omitted from the reaction with Taq polymerase (lane
2). (C) DNA primer synthesis and their elongation. The primase
activities of Rep245 and Rep516 proteins were assayed between 5
and 90 °C for 30 min on M13 single-stranded DNA, with dNTPs
including [
32
P]dATP[aP] as substrates. The approximate size of the
bands (in nucleotides) is indicated on the right-hand side of each
panel.
S. Prato et al. Analysis of the pIT3 prim–pol domain
FEBS Journal 275 (2008) 4389–4402 ª 2008 The Authors Journal compilation ª 2008 FEBS 4395
when ribonucleotides were included in the reaction
mixtures, Rep245 was able to elongate synthetic oligo-
nucleotides, although it showed no preferential use of
any rNTPs in the transferase activity (Fig. 5B,D). The
longer variant Rep516 was also tested for nucleotidyl
transferase activity under identical experimental condi-
tions. As already described for DNA and RNA syn-
thesis, Rep516 proved more efficient than Rep245 in
elongating the 3¢-ends of synthetic oligonucleotides
(data not shown).
Because our enzymatic assays were conducted at
60 °C, a temperature at which hairpin loop-like DNA
structures are likely to be fairly unstable, we were able
to rule out that the elongation products observed had

its length and within-domain location, the loop
region of the pIT3 prim–pol domain which replaces
the Zn-stem motif could play a comparable role to
that ascribed to the Zn-stem motif in DNA interac-
tion [24]. A sequence–structure comparison of the
Rep245 model with other archaeal primase–polyme-
rases revealed the conservation of motifs which were
either absent from the pRN1 prim–pol domain or
slightly different from those occurring therein. These
differences may account for the fairly different func-
tions performed by the prim–pol domain of the pIT3
plasmid in vitro, i.e. DNA and RNA synthesis and
3¢-terminal nucleotidyl transferase activity.
Accordingly, we used the modeled pIT3 prim–pol
structure in designing the truncated Rep245 protein
containing the residues predicted to be responsible for
polymerase and primase catalysis, and reported on the
functional characterization of the main functions of
this protein.
C
G
U
A
dC
dG dT
dAAB
CD
28-mer
0312 4 0 312 4
0312 4 0 312 4

for in vivo catalysis [1]. Similarly, the DNA polymerase
activity of Rep245 was found to be dependent on diva-
lent cations, especially Mg
2+
ions which probably act
as physiological metal activators, in a broad optimum
concentration range between 5 and 10 mm. By con-
trast, polymerase activity is stimulated by Mn
2+
ions
at low concentrations (1.0–2.5 mm) and strongly inhib-
ited at higher concentrations. The ability of polymeras-
es to use Mn
2+
instead of Mg
2+
as a required
cofactor is well established [25]. However, the bio-
chemical properties of polymerases are altered as a
result of replacing Mg
2+
with Mn
2+
, which reduces
substrate selection stringency and incorporation fidelity
[26].
Thermal activity analysis of Rep245 revealed an
optimal temperature of 65 °C, i.e.  10 °C lower than
the growth temperature of the natural host S. solfatari-
cus strain IT3 harboring the pIT3 plasmid. Hence,

inherently able to count the number of bases
incorporated.
A reasonable structural interpretation of the primase
activity of Rep245 suggests involvement of the K135
and R186 residues, which have counterparts in Pfu-
primase, although not in the pRN1 prim–pol protein.
In archaeal and eukaryotic primases, the K135 residue
(the counterpart of R148 in Pfu-primase) is part of a
highly conserved motif which is absent from the pRN1
prim–pol domain (see alignment in Fig. 1A). The
sequence similarity observed reflects a similar spatial
arrangement, because this motif is part of a b-strand-
loop situated close to the active site in either protein.
Similarly, both the R186 residue in the Rep245 domain
and K300, its counterpart in Pfu-primase, were
contained in a loop that is plausibly involved in DNA
recognition and binding and is positioned left of
the active site [14].
Rep245 is both capable of de novo synthesis of
DNA primers and of elongating them. Long DNA
extension products were observed on the ssDNA tem-
plate when dNTPs were used as substrates, although
primase activity was found to prevail over DNA elon-
gation at higher temperatures. Such reduced DNA
elongation activity might either depend on dissociation
of the Rep245 prim–pol ⁄ ssDNA template complex or
on the fact that Rep245 translocation along the
substrate is probably hindered by the absence of the
additional amino acids needed to stabilize the enzyme–
DNA complex. This explanation seems to be

cules in a non-templated manner. To our knowledge,
this is the first evidence that a prim–pol domain
encoded by a crenarchaeal plasmid is intrinsically able
to perform 3¢-terminal nucleotidyl transferase activity.
Similarly, DNA primase from the S. solfataricus
crenarcheon has been shown to synthesize DNA in a
template-independent manner [7,8]. Interestingly, this
property is shared by the X family of human DNA
polymerases, which includes the TdT enzymes and two
additional members, Pol k [29] and Pol l [30]. The
latter two enzymes are functionally malleable to the
point of carrying out various nucleic acid synthesis
reactions on a wide range of substrates [31–33]. Fur-
thermore, like the TdT enzyme [34], the Rep245 protein
can incorporate ribo- and deoxynucleotides in vitro.
A noteworthy finding is that this functional equivalence
is matched by structural relationships between the
catalytic subunit of archaeal primases and the active
site of the X family of polymerases [23]. Indeed, unlike
the pRN1 prim–pol protein whose motif is DXE D,
the Rep245 protein, the X family of DNA polymerases
and the TdT enzymes have the DXD D motif in the
carboxylate triad in common. An additional major
finding reported previously in the literature is a drastic
reduction in enzymatic activity observed when the sec-
ond aspartic residue in the human TDT enzyme motif
is mutated to glutamate [22].
Thanks to the modular architecture of the replication
protein from the pIT3 plasmid, we were able to design
Rep245 and Rep516 truncated proteins and to charac-

P]ATP[aP] (3000 CiÆmmol
)1
) and
[
32
P]ATP[cP] (3000 CiÆmmol
)1
) were purchased from Per-
kin–Elmer (Waltham, MA, USA). The expression vector
pET-30c(+) was supplied by Novagen (Milan, Italy).
Homology modeling and MD calculations
Sequence search against PDB using psi-blast [35] identified
the crystallographic structure of ORF904 bifunctional
DNA primase–polymerase from the archaeal plasmid
pRN1 at 1.85 A
˚
of resolution (PDB entry 1RNI) [13], as
the best template for Rep245 (32–103, 29% of identity). A
sequence search by fold recognition as implemented in the
FUGUE server [36] also identified the same protein which
was then selected as the best template (Z-score 12.41). To
build the Rep245 model, 16 pairwise and multiple align-
ments between the template and target sequences were
proved, also using modified versions of template structure.
The alignments were carried out with clustal w v. 1.83
[37] and manually edited in order to better align secondary
structure elements of the template with the consensus for
the target sequence deriving from phd and prof secondary
structure prediction programs [38], along with the structural
alignment deriving from FUGUE server. For each align-

)1
) at constant volume, gradually heating to
300 K, followed by 60 ps restrained MD (5 kcalÆmol
)1
ÆA
˚
)1
)
at constant pressure to adjust the system density. The
Analysis of the pIT3 prim–pol domain S. Prato et al.
4398 FEBS Journal 275 (2008) 4389–4402 ª 2008 The Authors Journal compilation ª 2008 FEBS
production MD simulation was carried out at 300 K at
constant pressure for 1.5 ns, with a time-step of 1.5 fs. The
bonds involving hydrogens were constrained using the
shake algorithm [43]. The snapshots were saved every
10 000 steps and analyzed with molmol [44].
Construction of bacterial expression plasmids
Truncated variants of the orf915 gene encoding a putative
replication protein (RepA) were amplified by PCR from
S. solfataricus plasmid pIT3 as a template [18], using
HF Taq DNA polymerase (Roche Applied Science). A
deletion mutant, which contains the N-terminal residues
1–245, was amplified with primers F-245 (5¢-CGGTGCC
GC
CATATGGATAGTTTC-3¢) and R-245 (5¢-CTCGAG
CTGTTCTTTCCT-3). A N-terminal variant comprising
residues 1–516 was obtained with primers F-245 (see above)
and R-516 (5¢-
CTCGAGAGGCTCACGGGC-3¢). The
primers F-245, R-245 and R-516 introduce NdeI and XhoI

centrated (Amicon, Millipore Corp., Bedford, MA, USA)
and loaded on to a HiTrapÒ affinity column (GE Health-
care Europe GmbH, Milan, Italy) pre-equilibrated in
20 mm sodium phosphate pH 7.4, 500 mm NaCl, 10 mm
imidazole (buffer A). The column was equilibrated with
buffer A containing 20 mm imidazole and the recombinant
proteins were eluted with buffer A supplemented with
250 mm imidazole. The active fractions were pooled and
dialyzed against 20 m m Tris ⁄ HCl pH 9.0 (buffer B). The
dialyzed sample was loaded on to a Resource Q column
(GE Healthcare) developed with a linear gradient of 0–
400 mm NaCl in 20 mm Tris ⁄ HCl pH 9.0. Pooled fractions
were extensively dialyzed against 20 mm sodium phosphate
pH 7.4 and 20% glycerol (buffer C). The mutant Rep516
was purified by the same procedure used for the Rep245.
The purification was monitored after each step by Coomas-
sie Brillant Blue and silver-stained SDS ⁄ PAGE gels. Protein
concentrations were determined by the Bradford assay, and
enzyme stocks (typically 1 mgÆmL
)1
in buffer C) were
stored at )20 °C.
DNA substrates
DNA oligonucleotides were synthesized by MWG-Biotech-
nologies AG (Ebersberg, Germany). When appropriate,
labeling at the 5¢-end was performed using [
32
P]ATP[cP] and
T4 polynucleotide kinase (Roche Applied Science). Unincor-
porated nucleotide was removed with a NickÔ column (GE

). Protein (0.5 lm) was incubated
with TP, 10 lm dNTPs and [
32
P]dATP[aP] in 25 mm
Tris ⁄ HCl (pH 7.5), 1 mm dithiothreitol, 5 mm MgCl
2
in a
10 lL reaction. Aliquots of the reactions were pipetted
onto a DE81 filter; unincorporated dNTPs were removed
by washing with 0.5 m sodium phosphate, pH 7.0, and
filters were counted.
S. Prato et al. Analysis of the pIT3 prim–pol domain
FEBS Journal 275 (2008) 4389–4402 ª 2008 The Authors Journal compilation ª 2008 FEBS 4399
Determination of pH and divalent ion optima for
polymerase activity
The influence of pH on Rep245 polymerase activity was
determined using the standard polymerase assay in buffer
solutions whose pH was adjusted to the desired value at
65 °C. The buffer systems used (25 mm final concentra-
tion) were as follows: Na-acetate (pH 5.0, 5.4 and 5.8),
Tris ⁄ HCl (pH 6.5, 7.0, 7.5 and 8.0) and glycine ⁄ NaOH
(pH 8.6, 9.0 and 9.6). Reactions were carried out for
30 min at 65 °C and the products resolved by denaturing
gel electrophoresis, as previously described. The amount
of radioactivity in each lane was quantified by Phosphor-
Imager.
The dependence of the Rep245 polymerase activity on
divalent cations was determined using Mg
2+
,Mn

of M13 mp18 ssDNA, 100 lm each of dCTP, dGTP and
dTTP, and 10 lm [
32
P]dATP[aP], in the temperature range
5–90 °C for 30 min. The primase reactions were resolved
onto 10 and 20% denaturing polyacrylamide gel, followed
by autoradiography.
Terminal transferase activity
The 3¢-terminal nucleotidyl-transferase activity was assayed
on 5¢-labeled oligo(dT)
28
and a random 20-mer oligonucleo-
tide (Table 1), with 0.1 mm unlabeled (d)NTPs and 1.7 lm
Rep245 or 0.8 lm Rep516 in the polymerase assay buffer.
Samples were incubated at 60 ° C for 30 min, the aliquots
were analyzed by electrophoresis using denaturing 20% poly-
acrylamide gels and the radioactivity was detected by using a
PhosphorImager. Commercial TdT was used according to
the manufacturer’s protocol (Roche Applied Science).
CD spectrum
CD in the far-UV region was performed with a thermo-
stated Jasco J-815 spectropolarimeter using 0.1 cm path
length quartz cuvettes. The concentration of Rep245 pre-
pared in 10 mm sodium phosphate (pH 7.4) was 5 lm. CD
spectra were recorded at 60, 70 and 80 °C between 190 and
260 nm with a step increase of 0.2 nm, and a bandwidth of
1 nm. Thermal stability of Rep245 was measured by incu-
bating the protein at 60, 70 and 80 °C for 30 min and then
recording the CD spectra of the incubated samples at indi-
cated temperatures.

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Analysis of the pIT3 prim–pol domain S. Prato et al.
4400 FEBS Journal 275 (2008) 4389–4402 ª 2008 The Authors Journal compilation ª 2008 FEBS
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